Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Emergence of β-lactamase- and carbapenemase- producing Enterobacteriaceae at integrated fish farms

Emergence of β-lactamase- and carbapenemase- producing Enterobacteriaceae at integrated fish farms Background: Epidemiological studies suggested that determinants for antibiotic resistance have originated in aquaculture. Recently, the integrated agriculture-aquaculture system has been implemented, where fish are raised in ponds that receive agriculture drainage water. The present study aims to investigate the occurrence of β- lactamase and carbapenemase-producing Enterobacteriaceae in the integrated agriculture-aquaculture and the consequent public health implication. Methods: Samples were collected from fish, fishpond water inlets, tap water, outlet water, and workers at sites of integrated agriculture-aquacultures. Samples were also taken from inhabitants of the aquaculture surrounding areas. All samples were cultured on MacConkey agar, the Enterobacteriaceae isolates were tested for susceptibility to cephalosporins and carbapenems, and screened for bla , bla , bla , bla , bla , bla , bla ,and bla .Strains CTX-M-15 SHV OXA-1 TEM PER-1 KPC OXA-48 NDM having similar resistance phenotype and genotype were examined for the presence of Incompatible (Inc) plasmids. Results: A major proportion of the Enterobacteriaceae isolates were resistant to cephalosporins and carbapenems. Among the 66 isolates from fish, 34 were resistant to both cephalosporin and carbapenem groups, 26 to carbapenems alone, and 4 to cephalosporins alone. Of the 15 isolates from fishpond water inlets, 8 showed resistance to both groups, 1 to carbapenems alone, and 5 to cephalosporins alone. Out of the 33 isolates from tap water, 17 were resistant to both groups, and 16 to cephalosporins alone. Similarly, of the 16 outlet water isolates, 10 were resistant to both groups, and 6 to cephalosporins alone. Furthermore, of the 30 examined workers, 15 carried Enterobacteriaceae resistant strains, 10 to both groups, and 5 to cephalosporins alone. Similar strains were isolated from the inhabitants of the aquaculture surrounding areas. Irrespective of source of samples, strains resistant to all examined antibiotics, carried predominantly the carbapenemase gene bla either alone or with the β-lactamase genes (bla , bla , bla ,and bla ). The KPC CTX-M-15 SHV TEM PER-1 isolates from fish, water, and workers harboured a wide-range of multi-drug-resistance Inc. plasmids, which were similar among all isolates. Conclusion: The present findings suggest transmission of the resistance genes among Enterobacteriaceae strains from different sources. This reiterates the need for control strategies that focus on humans, animals, water, and sewage systems to solve the antibiotic resistance problem. Keywords: Egypt, Aquaculture-agriculture-farms, Enterobacteriaceae, β lactam-resistance, Inc. plasmids, PBRT kits * Correspondence: e.hamza@gmx.ch Department of Zoonoses, Faculty of Veterinary Medicine, Cairo University, Giza square, PO Box 12211, Cairo, Egypt Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 2 of 12 Introduction replicons are now recognised, which represent the major A link between aquaculture and the development of replicase genes of the Inc. plasmids circulating among antibiotic resistance has been demonstrated by an in- Enterobacteriaceae, including HI1, HI2, I1, I2, X1, X2, creasing body of research [1, 2]. Worldwide, there is a X3, X4, L, M, N, FIA, FIB, FIC, FII, FIIS, FIIK, FIB-KN, massive increase in fish farming, which is associated with FIB-KQ, W, Y, P1, A/C, T, K, U, R, B/O, HIB-M, and intensive use of antibiotics to combat bacterial infections FIB-M [21]. [3]. Other potential explanations are contamination of Recently in Egypt, small scale use of the integrated aquaculture with antibiotic residues and antibiotic resist- agriculture-aquaculture system began to be used. The ant bacteria coming from animals through using the so- aims of the present study are, therefore, to investigate called livestock integrated aquaculture system [4–6]. Re- the extent of antibiotic resistance in aquacultures inte- cently, an integrated agriculture-aquaculture system has grated with agriculture in Egypt and its public health sig- been implemented to save water resources [7]. In such nificance. Accordingly, this was performed through (i) system, fish are reared in ponds receiving water from assessing the prevalence of β-lactamase (βLPE) and crop farms through irrigation canals. carbapenemase-producing Enterobacteriaceae (CPE) in The major concern with the use of antibiotics in aqua- the water inlets of aquaculture originating from agricul- culture is that fish do not effectively metabolize antibi- ture and supply fishponds, in fish, tap water, and the otics and pass them largely unused in faeces [8]. This outlet water of the aquaculture; (ii) determination of the encourages the development of antibiotic resistance in occurrence of βLPE and CPE in aquaculture workers bacteria present in fish and the surrounding environ- and inhabitants of the aquaculture surrounding areas, ment [1, 2, 9]. Fishponds are not subject to frequent those having a history of eating fish from these aquacul- water exchange, resulting in accumulation of antibiotic- ture and also use the same tap water source as the aqua- residues and -resistant bacteria, which are then dissemi- culture (iii) identifying the presence and type of Inc. nated at harvest time. plasmids carried by the resistant strains isolated from Fish bacteria particularly Enterobacteriaceae can ex- different sources. change antibiotic resistance genes with human and ani- mal bacteria [10, 11]. Among these, β-lactam resistant Materials and methods bacteria are of great concern, as they are becoming non- Sample collection susceptible to nearly all available antibiotics [12, 13]. Four integrated agriculture-aquaculture farms were ran- They include extended-spectrum β-lactam (ESBL) and domly selected from the Giza governorate, the third- carbapenem-resistant (CRE) Enterobacteriaceae. ESBL largest city in Egypt. The selected farms are located near carry a broad spectrum of β-lactamase enzymes that each other (approx. 5 km away from each other), they hydrolyse a wide range of penicillin and cephalosporin combine agriculture with fish farming As illustrated in antibiotics, but not carbapenems [14]. However, CRE Fig. 1, the fish are raised in ponds, each fishpond re- carry carbapenemase enzymes that confer resistance to ceives water from agriculture drainage water through a nearly all β-lactam antibiotics, including carbapenems separate water pipe (Water inlet). Tap water is another [15]. The β-lactamase enzymes encompass members of source of water for the aquaculture, which is used to dis- the TEM and SHV families and other groups, such as solve medicines given to the fish and sometimes as a CTX-M, OXA, and PER β-lactamases [16]. The carbape- supply for the fishponds in case of shortage of agricul- nemase enzymes are a diverse group of β-lactamases, the ture water. Tap water is also used by the workers for most remarkable of which are the big five enzymes KPC, drinking and hand washing. Outlet water represents the NDM, OXA-48, IMP, and VIM [17]. There is strong evi- waste drained from fishponds through pipes. dence that the β-lactamase and carbapenemase encoding Samples were aseptically collected from internal organs genes are found on plasmids that facilitate their transfer (liver, spleen, and kidney) from 105 apparently healthy among bacteria of different genera and kingdoms [15, fresh Tilapia fish (Oreochromis niloticus). Additionally, 16]. Plasmids are extra-chromosomal, self-replicating samples were collected from fishpond water inlets (n = genetic elements, carried by the bacteria. Plasmids hav- 30), tap water (n = 44), and outlet water (n =26). The ing the same origin of replication cannot stably co-reside water samples were placed in sterile glass bottles contain- within the same bacterial cells, are known as incompat- ing sodium thiosulphate and were filtered through 0.4 μm ible (Inc) [18]. The Inc. plasmids can positively select for pore size nitrocellulose filters (Sartorius, Aubagne, France) the presence of resistance genes [19]. Identification of placed on tubes containing peptone broth [22]. the Inc. plasmids in the bacterial isolates helps to trace Faecal and hand swab samples were collected from the transfer of the resistance genes. Currently, a PCR humans working in the aquaculture farms (n = 30), hav- method is accepted to classify the Inc. resistance plas- ing contact with the fish, and who agreed to participate mids based on their type of replicon [20]. Thirty in the study. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 3 of 12 Fig. 1 Illustration of the setup of the integrated agriculture-aquaculture farms included in the present study. Locations where samples were collected are indicated with an asterisk (*) Additionally, faecal samples were collected from in- instructions. The accuracy of API 20E results was 100% as habitants of the aquaculture surrounding areas (n = 45), determined by API web software (BioMérieux). who use the same source of tap water as the aquacul- tures and were admitted to the local medical centre with Antibiotic susceptibility test diarrhoea after eating fish from the tested aquacultures. Confirmed strains of Enterobacteriaceae were examined for susceptibility to different antibiotics (Oxoid) using the disk diffusion test on Mueller-Hinton agar (Oxoid). Bacterial isolation and identification The antibiotics included were cephalosporins [Cefoxitin All samples were inoculated into Trypticase soy broth (FOX, 30 μg), Ceftazidime (CAZ, 30 μg) Cefotaxime tubes and were incubated at 37 °C for 24 h. A loop-ful (CTX, 30 μg), Ceftriaxone (CRO, 30 μg)] and the carba- from the previously incubated tubes were streaked onto penems [Imipenem (IPM, 10 μg), Meropenem (MEM, MacConkey agar plates (Oxoid Ltd., Cairo, Egypt) and 10 μg), Ertapenem (ETP, 10 μg)] groups. The results Eosin methylene blue agar (EMB) (Oxoid) and were in- were interpreted according to the clinical breakpoints cubated aerobically at 37 °C for 24 h. Suspected colonies recommended by CLSI (Clinical laboratory Standard were purified through subculture on MacConkey agar Institute) [24]. plates and were examined for morphology and pheno- Based on CDC definitions [25], Enterobacteriaceae iso- type traits according to Collee et al. [23]. lates resistant to at least one of the carbapenem drugs (Imipenem, Meropenem, Ertapenem, or Doripenem) or Biochemical identification produce a carbapenemase gene are called carbapenem- Pure colonies were subjected to API 20E kits (BioMerieux, resistant Enterobacteriaceae (CRE). Accordingly, in the Marcy-l’Étoile, France) according to the manufacturer’s present study, isolates resistant to at least one of the Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 4 of 12 tested carbapenem drugs were considered CRE and were isolated Enterobacteriaceae strains and type of samples tested for the presence of carbapenemase genes. (fish, fishpond water inlets, tap water, and outlet water) (Table 1). Chi-square (X ) test was also performed to PCR for detection of β-lactamase- and carbapenemase- examine whether the prevalence of antibiotic resistance encoding genes is influenced by the type of samples or species of bac- Genomic DNA from resistant isolates was extracted teria, or both combined. On each occasion, the signifi- using a QIAamp® mini kit (Qiagen, Hombrechtikon, cance of differences was set at p value < 0.05. Switzerland). PCR was performed to identify β- lactamase-encoding genes (bla , bla , bla , Results CTX-M-15 SHV TEM bla ) using specific oligonucleotide primers as previ- Prevalence of Enterobacteriaceae in apparently healthy PER-1 ously described [26]. The PCR for bla was done Nile tilapia fish and water from integrated agriculture- OXA-1 using specific oligonucleotide primers according to [27]. aquacultures Multiplex PCR [28] was performed to identify As demonstrated in Table 1, of the 105 tested apparently carbapenemase-encoding genes (bla bla , and healthy Nile tilapia fish, 66 (63%) showed the presence KPC, OXA-48 bla ) using specific oligonucleotide primers [29]. The of Enterobacteriaceae. Additionally, the occurrence of NDM three genes were chosen to include one representative Enterobacteriaceae was also high among the collected candidate from each of the three carbapenemase groups samples from fishpond water inlets (15/30; 50%), tap (Serine, OXA, and MLBs) and the selection was based water (33/44; 75%), and outlet water (16/26; 62%) from on previous findings in Egypt [28]. The sequences of the the aquacultures. E. coli represented the major species of primers used in the present study are shown in S1. Enterobacteriaceae isolated from fish and water. Entero- bacter cloacae complex (ECC) and Klebsiella (K.) pneu- Characterisation of Inc. plasmids present in the resistant moniae were also detected, but with a significantly lower isolates using PCR-based-replicon-typing prevalence than E. coli. Genomic DNA from the isolates that were resistant to all tested antibiotics and showed similar resistance geno- Occurrence of cephalosporin- and carbapenem-resistant types (n = 30) were subjected to plasmid typing using Enterobacteriaceae in the integrated agriculture- the PBRT 2.0 kit (PCR based replicon typing Ver.01/06/ aquacultures 2017, Diatheva, Fano, Italy). The kit contains eight The Enterobacteriaceae isolates from fish and water multiplex PCR, each of which amplifies three to four samples of the aquacultures were screened for resistance targets, allowing detection of a total of 30 replicons. against carbapenems (CRE) and cephalosporins (CEPH). It also includes positive control plasmids for the 8 The resistant strains were examined for carbapenemase multiplex PCR. The kit was used following the manu- and β-lactamase genes (Figs. 2, 3, 4). facturer’s instructions. Interpretation of the results The Chi-square test showed that resistance to carbapen- was performed according to the manufacture instruc- ems and CEPH was significantly influenced by the source tion with IncF plasmids designated as multi-replicons of samples (fish or water), but not by species of bacteria when they showed a combination of FII replicons (E. coli, ECC,and K. pneumoniae). Indeed, Figs. 2, 3, 4 with FIA or FIB [30, 31]. demonstrated that the resistance phenotype was similar between the three species of bacteria. Statistical analysis As displayed in Figs. 2, 3, 4, the isolates were grouped PASW Version 18 software (SPSS Inc., Chicago, IL, according to the resistance phenotype into; CRE and all USA) was used for statistical analysis. Descriptive statis- CEPH (isolates resistant to at least one of the tested car- tics were run and showed that the data are normally dis- bapenems and all tested cephalosporins); CRE and 2 tributed. A Chi square (X ) test was used to determine CEPH (isolates resistant to at least one of the tested car- whether there is a difference in the prevalence of the bapenems and two of the tested cephalosporins); CRE Table 1 Prevalence of Enterobacteriaceae strains in Tilapia fish and water at four different aquacultures Source No. of Enterobacteriaceae-positive samples (No. examined) Total E. coli ECC K. pneumoniae Fish (105) 66 (63%) 45* 12 9 Fishpond water inlets (30) 15 (50%) 9* 42 Tap water (44) 33 (75%) 22* 65 Outlet water (26) 16 (62%) 12* 22 * indicates a significant difference in the prevalence of E. coli from that of ECC and K. pneumoniae Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 5 of 12 Fig. 2 Number of carbapenem- and cephalosporin- resistant Enterobacteriaceae isolates among fish. The Enterobacteriaceae isolates from fish (n = 66), including E. coli (n = 45), ECC (n = 12), and K. pneumoniae (n = 9) were screened for resistance against carbapenems (CRE) and cephalosporins (CEPH: FOX, CAZ, CTX, and CRO). The strains are grouped according to their resistance phenotypes as follow: CRE and all CEPH (isolates resistant to at least one of the tested carbapenems and all tested cephalosporins); CRE and 2 CEPH (isolates resistant to at least one of the tested carbapenems and two of the tested cephalosporins); CEPH (isolates resistant to 2 or more of the tested cephalosporins but not to carbapenems); CRE (isolates resistant to at least one of the tested carbapenems but not to any of the tested cephalosporins). Each phenotype is marked with colour and its resistance genotypes are also provided; carbapenemase (bla , bla , and bla ) and β-lactamase (bla , bla , bla , KPC OXA-48 NDM CTX-M-15 SHV OXA-1 bla , and bla ) genes. (n) represents numbers of resistant strains TEM PER-1 (isolates resistant to at least one of the tested carbapen- carbapenemase genes (bla , bla , bla ). Of KPC OXA-48 NDM ems, but not to any of the tested cephalosporins); CEPH the 26 fish isolates resistant only to carbapenems, 17 (isolates resistant to 2 or more of the tested cephalospo- were carbapenemase producing, while 9 were negative to rins, but not to carbapenems). the three screened carbapenemase genes (Figs. 2, 3, 4). Of the 66 fish isolates, 64 (45 E. coli,12 ECC,7 K. pneumoniae) showed resistance as follow; 29 to carba- High numbers of aquaculture workers carried penems and all tested cephalosporins, 5 to carbapenems cephalosporin- and carbapenem-resistant and two of the tested cephalosporins, 26 to carbapenems Enterobacteriaceae alone, and 4 to cephalosporins alone (Fig. 2). Addition- Hand swab and faecal samples were collected from ally, of the 15 isolates from fishpond water inlets, 7 were humans working in the integrated agriculture- resistant to carbapenems and all cephalosporins, 1 to aquacultures. A relatively high proportion of the workers carbapenems and two cephalosporins, 1 to carbapenems showed the presence of Enterobacteriaceae in hand alone, and 5 to cephalosporins alone (Fig. 3a). All the 33 swabs (20 / 30) and faecal samples (15 / 30) (Fig. 5, isolates from tap water were resistant. Among them, 17 Panel I). Like in fish and water isolates, E. coli was the were resistant to carbapenems and all cephalosporins, predominant species isolated from the human samples and 16 were resistant to cephalosporins alone (Fig. 3b). (Panel I A and B). Among the 20 isolates from hand Similarly, the 16 outlet water isolates were resistant, 9 to swabs, 5 were resistant to carbapenems and all cephalo- carbapenems and all cephalosporins, 1 to carbapenems sporins, 2 to carbapenems and two of the tested cepha- and two cephalosporins drugs, and 6 to cephalosporins losporins, and 3 to cephalosporins alone (Panel I C). Of alone (Fig. 4). the 15 faecal isolates, 10 were resistant to carbapenems Irrespective of the source of samples or species of bac- and all cephalosporins, and 5 to cephalosporins alone teria, we found that strains resistant to all examined an- (Panel I D). Additionally, the resistance phenotype and tibiotics, carry predominantly bla either alone or genotype were similar among E. coli, ECC, and K. pneu- KPC with the β-lactamase genes (bla , bla , bla , moniae. Strains resistant to all examined antibiotics har- CTX-M-15 SHV TEM bla , and bla ). However, strains resistant to boured predominantly bla either with the other OXA-1 PER-1 KPC carbapenems and two cephalosporins harbour only carbapenemase genes (bla and bla ) or with NDM OXA-48 Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 6 of 12 Fig. 3 Number of carbapenem- and cephalosporin- resistant Enterobacteriaceae isolates among fishpond water inlets and tap water. The Enterobacteriaceae isolates from a fishpond water inlets (n = 14), including E. coli (n = 9), ECC (n = 3), and K. pneumoniae (n = 2), as well as from b tap water (n = 33), including E. coli (n = 22), ECC (n = 6), and K. pneumoniae (n = 5) were screened for resistance against carbapenems (CRE) and cephalosporins (CEPH: FOX, CAZ, CTX, and CRO). The strains are grouped according to their resistance phenotypes as follow: CRE and all CEPH (isolates resistant to at least one of the tested carbapenems and all tested cephalosporins); CRE and 2 CEPH (isolates resistant to at least one of the tested carbapenems and two of the tested cephalosporins); CEPH (isolates resistant to 2 or more of the tested cephalosporins but not to carbapenems); CRE (isolates resistant to at least one of the tested carbapenems but not to any of the tested cephalosporins). Each phenotype is marked with colour and its resistance genotypes are also provided; carbapenemase (bla , bla , and bla ) and β-lactamase (bla , KPC OXA-48 NDM CTX-M-15 bla , bla , bla , and bla ) genes. (n) represents numbers of resistant strains SHV OXA-1 TEM PER-1 Fig. 4 Number of carbapenem- and cephalosporin- resistant Enterobacteriaceae isolates among outlet water. The Enterobacteriaceae isolates from outlet water (n = 16), including E. coli (n = 12), ECC (n = 2), and K. pneumoniae (n = 2) were screened for resistance against carbapenems (CRE) and cephalosporins (CEPH: FOX, CAZ, CTX, and CRO). The strains are grouped according to their resistance phenotypes as follow: CRE and all CEPH (isolates resistant to at least one of the tested carbapenems and all tested cephalosporins); CRE and 2 CEPH (isolates resistant to at least one of the tested carbapenems and two of the tested cephalosporins); CEPH (isolates resistant to 2 or more of the tested cephalosporins but not to carbapenems). Each phenotype is marked with colour and its resistance genotypes are also provided; carbapenemase (bla , bla , and KPC OXA-48 bla ) and β-lactamase (bla , bla , bla , bla , and bla ) genes. (n) represents numbers of resistant strains NDM CTX-M-15 SHV OXA-1 TEM PER-1 Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 7 of 12 Fig. 5 Number of cephalosporins- and carbapenems-resistant Enterobacteriaceae isolates from aquaculture workers and inhabitants of aquaculture surrounding areas. Panel I: Hand swab and faecal samples from aquaculture workers (n = 30) were examined. a and b illustrate the number (n) and species of Enterobacteriaceae isolated from hand swabs and faecal samples, respectively. c and d illustrate the number (n) of resistant Enterobacteriaceae isolates from hand swabs and faecal samples, respectively. Panel II: Faecal samples from inhabitants (n = 45) were examined. e illustrates the number (n) and species of Enterobacteriaceae isolated. f shows the number (n) of resistant Enterobacteriaceae. The strains are grouped based on resistance against carbapenems (CRE) and cephalosporins (CEPH: FOX, CAZ, CTX, and CRO) as follow: CRE and all CEPH (isolates resistant to at least one of the tested carbapenems and all tested cephalosporins); CRE and 2 CEPH (isolates resistant to at least one of the tested carbapenems and two of the tested cephalosporins); CEPH (isolates resistant to 2 or more of the tested cephalosporins but not to carbapenems). Each phenotype is marked with colour and its resistance genotypes are also provided; carbapenemase (bla , bla , and KPC OXA-48 bla ) and β-lactamase (bla , bla , bla , bla , and bla ) genes. (n) represents numbers of resistant strains NDM CTX-M-15 SHV OXA-1 TEM PER-1 the β-lactamase genes (bla , bla , bla , and for the presence and type of Inc. plasmids using PCR- CTX-M-15 SHV TEM bla ) (Panel I C and D). Based-Replicon-Typing (S2, Fig. 6). PER-1 Faecal samples were also collected from inhabitants of Therewerenodifferences in thetypeof Inc.plas- the aquaculture surrounding areas. Enterobacteriaceae mids, based on the source of the isolates (fish, fish- was isolated from 15 of the examined 45 samples with a pond water inlets, tap water, outlet water, and predominance of E. coli (Fig. 5, Panel II A). Of the 15 iso- workers) (S2). As demonstrated in Fig. 6a, of the 30 lates, 6 were resistant to carbapenems and all cephalospo- examined resistant strains, 29 showed the presence of rins, and 7 were resistant to cephalosporins alone (Fig. 5, the IncF and the IncHI groups of plasmids, 28 carried Panel II B). Consistent with the results of the aquaculture the IncX, 21 harboured the IncI complex group, 17 workers, the strains from the inhabitants were resistant to had the epidemic plasmid IncA/C, and 14 carried the carbapenems and all cephalosporins carried predomin- wide-host range IncP and the IncW groups. Other antly bla with other carbapenemase genes (bla Inc. plasmids found in minority of the isolates were KPC NDM, bla )orwith the β-lactamase genes (bla , as follows; IncN (n =9), IncN2 (n =7), IncR (n =5), OXA-48 CTX-M-15 bla , bla ,and bla )(Fig. 5, Panel II B). IncU (n =4), and IncT (n =4). SHV TEM PER-1 It is important to note that, the majority of the IncF plasmids present (22 / 29) were multi-replicon (Fig. 6a), Similar types of incompatible (Inc) plasmids were carried carrying the FII with FIA and or FIB replicons (S2)[30, by the Enterobacteriaceae resistant strains isolated from 31]. A divergence in FIB (FIB-KN and FIB-KQ) and FII different sources as determined by the PBRT-kits (FIIS and FIIK) replicons was observed. The FIB-KN was CRE isolates that were resistant to all tested antibiotics mainly found in the K. pneumoniae isolates, while the and showed similar resistance genotypes were examined FIB-KQ was present in K. pneumoniae and E. coli isolates. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 8 of 12 Fig. 6 Types of Incompatible (Inc) plasmids carried by the Enterobacteriaceae-resistant isolates. a illustrates the types of Inc. plasmids found in the examined CRE isolates (n = 30) that were resistant to all tested CEPH and showed similar genotypes. The red colour in the histogram of the IncF plasmid indicates number of isolates that harbour multi-replicon. b shows the differences between the CRE isolates producing-carbapenemase genes alone (CPE, n = 11) and those producing both carbapenamese and β-lactamase (CPE + βLPE, n = 19) genes, in the proportion and types of Inc. plasmids Moreover, the FIIS and FIIK divergences were found in E. were collected from inhabitants of the aquaculture sur- coli, K. pneumoniae,and ECC isolates (S2). rounding areas. The prevalence of Enterobacteriaceae iso- Some differences were noticed in the type of Inc. plas- lated from apparently healthy fresh Nile Tilapia fish was mids between strains producing carbapenemase genes 63%, which is higher than that reported by previous stud- alone (CPE) and those producing both carbapenemase ies performed on fish raised in earthen pond in Egypt and β-lactamase genes (CPE + βLPE) (Fig. 6b). Among (44–53%) [35–37]. Like in fish, the aquaculture water them, the IncI complex plasmid was found in the higher showed a high prevalence of Enterobacteriaceae, particu- proportion of the CPE + βLPE strains (15/19) than the larly the tap water (75%) as compared to fishpond water CPE strains (6/11). The IncI complex contains five groups inlets (50%) and outlet water (62%). Furthermore, of the IncI, IncL, IncM, IncK, and IncB/O combined, or at least 30 examined workers, 15 showed the presence of Entero- two of them. Another difference was the presence of the bacteriaceae in the hand and faecal samples, whereas, 5 IncW plasmid only in CPE + βLPE strains (14 /19). had Enterobacteriaceae only in the hand swabs. The spe- cies of Enterobacteriaceae isolated from fish, water sam- Discussion ples and the workers included E. coli, ECC,and K. For the past 60 years, β-lactam antibiotics have been pneumoniae,with E. coli being the predominant species. amongst the most successful drugs used for the treatment Although the species of Enterobacteriaceae found could of bacterial infections in humans, animals, and fish [32– be fish pathogens [35–40], it is not the most common 34]. In the current study, we investigated the occurrence found bacteria in fish [2]. This together with the high of β-lactamase (βLPE)- and carbapenemase (CPE)- produ- prevalence of Enterobacteriaceae found in water, suggests cing Enterobacteriaceae in fresh fish, fishpond water inlets, faecal contamination in the aquaculture environment. tap water, outlet water, and workers at four integrated A high proportion of the Enterobacteriaceae isolates agriculture-aquacultures. Additionally, faecal samples from the aquaculture showed resistance to the tested Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 9 of 12 antibiotics, as found in fish (64 / 66), water inlets (14 / shown to be commonly associated with bla on the TEM 15), tap water (33 / 33), outlet water (16 /16), and the same plasmid [52], and possibly with bla and the SHV workers (15 / 20). Regardless of the source of samples, a broad spectrum bla [15, 51]. Our findings then OXA-1 major number of these isolates were CRE, which encom- raised the question of how such CRE strains have pass predominantly strains resistant to carbapenems and reached the aquacultures. The most likely explanation is all tested cephalosporins, followed by strains resistant to a co-resistance to carbapenems by selective pressure carbapenems and some cephalosporins, while a few from overuse of other antibiotics, for which resistance strains were resistant to carbapenems alone. The three determinants are co-localised with carbapenemase on phenotypes of CRE isolates had different resistance ge- the same plasmid [53]. Another possible explanation notypes. However, CRE isolates resistant to all cephalo- might be unrestricted use of carbapenems to treat fish, sporins carried predominantly both carbapenemase and which is consistent with our results that strains resistant β-lactamase genes, CRE isolates resistant to some cepha- only to carbapenems were found mainly among fish iso- losporins or to carbapenems alone harboured only car- lates. Unfortunately, this cannot be clarified, as no data bapenemase genes. According to CLSI 2020, CRE is available on quantities and types of antibiotics used in isolates that produce one or more carbapenemase genes aquaculture in Egypt. Furthermore, the resistant strains usually test resistant to one or more of cephalosporins, might access the aquaculture from agricultural sources, however, some CPE isolates can still test susceptible to where animal manure is known to be used as fertilizers cephalosporins [41]. This can be a possible explanation [6, 54, 55]. CRE has been isolated from dairy farms in for our finding of CRE isolates which carry one or more Egypt [56]. Another possible source of the resistant of the carbapenemase genes (bla , bla , bla ), strains could be tap water, which showed the presence KPC OXA-48 NDM but susceptible to the tested cephalosporins (Figs. 2, 3). of high amounts of CRE and cephalosporins-resistant In this regard, a study from South Africa also found Enterobacteriaceae, indicating faecal contamination. The some CPE-CRE isolates which were susceptible to ceph- main source of tap water in Egypt [57] is from surface alosporins [42]. water like rivers, lakes or canals. In many Egyptian gov- It is important to note that among the 26 CRE fish iso- ernorates, sewage is usually discharged untreated into lates that showed resistance only to carbapenems, 9 iso- the surface water [58, 59]. Since sewage act as a pool of lates did not carry any of the tested carbapenemase genes, antibiotic residues as well as resistant bacteria from dif- suggesting that they might harbour other carbapenemases ferent sources [58, 59], this can result in contamination which were not screened in the present study. of tap water. This is supported by our results that Enterobacteriaceae isolates resistant to cephalosporins showed the occurrence of Enterobacteriaceae in faecal alone were also found but in a lower proportion than samples from inhabitants of the aquaculture surrounding the CRE. Slight differences were noticed between the areas, which have similar resistance phenotype and four examined aquacultures, in the numbers of CRE and genotype as the isolates from the aquaculture workers cephalosporins-resistant Enterobacteriaceae, which were with a predominance of bla and bla . In this KPC CTX-M-15 not statistically different. Cephalosporins-resistant En- regard, previous studies reported preponderance of En- terobacteriaceae isolates have previously been found in terobacteriaceae carrying bla and bla among KPC CTX-M-15 fish and aquatic environments from different countries, humans in Middle East [60] and North Africa [61, 62]. including Egypt [40, 43–48]. However, a few recent stud- Characterisation of the Inc. plasmid types harboured ies showed the presence of CRE strains among fish [49, by the CRE isolates that were resistant to all tested ceph- 50], indicating an increase in the prevalence of CRE in alosporins, revealed a similar profile among the isolates aquaculture. Irrespective of the species of bacteria or from fish, water, and workers. The major plasmid groups source of samples, the carbapenemase-producing isolates found were as follow; IncF, IncHI, IncX, IncI-complex, showed a predominance of bla over the other two IncA/C, IncP, and IncW, which agrees with findings that KPC genes bla and bla , which agrees with other IncF, IncHI, IncX, IncI-complex, and IncA/C are com- OXA-48 NDM study that detected the presence of bla in seafood monly associated with CRE isolated from humans and KPC isolates [51]. Furthermore, all the isolates that were posi- animals [19–21]. Crucially, the IncA/C is considered as tive for β-lactamase genes harboured bla in an epidemic plasmid, as it has been detected in different CTX-M-15 combination with one or more of the other β-lactamase countries and has a wide-host range of different sources genes (bla , bla , bla and bla ). This is [63]. IncP and IncW have been categorised among rarely OXA-1 SHV TEM, PER-1 consistent with findings that showed a predominance of reported plasmids [21, 64], however, we found them in a bla among β-lactam-resistant Enterobacteria- relatively high number of the isolates from different CTX-M-15 ceae isolated from wild fish in Algeria [44], from frozen sources. Since these two plasmids have a broad-host Mackerel in Saudi Arabia [45], and water collected from range and can carry multiple resistance genes [64, 65], fish farms in Egypt [46]. Moreover, the bla was their presence in our isolates indicates a role in the CTX-M Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 10 of 12 spread of resistance genes among bacteria of different gen- Supplementary information Supplementary information accompanies this paper at https://doi.org/10. era and sources. Other plasmids like IncN, IncN2, IncL, 1186/s13756-020-00736-3. IncU, IncT, and IncR were also present, but in minority of the current isolates. This is consistent with [19], who Additional file 1. stated that these plasmids are not often reported. One of the interesting results is a predominance of the Abbreviations multi-replicon IncF among the tested CRE isolates, with βLPE: β-lactamase-producing Enterobacteriaceae; CAZ: Ceftazidime; divergence in FII (FIIS, FIIK) and FIB (FIB-KQ, FIB-KN), CDC: Centre for Disease Control and Prevention; CEPH: Cephalosporins; but not in FA (S2), suggesting that such plasmids are rap- CLSI: Clinical and Laboratory Standards Institute; CPE: Carbapenemase- producing Enterobacteriaceae; CRE: Carbapenem-resistant Enterobacteriaceae; idly emerging with the subsequent increase in dissemin- CRO: Ceftriaxone; CTX: Cefotaxime; ECC: Enterobacter cloacae complex; ation of resistance genes. IncF plasmid is unique, as it can EMB: Eosin methylene blue agar; ESBL: Extended-spectrum β-lactam; carry many replicons, one replicon is strongly conserved ETP: Ertapenem; FOX: Cefoxitin; Inc. plasmids: Incompatible plasmids; IPM: Imipenem; K. pneumoniae: Klebsiella pneumoniae; MEM: Meropenem for plasmid replication, while the others are free to diverge as a mechanism of evolution [19, 30]. Additionally, the IncF plasmid can co-integrate with other plasmids found Acknowledgements We are grateful to the aquaculture workers and people living in the areas in the current study, like IncI1, IncA/C, and IncN forming surrounding the fish farms for participating in the current study. The a mega-plasmid with multi-resistance regions [19, 31, 66, cooperation of the aquaculture owners is highly appreciated. 67]. Taken together, the current CRE isolates carry a wide range of Inc. plasmids which are known to be associated Authors’ contributions with multi-drug resistance determinants. The profile of Dalia Hamza participated in the design and coordination of the study, performed the experiments, and assisted in the editing of the manuscript. these plasmids was similar between isolates from fish, Sohad Dorgham participated in the design of the study, collection of water, and humans working in contact with fish, suggest- samples and performed the experiments. Elshaima Ismael performed the ing a possible transfer among bacteria from different statistical analysis and preparation of tables. Sherein Ismail Abd El-Moez par- ticipated in the design of the study and collection of samples. Rehab Elhelw sources. To our knowledge, no data is available regarding and Mahmoud Elhariri performed the experiments and preparation of the the prevalent type of Inc. plasmids in Egypt. manuscript. Eman Hamza participated in the design of the study, assisted in the statistical analysis and preparation of the figures, and edited the manu- script. All the authors read and approved the final manuscript. Conclusion The present study reveals the occurrence of CRE which are resistant to all tested cephalosporins among fish, Funding This research did not receive any grant from funding agencies in the public, water, and workers at aquacultures integrated with agri- commercial, or not-for-profit sectors. culture systems in Egypt. The fish included in the study were apparently healthy, indicating they could act as res- Availability of data and materials ervoirs for potentially pathogenic bacteria in humans. All data generated or analysed during this study are included in this Such resistant strains might have occurred in the aqua- published article. culture due to unregulated use of antibiotics to treat fish, or through agricultural sources, faecal contaminated tap Ethics approval and consent to participate water, or the workers. The resistant strains carry a var- Protocols for collection of samples as well as the experiment plan and all methods were performed in accordance with the guidelines and regulations iety of resistance Inc. plasmids which are similar be- of Cairo University Council and were approved by the Scientific Research tween the strains from different sources. Most of these Committee and Bioethics Board of Cairo University, Faculty of Veterinary plasmids have a broad-host range and pose a great risk Medicine, Cairo, Egypt. Ethical clearance to use human subjects was obtained from the designated health facility (National Research Centre, Giza, for the possible spread of resistance. Therefore, we rec- Egypt) and written informed consent was obtained from each person upon ommend that studies on antimicrobial resistance should information of the use of samples. consider thorough, systematic studies of animals, fish, humans, tap water, and sewage, as all these factors are Consent for publication linked together. The current study has some limitations, Not applicable. including examination of integrated aquaculture from only one governorate in Egypt and the limited number Competing interests of the tested carbapenemase genes (bla , bla , The authors have no competing interests. KPC NDM-1 and bla ). Therefore, further work is required to OXA-48 Author details elucidate the extent of antibiotic resistance in more inte- 1 Department of Zoonoses, Faculty of Veterinary Medicine, Cairo University, grated aquaculture from other governorates. Particularly, Giza square, PO Box 12211, Cairo, Egypt. Department of Microbiology and Immunology, National Research Centre, Giza, Egypt. Department of to screen for carbapenems and extended-spectrum- Veterinary Hygiene and Management, Faculty of Veterinary Medicine, Cairo cephalosporins, with including a wide panel of carbape- 4 University, Giza, Egypt. Department of Microbiology, Faculty of Veterinary nemase genes. Medicine, Cairo University, Giza, Egypt. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 11 of 12 Received: 31 October 2019 Accepted: 9 May 2020 25. CDC, Centre for Disease Control and Prevention. Patient safety atlas. 2018. https://www.cdc.gov/hai/surveillance/ar-patient-safety-atlas.html. Accessed 13 July 2018. 26. Elhariri M, Hamza D, Elhelw R, Dorgham SM. Extended-spectrum beta- lactamase -producing Pseudomonas aeruginosa in camel in Egypt: potential References human hazard. Ann Clin Microbiol Antimicrob. 2017;16:1–6. 1. Cabello FC, Godfrey HP, Buschmann AH, Dölz HJ. Aquaculture as yet 27. Sugumar M, Kumar KM, Manoharan A, Anbarasu A, Ramaiah S. Detection of another environmental gateway to the development and globalization of OXA-1 β-lactamase gene of Klebsiella pneumoniae from blood stream antimicrobial resistance. Lancet Infect Dis. 2016;16:127–33. infections by conventional PCR and in-Silico analysis to understand the 2. Schmidt AS, Bruun MS, Dalsgaard I, Larsen JL. Incidence, distribution, and mechanism of OXA mediated resistance. PLoS One. 2014. https://doi.org/10. spread of tetracycline resistance determinants and integron-associated 1371/journal.pone.0091800. antibiotic resistance genes among motile aeromonads from a fish farming 28. Hamza E, Dorgham SM, Hamza DA. Carbapenemase-producing Klebsiella environment. Appl Environ Microbiol. 2001;67:5675–82. pneumoniae in broiler poultry farming in Egypt. J Global Antimicrob Resist. 3. Defoirdt T, Sorgeloos P, Bossier P. Alternative to antibiotics for the control of 2016;7:8–10. bacterial disease in the aquaculture. Curr Opin Microbiol. 2011;14:251–8. 29. Li B, Yi Y, Wang Q, Woo PCY, Jing H, Gao GF. Analysis of drug resistance 4. Hoa PT, Managaki S, Nakada N, Takada H, Shimiz A, Anh DH. Antibiotic determinants in Klebsiella pneumoniae isolates from a tertiary-care hospital contamination and occurrence of antibiotic-resistant bacteria in aquatic in Beijing, China. PLoS One. 2012. https://doi.org/10.1371/journal.pone. environments of northern Vietnam. Sci Total Environ. 2011;409:2894–901. 5. Little DC, Edwards P. Integrated livestock-fish farming systems. Rome: Food 30. Villa L, Garcia-Fernandez A, Fortini D, Carattoli A. Replicon typing of IncF and Agriculture Organization of the United Nations; 2003. http://www.fao. plasmids carrying virulence and resistance determinants. J Antimicrob org/3/a-y5098e. Chemother. 2010;65:2518–29. 6. Neela FA, Banu NA, Rahman A, Rahman MH, Alam MF. Occurrence of 31. Osborn AM, da Silva Tatley FM, Steyn LM, Pickup RW, Saunders JR. Mosaic antibiotic resistant bacteria in pond water associated with integrated plasmids and mosaic replicons: evolutionary lessons from the analysis of poultry-fish farming in Bangladesh. Sains Malaysiana. 2015;44:371–7. genetic diversity in IncFII-related replicons. Microbiol. 2000;146:2267–75. 7. Seiler C, Berendonk TU. Heavy metal driven co-selection of antibiotic 32. Economou V, Gousia P. Agriculture and food animals as a source of resistance in soil and water bodies impacted by agriculture and antimicrobial-resistant bacteria. Infect Drug Resist. 2015;8:49–61. aquaculture. Front Microbiol. 2012;3:399. 33. Prestinaci F, Pezzoti P, Pantosti A. Antimicrobial resistance: a global 8. Burridge L, Weis JS, Cabello F, Pizarro J, Bostick K. Chemical use in salmon multifaceted phenomenon. Pathog Glob Health. 2015;109:309–18. aquaculture: a review of current practices and possible environmental 34. Sarmah AK, Meyer MT, Boxall AB. A global perspective on the use, sales, effects. Aquaculture. 2010;306:7–23. exposure pathways, occurrence, fate and effects of veterinary antibiotics 9. Buschmann AH, Tomova A, Lopez A, Maldonado MA, Henriquez LA, Ivanova (VAs) in the environment. Chemosphere. 2006;65:725–59. L, et al. Salmon aquaculture and antimicrobial resistance in the marine environment. PLoS One. 2012;7:42724. 35. Abdel-Latif HMR, Sedeek EK. Diversity of Enterobacteriaceae retrieved from diseased cultured Oreochromis niloticus. Int J Fish Aqua Stud. 2017;5:29–34. 10. Heuer OE, Kruse H, Grave K, Collignon P, Karunasagar I, Angulo FJ. 2009. 36. Aly SM, Nouh WG, Salem-Bekhit MM. Bacteriological and histopathological Human health consequences of use of antimicrobial agents in studies on Enterobacteriaceae in Nile Tilapia, Oreochromis niloticus. J Pharm aquagculture. Clin Infect Dis. 2009;49:1248–53. Biomed Sci. 2012;2:94–104. 11. Rhodes G, Huys G, Swings J, Mcgann P, Hiney M, Smith P, et al. Distribution 37. Elsherief MF, Mousa MM, Abd El-Galil H, El-Bahy EF. Enterobacteriaceae of oxytetracycline resistance plasmids between aeromonads in hospital and associated with farm fish and retailed ones. Alex J Vet Sci. 2014;42:99–104. aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Appl Environ Microbiol. 2000;66: 38. Hassan AHM, Noor-El Deen AE, Galal HM, Dorgham SM, Bakry MA, Hakim AS. Further characterization of Enterobacteriaceae isolated from cultured 3883–90. 12. Sheu CC, Chang YT, Lin SY, ChengYH HPR. Infections caused by freshwater fish in Kafr El Shiek governorate: clinical, biochemical and carbapenem-resistant Enterobacteriaceae: An update on therapeutic histopathological study with emphasis on treatment trials. Glob Veter. 2012; options. Front Microbiol. 2019;10:80. 9:617–29. 13. Wilson H, Török ME. Extended spectrum β-lactamase producing and 39. Saswathi R, Sumithra P, Sivakami R. Identification of Enterobacteriaceae carbapenamese producing Enterobacteriaceae. Microb Genom. 2018;4:7. studies in carps during rearing a freshwater pond. Int J Curr Microbiol Appl 14. Bush K, Jacoby GA. Updated functional classification of beta-lactamases. Sci. 2015;4:661–9. Antimicrob Agents Chemother. 2010;54:969–76. 40. Abgottspon H, Nüesch-Inderbinen MT, Zurfluh K, Althaus D, Hächler H, 15. Dandachi I, Chaddad A, Hanna J, Matta J, Daoud Z. Understanding the Stephan R. Enterobacteriaceae with extended-Spectrum- and pAmpC-type epidemiology of multi-drug resistant gram-negative bacilli in the Middle -lactamase-encoding genes isolated from freshwater fish from two lakes in East using a one health approach. Front Microbiol. 2019;10:1941. Switzerland. Antimicrob Agents Chemother. 2014;58:2482–4. 41. CLSI, Clinical and Laboratory Standards Institute. Performance standards for 16. Exner M, Bhattacharya S, Christiansen B, Gebel J, Goroncy-Bermes P. th antimicrobial susceptibility testing (M100, 30 Edition); 2020. Hartemann p. Antibiotic resistance: What is so special about multidrug- resistant gram-negative bacteria?. GMS Hyg Infect Control. 2017;12:1–24. 42. Singh-Moodley A, Perovic O. Antimicrobial susceptibility testing in 17. Canton R, Ruiz-Garbajosa P. Co-resistance: an opportunity for the bacteria predicting the presence of carbapenemase genes in Enterobacteriaceae in and 2 resistance genes. Curr Opin Pharmacol. 2011;11:477–85. South Africa. BMC Infect Dis. 2016;16:536. 18. Austin S, Nordström K. Partition-mediated incompatibility of bacterial 43. Akinbowale OL, Peng H, Barton MD. Antimicrobial resistance in bacteria plasmids. Cell. 1990;60:351–4. isolated from aquaculture sources in Australia. J Appl Microbiol. 2006;100: 19. Rozwandowicz M, Brouwer MSM, Fischer J, Wagenaar JA, Gonzalez-Zorn B, 1103–13. Guerrat B. Plasmids carrying antimicrobial resistance genes in 44. Brahmi S, Tuoati A, Dunyach-Remy C, Sotto A, Pantel A, Lavigne JP. High Enterobacteriaceae. J Antimicrob Chemother. 2018;73:1121–37. prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae 20. Carattoli A. Resistance plasmid families in Enterobacteriaceae. Antimicrob in wild fish from the Mediterranean Sea in Algeria. Microb Drug Resist. 2018; Agents Chemother. 2009;53:2227–38. 24:290–8. 21. Carloni E, Andreoni F, Omiccioli E, Villa L, Magnani M, Carratoli A. 45. Elhadi N, Alsamman K. Incidence and antimicrobial susceptibility pattern of Comparative analysis of the standard PCR-based replicon typing (PBRT) with extended-spectrum-β-lactamase-producing Escherichia coli isolated from the commercial PBRT-Kit. Plasmid. 2017;90:10–4. retail imported mackerel fish. Afr J Biotech. 2015;14:1954–60. 22. Bezuidenhout CC, Mthembu N, Puckree T, Lin J. Microbiological evaluation 46. Ishida M, Ahmed AM, Mahfouz NB, Kimura T, El-Khodery SA, Moawad AA. of the Mhlathuze River, Kwazulu-Natal (RSA). Water SA. 2002;28:281–6. Molecular analysis of antimicrobial resistance in gram-negative bacteria 23. Collee JG, Fraser AG, Marmion BP, Simmons A. Practical medical isolated from fish farms in Egypt. J Vet Med Sci. 2010;72:727–34. microbiology. New York: Churchill Livingstone; 1996. 47. Sarter S, Nguyen K, Hung LT, Lazart J, Montet D. Antibiotic resistance in 24. CLSI, Clinical and Laboratory Standards Institute. Performance standards for gram-negative bacteria isolated from farmed catfish. Food Control. 2007;18: antimicrobial susceptibility testing (M100-S24);2016. 1391–6. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 12 of 12 48. Singh AS, Lekshmi M, Prakasan S, Nayak BB, Kumar S. Multiple antibiotic- resistant, Extended Spectrum-Lactamase (ESBL)-producing Enterobacteriaceae in fresh seafood. Microorganisms. 2017;5(53):1–10. 49. Janecko N, Martz SL, Avery BP, Daignault D, Desruisseau A, Boyd D. Carbapenem-resistant Enterobacter spp. in retail seafood imported from Southeast Asia to Canada. Emerg Infect Dis. 2016;22:1675–7. 50. Mangat CS, Boyd D, Janecko N, Martz SL, Desruisseau A, Carpenter M. Characterization of VCC-1, a novel ambler class a carbapenemase from Vibrio cholerae isolated from imported retail shrimp sold in Canada. Antimicrob Agents Chemother. 2016;60:1819–25. 51. Sabate M, Tarrago R, Navarro F, Miro E, Verges C. Barbe, et al. cloning and sequence of the gene encoding a novel cefotaxime-hydrolysing β- lactamase (CTX-M-9) from Escherichia coli in Spain. Antimicrob Agents Chemother. 2000;44:1970–3. 52. Karim A, Poirel L, Nagarajan S, Nordmann P. Plasmid-mediated extended- spectrum β-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiol Banner. 2006;201:237–41. 53. Barron SA, Diene SM, Rolain JM. Human microbiomes and antibiotic resistance. Hum Microb J. 2018;10:43–52. 54. Dang STT, Petersen A, Truong DV, Chu HTT, Dalsgaard A. Resistance in bacteria at integrated pig-fish farms in Vietnam. Appl Environ Microbiol. 2011;77:4494–8. 55. Petersen A, Andersen JS, Kaewmak T, Somsiri T, Dalsgaard A. Impact of integrated fish farming on antimicrobial resistance in a pond environment. Appl Environ Microbiol. 2002;68:6036–42. 56. Braun S, Ahmed MFE, El-Adway H, Hotzel H, Engelmann I, Weiss D, et al. Surveillance of extended Spectrum β-lactamase-producing Escherichia coli in dairy cattle farms in the Nile Delta, Egypt. Front Microbiol. 2016;7(1020):1–14. 57. El-Kowrany SI, El-Zamarany EA, El-Nouby KA, El-Mehy DA, Abo Ali EA, Othman AA. Water pollution in the middle Nile Delta, Egypt: an environmental study. J Adv Res. 2016;7:781–9. 58. Ezzat SM, Mahdy HM, Abo-State MA, Abd-Elshakour EH, El-Bahnasawy MA. Water quality assessment of River Nile at Rosetta branch Middle East. J Sci Res. 2012;12:413–23. 59. Amine AEK. Extended spectrum Beta-lactamase producing bacteria in sewage water, Alexandria, Egypt. Int J Biosc Bioch Bioinf. 2013;3:605–8. 60. Sonnevend A, Al-Dhaheri K, Mag T, Herpay M, Kolodziejek J, Nowotny N. CTX-M-15-producing multidrug-resistant enteroaggregative Escherichia coli in the United Arab Emirates. Clin Microbiol Infect. 2006;12:582–285. 61. Iabadene H, Messai Y, Ammari H, Alouache S, Verdet C, Bakour R, Arlet G. Prevalence of plasmid-mediated AmpC β-lactamases among Enterobacteriaceae in Algiers hospitals. Int J Antimicrob Agents. 2009;34: 340–2. 62. Mamlouk K, Boubaker IB, Gautier V, Vimont S, Picard B, Ben RS. Emergence and outbreaks of CTX-M β-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital. J Clin Microbiol. 2006;44: 4049–56. 63. Hancok S, Phan MD, Peters KM, Forde BM, Chong TM, Yin WF, et al. Identification of IncA/C plasmid replication and maintenance genes and development of a plasmid multi-locus sequence typing scheme. Antimicrob Agents Chemother. 2017;61(2):1–17. 64. Almeida AC. de Sa Cavalcanti FL, Vilela MA, gales AC, de Morais MA, Jr Camargo de Morais MM. Escherichia coli ST502 and Klebsiella pneumoniae ST11 sharing an IncW plasmid harbouring the bla gene in an intensive KPC-2 care unit patient. Int J Antimicrob Agents. 2012;40:374–6. 65. Yano H, Rogers LM, Knox MG, Heuer H, Smalla K, Brown CJ, et al. Host range diversification within the IncP1 plasmid group. Microbiol. 2013;159:2303–15. 66. Froehlich B, Parkhill J, Sanders M, Quail MA, Scott JR. The pCoo plasmid of enterotoxigenic Escherichia coli is a mosaic conitegrate. J Bacteriol. 2005; 187:6509–16. 67. Desmet S, Nepal S, van Dijl JM, van Ranst M, Chlebowicz MA, Rossen JW, et al. Antibiotic resistance plasmids cointegrated into a megaplasmid harbouring the bla carbapenemase gene. Antimicrob Agents OXA-427 Chemother. 2018;62(3):1–6. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antimicrobial Resistance & Infection Control Springer Journals

Emergence of β-lactamase- and carbapenemase- producing Enterobacteriaceae at integrated fish farms

Download PDF
12 pages

Loading next page...
 
/lp/springer-journals/emergence-of-lactamase-and-carbapenemase-producing-enterobacteriaceae-v3tLmMPNbU

References (78)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2020
eISSN
2047-2994
DOI
10.1186/s13756-020-00736-3
Publisher site
See Article on Publisher Site

Abstract

Background: Epidemiological studies suggested that determinants for antibiotic resistance have originated in aquaculture. Recently, the integrated agriculture-aquaculture system has been implemented, where fish are raised in ponds that receive agriculture drainage water. The present study aims to investigate the occurrence of β- lactamase and carbapenemase-producing Enterobacteriaceae in the integrated agriculture-aquaculture and the consequent public health implication. Methods: Samples were collected from fish, fishpond water inlets, tap water, outlet water, and workers at sites of integrated agriculture-aquacultures. Samples were also taken from inhabitants of the aquaculture surrounding areas. All samples were cultured on MacConkey agar, the Enterobacteriaceae isolates were tested for susceptibility to cephalosporins and carbapenems, and screened for bla , bla , bla , bla , bla , bla , bla ,and bla .Strains CTX-M-15 SHV OXA-1 TEM PER-1 KPC OXA-48 NDM having similar resistance phenotype and genotype were examined for the presence of Incompatible (Inc) plasmids. Results: A major proportion of the Enterobacteriaceae isolates were resistant to cephalosporins and carbapenems. Among the 66 isolates from fish, 34 were resistant to both cephalosporin and carbapenem groups, 26 to carbapenems alone, and 4 to cephalosporins alone. Of the 15 isolates from fishpond water inlets, 8 showed resistance to both groups, 1 to carbapenems alone, and 5 to cephalosporins alone. Out of the 33 isolates from tap water, 17 were resistant to both groups, and 16 to cephalosporins alone. Similarly, of the 16 outlet water isolates, 10 were resistant to both groups, and 6 to cephalosporins alone. Furthermore, of the 30 examined workers, 15 carried Enterobacteriaceae resistant strains, 10 to both groups, and 5 to cephalosporins alone. Similar strains were isolated from the inhabitants of the aquaculture surrounding areas. Irrespective of source of samples, strains resistant to all examined antibiotics, carried predominantly the carbapenemase gene bla either alone or with the β-lactamase genes (bla , bla , bla ,and bla ). The KPC CTX-M-15 SHV TEM PER-1 isolates from fish, water, and workers harboured a wide-range of multi-drug-resistance Inc. plasmids, which were similar among all isolates. Conclusion: The present findings suggest transmission of the resistance genes among Enterobacteriaceae strains from different sources. This reiterates the need for control strategies that focus on humans, animals, water, and sewage systems to solve the antibiotic resistance problem. Keywords: Egypt, Aquaculture-agriculture-farms, Enterobacteriaceae, β lactam-resistance, Inc. plasmids, PBRT kits * Correspondence: e.hamza@gmx.ch Department of Zoonoses, Faculty of Veterinary Medicine, Cairo University, Giza square, PO Box 12211, Cairo, Egypt Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 2 of 12 Introduction replicons are now recognised, which represent the major A link between aquaculture and the development of replicase genes of the Inc. plasmids circulating among antibiotic resistance has been demonstrated by an in- Enterobacteriaceae, including HI1, HI2, I1, I2, X1, X2, creasing body of research [1, 2]. Worldwide, there is a X3, X4, L, M, N, FIA, FIB, FIC, FII, FIIS, FIIK, FIB-KN, massive increase in fish farming, which is associated with FIB-KQ, W, Y, P1, A/C, T, K, U, R, B/O, HIB-M, and intensive use of antibiotics to combat bacterial infections FIB-M [21]. [3]. Other potential explanations are contamination of Recently in Egypt, small scale use of the integrated aquaculture with antibiotic residues and antibiotic resist- agriculture-aquaculture system began to be used. The ant bacteria coming from animals through using the so- aims of the present study are, therefore, to investigate called livestock integrated aquaculture system [4–6]. Re- the extent of antibiotic resistance in aquacultures inte- cently, an integrated agriculture-aquaculture system has grated with agriculture in Egypt and its public health sig- been implemented to save water resources [7]. In such nificance. Accordingly, this was performed through (i) system, fish are reared in ponds receiving water from assessing the prevalence of β-lactamase (βLPE) and crop farms through irrigation canals. carbapenemase-producing Enterobacteriaceae (CPE) in The major concern with the use of antibiotics in aqua- the water inlets of aquaculture originating from agricul- culture is that fish do not effectively metabolize antibi- ture and supply fishponds, in fish, tap water, and the otics and pass them largely unused in faeces [8]. This outlet water of the aquaculture; (ii) determination of the encourages the development of antibiotic resistance in occurrence of βLPE and CPE in aquaculture workers bacteria present in fish and the surrounding environ- and inhabitants of the aquaculture surrounding areas, ment [1, 2, 9]. Fishponds are not subject to frequent those having a history of eating fish from these aquacul- water exchange, resulting in accumulation of antibiotic- ture and also use the same tap water source as the aqua- residues and -resistant bacteria, which are then dissemi- culture (iii) identifying the presence and type of Inc. nated at harvest time. plasmids carried by the resistant strains isolated from Fish bacteria particularly Enterobacteriaceae can ex- different sources. change antibiotic resistance genes with human and ani- mal bacteria [10, 11]. Among these, β-lactam resistant Materials and methods bacteria are of great concern, as they are becoming non- Sample collection susceptible to nearly all available antibiotics [12, 13]. Four integrated agriculture-aquaculture farms were ran- They include extended-spectrum β-lactam (ESBL) and domly selected from the Giza governorate, the third- carbapenem-resistant (CRE) Enterobacteriaceae. ESBL largest city in Egypt. The selected farms are located near carry a broad spectrum of β-lactamase enzymes that each other (approx. 5 km away from each other), they hydrolyse a wide range of penicillin and cephalosporin combine agriculture with fish farming As illustrated in antibiotics, but not carbapenems [14]. However, CRE Fig. 1, the fish are raised in ponds, each fishpond re- carry carbapenemase enzymes that confer resistance to ceives water from agriculture drainage water through a nearly all β-lactam antibiotics, including carbapenems separate water pipe (Water inlet). Tap water is another [15]. The β-lactamase enzymes encompass members of source of water for the aquaculture, which is used to dis- the TEM and SHV families and other groups, such as solve medicines given to the fish and sometimes as a CTX-M, OXA, and PER β-lactamases [16]. The carbape- supply for the fishponds in case of shortage of agricul- nemase enzymes are a diverse group of β-lactamases, the ture water. Tap water is also used by the workers for most remarkable of which are the big five enzymes KPC, drinking and hand washing. Outlet water represents the NDM, OXA-48, IMP, and VIM [17]. There is strong evi- waste drained from fishponds through pipes. dence that the β-lactamase and carbapenemase encoding Samples were aseptically collected from internal organs genes are found on plasmids that facilitate their transfer (liver, spleen, and kidney) from 105 apparently healthy among bacteria of different genera and kingdoms [15, fresh Tilapia fish (Oreochromis niloticus). Additionally, 16]. Plasmids are extra-chromosomal, self-replicating samples were collected from fishpond water inlets (n = genetic elements, carried by the bacteria. Plasmids hav- 30), tap water (n = 44), and outlet water (n =26). The ing the same origin of replication cannot stably co-reside water samples were placed in sterile glass bottles contain- within the same bacterial cells, are known as incompat- ing sodium thiosulphate and were filtered through 0.4 μm ible (Inc) [18]. The Inc. plasmids can positively select for pore size nitrocellulose filters (Sartorius, Aubagne, France) the presence of resistance genes [19]. Identification of placed on tubes containing peptone broth [22]. the Inc. plasmids in the bacterial isolates helps to trace Faecal and hand swab samples were collected from the transfer of the resistance genes. Currently, a PCR humans working in the aquaculture farms (n = 30), hav- method is accepted to classify the Inc. resistance plas- ing contact with the fish, and who agreed to participate mids based on their type of replicon [20]. Thirty in the study. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 3 of 12 Fig. 1 Illustration of the setup of the integrated agriculture-aquaculture farms included in the present study. Locations where samples were collected are indicated with an asterisk (*) Additionally, faecal samples were collected from in- instructions. The accuracy of API 20E results was 100% as habitants of the aquaculture surrounding areas (n = 45), determined by API web software (BioMérieux). who use the same source of tap water as the aquacul- tures and were admitted to the local medical centre with Antibiotic susceptibility test diarrhoea after eating fish from the tested aquacultures. Confirmed strains of Enterobacteriaceae were examined for susceptibility to different antibiotics (Oxoid) using the disk diffusion test on Mueller-Hinton agar (Oxoid). Bacterial isolation and identification The antibiotics included were cephalosporins [Cefoxitin All samples were inoculated into Trypticase soy broth (FOX, 30 μg), Ceftazidime (CAZ, 30 μg) Cefotaxime tubes and were incubated at 37 °C for 24 h. A loop-ful (CTX, 30 μg), Ceftriaxone (CRO, 30 μg)] and the carba- from the previously incubated tubes were streaked onto penems [Imipenem (IPM, 10 μg), Meropenem (MEM, MacConkey agar plates (Oxoid Ltd., Cairo, Egypt) and 10 μg), Ertapenem (ETP, 10 μg)] groups. The results Eosin methylene blue agar (EMB) (Oxoid) and were in- were interpreted according to the clinical breakpoints cubated aerobically at 37 °C for 24 h. Suspected colonies recommended by CLSI (Clinical laboratory Standard were purified through subculture on MacConkey agar Institute) [24]. plates and were examined for morphology and pheno- Based on CDC definitions [25], Enterobacteriaceae iso- type traits according to Collee et al. [23]. lates resistant to at least one of the carbapenem drugs (Imipenem, Meropenem, Ertapenem, or Doripenem) or Biochemical identification produce a carbapenemase gene are called carbapenem- Pure colonies were subjected to API 20E kits (BioMerieux, resistant Enterobacteriaceae (CRE). Accordingly, in the Marcy-l’Étoile, France) according to the manufacturer’s present study, isolates resistant to at least one of the Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 4 of 12 tested carbapenem drugs were considered CRE and were isolated Enterobacteriaceae strains and type of samples tested for the presence of carbapenemase genes. (fish, fishpond water inlets, tap water, and outlet water) (Table 1). Chi-square (X ) test was also performed to PCR for detection of β-lactamase- and carbapenemase- examine whether the prevalence of antibiotic resistance encoding genes is influenced by the type of samples or species of bac- Genomic DNA from resistant isolates was extracted teria, or both combined. On each occasion, the signifi- using a QIAamp® mini kit (Qiagen, Hombrechtikon, cance of differences was set at p value < 0.05. Switzerland). PCR was performed to identify β- lactamase-encoding genes (bla , bla , bla , Results CTX-M-15 SHV TEM bla ) using specific oligonucleotide primers as previ- Prevalence of Enterobacteriaceae in apparently healthy PER-1 ously described [26]. The PCR for bla was done Nile tilapia fish and water from integrated agriculture- OXA-1 using specific oligonucleotide primers according to [27]. aquacultures Multiplex PCR [28] was performed to identify As demonstrated in Table 1, of the 105 tested apparently carbapenemase-encoding genes (bla bla , and healthy Nile tilapia fish, 66 (63%) showed the presence KPC, OXA-48 bla ) using specific oligonucleotide primers [29]. The of Enterobacteriaceae. Additionally, the occurrence of NDM three genes were chosen to include one representative Enterobacteriaceae was also high among the collected candidate from each of the three carbapenemase groups samples from fishpond water inlets (15/30; 50%), tap (Serine, OXA, and MLBs) and the selection was based water (33/44; 75%), and outlet water (16/26; 62%) from on previous findings in Egypt [28]. The sequences of the the aquacultures. E. coli represented the major species of primers used in the present study are shown in S1. Enterobacteriaceae isolated from fish and water. Entero- bacter cloacae complex (ECC) and Klebsiella (K.) pneu- Characterisation of Inc. plasmids present in the resistant moniae were also detected, but with a significantly lower isolates using PCR-based-replicon-typing prevalence than E. coli. Genomic DNA from the isolates that were resistant to all tested antibiotics and showed similar resistance geno- Occurrence of cephalosporin- and carbapenem-resistant types (n = 30) were subjected to plasmid typing using Enterobacteriaceae in the integrated agriculture- the PBRT 2.0 kit (PCR based replicon typing Ver.01/06/ aquacultures 2017, Diatheva, Fano, Italy). The kit contains eight The Enterobacteriaceae isolates from fish and water multiplex PCR, each of which amplifies three to four samples of the aquacultures were screened for resistance targets, allowing detection of a total of 30 replicons. against carbapenems (CRE) and cephalosporins (CEPH). It also includes positive control plasmids for the 8 The resistant strains were examined for carbapenemase multiplex PCR. The kit was used following the manu- and β-lactamase genes (Figs. 2, 3, 4). facturer’s instructions. Interpretation of the results The Chi-square test showed that resistance to carbapen- was performed according to the manufacture instruc- ems and CEPH was significantly influenced by the source tion with IncF plasmids designated as multi-replicons of samples (fish or water), but not by species of bacteria when they showed a combination of FII replicons (E. coli, ECC,and K. pneumoniae). Indeed, Figs. 2, 3, 4 with FIA or FIB [30, 31]. demonstrated that the resistance phenotype was similar between the three species of bacteria. Statistical analysis As displayed in Figs. 2, 3, 4, the isolates were grouped PASW Version 18 software (SPSS Inc., Chicago, IL, according to the resistance phenotype into; CRE and all USA) was used for statistical analysis. Descriptive statis- CEPH (isolates resistant to at least one of the tested car- tics were run and showed that the data are normally dis- bapenems and all tested cephalosporins); CRE and 2 tributed. A Chi square (X ) test was used to determine CEPH (isolates resistant to at least one of the tested car- whether there is a difference in the prevalence of the bapenems and two of the tested cephalosporins); CRE Table 1 Prevalence of Enterobacteriaceae strains in Tilapia fish and water at four different aquacultures Source No. of Enterobacteriaceae-positive samples (No. examined) Total E. coli ECC K. pneumoniae Fish (105) 66 (63%) 45* 12 9 Fishpond water inlets (30) 15 (50%) 9* 42 Tap water (44) 33 (75%) 22* 65 Outlet water (26) 16 (62%) 12* 22 * indicates a significant difference in the prevalence of E. coli from that of ECC and K. pneumoniae Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 5 of 12 Fig. 2 Number of carbapenem- and cephalosporin- resistant Enterobacteriaceae isolates among fish. The Enterobacteriaceae isolates from fish (n = 66), including E. coli (n = 45), ECC (n = 12), and K. pneumoniae (n = 9) were screened for resistance against carbapenems (CRE) and cephalosporins (CEPH: FOX, CAZ, CTX, and CRO). The strains are grouped according to their resistance phenotypes as follow: CRE and all CEPH (isolates resistant to at least one of the tested carbapenems and all tested cephalosporins); CRE and 2 CEPH (isolates resistant to at least one of the tested carbapenems and two of the tested cephalosporins); CEPH (isolates resistant to 2 or more of the tested cephalosporins but not to carbapenems); CRE (isolates resistant to at least one of the tested carbapenems but not to any of the tested cephalosporins). Each phenotype is marked with colour and its resistance genotypes are also provided; carbapenemase (bla , bla , and bla ) and β-lactamase (bla , bla , bla , KPC OXA-48 NDM CTX-M-15 SHV OXA-1 bla , and bla ) genes. (n) represents numbers of resistant strains TEM PER-1 (isolates resistant to at least one of the tested carbapen- carbapenemase genes (bla , bla , bla ). Of KPC OXA-48 NDM ems, but not to any of the tested cephalosporins); CEPH the 26 fish isolates resistant only to carbapenems, 17 (isolates resistant to 2 or more of the tested cephalospo- were carbapenemase producing, while 9 were negative to rins, but not to carbapenems). the three screened carbapenemase genes (Figs. 2, 3, 4). Of the 66 fish isolates, 64 (45 E. coli,12 ECC,7 K. pneumoniae) showed resistance as follow; 29 to carba- High numbers of aquaculture workers carried penems and all tested cephalosporins, 5 to carbapenems cephalosporin- and carbapenem-resistant and two of the tested cephalosporins, 26 to carbapenems Enterobacteriaceae alone, and 4 to cephalosporins alone (Fig. 2). Addition- Hand swab and faecal samples were collected from ally, of the 15 isolates from fishpond water inlets, 7 were humans working in the integrated agriculture- resistant to carbapenems and all cephalosporins, 1 to aquacultures. A relatively high proportion of the workers carbapenems and two cephalosporins, 1 to carbapenems showed the presence of Enterobacteriaceae in hand alone, and 5 to cephalosporins alone (Fig. 3a). All the 33 swabs (20 / 30) and faecal samples (15 / 30) (Fig. 5, isolates from tap water were resistant. Among them, 17 Panel I). Like in fish and water isolates, E. coli was the were resistant to carbapenems and all cephalosporins, predominant species isolated from the human samples and 16 were resistant to cephalosporins alone (Fig. 3b). (Panel I A and B). Among the 20 isolates from hand Similarly, the 16 outlet water isolates were resistant, 9 to swabs, 5 were resistant to carbapenems and all cephalo- carbapenems and all cephalosporins, 1 to carbapenems sporins, 2 to carbapenems and two of the tested cepha- and two cephalosporins drugs, and 6 to cephalosporins losporins, and 3 to cephalosporins alone (Panel I C). Of alone (Fig. 4). the 15 faecal isolates, 10 were resistant to carbapenems Irrespective of the source of samples or species of bac- and all cephalosporins, and 5 to cephalosporins alone teria, we found that strains resistant to all examined an- (Panel I D). Additionally, the resistance phenotype and tibiotics, carry predominantly bla either alone or genotype were similar among E. coli, ECC, and K. pneu- KPC with the β-lactamase genes (bla , bla , bla , moniae. Strains resistant to all examined antibiotics har- CTX-M-15 SHV TEM bla , and bla ). However, strains resistant to boured predominantly bla either with the other OXA-1 PER-1 KPC carbapenems and two cephalosporins harbour only carbapenemase genes (bla and bla ) or with NDM OXA-48 Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 6 of 12 Fig. 3 Number of carbapenem- and cephalosporin- resistant Enterobacteriaceae isolates among fishpond water inlets and tap water. The Enterobacteriaceae isolates from a fishpond water inlets (n = 14), including E. coli (n = 9), ECC (n = 3), and K. pneumoniae (n = 2), as well as from b tap water (n = 33), including E. coli (n = 22), ECC (n = 6), and K. pneumoniae (n = 5) were screened for resistance against carbapenems (CRE) and cephalosporins (CEPH: FOX, CAZ, CTX, and CRO). The strains are grouped according to their resistance phenotypes as follow: CRE and all CEPH (isolates resistant to at least one of the tested carbapenems and all tested cephalosporins); CRE and 2 CEPH (isolates resistant to at least one of the tested carbapenems and two of the tested cephalosporins); CEPH (isolates resistant to 2 or more of the tested cephalosporins but not to carbapenems); CRE (isolates resistant to at least one of the tested carbapenems but not to any of the tested cephalosporins). Each phenotype is marked with colour and its resistance genotypes are also provided; carbapenemase (bla , bla , and bla ) and β-lactamase (bla , KPC OXA-48 NDM CTX-M-15 bla , bla , bla , and bla ) genes. (n) represents numbers of resistant strains SHV OXA-1 TEM PER-1 Fig. 4 Number of carbapenem- and cephalosporin- resistant Enterobacteriaceae isolates among outlet water. The Enterobacteriaceae isolates from outlet water (n = 16), including E. coli (n = 12), ECC (n = 2), and K. pneumoniae (n = 2) were screened for resistance against carbapenems (CRE) and cephalosporins (CEPH: FOX, CAZ, CTX, and CRO). The strains are grouped according to their resistance phenotypes as follow: CRE and all CEPH (isolates resistant to at least one of the tested carbapenems and all tested cephalosporins); CRE and 2 CEPH (isolates resistant to at least one of the tested carbapenems and two of the tested cephalosporins); CEPH (isolates resistant to 2 or more of the tested cephalosporins but not to carbapenems). Each phenotype is marked with colour and its resistance genotypes are also provided; carbapenemase (bla , bla , and KPC OXA-48 bla ) and β-lactamase (bla , bla , bla , bla , and bla ) genes. (n) represents numbers of resistant strains NDM CTX-M-15 SHV OXA-1 TEM PER-1 Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 7 of 12 Fig. 5 Number of cephalosporins- and carbapenems-resistant Enterobacteriaceae isolates from aquaculture workers and inhabitants of aquaculture surrounding areas. Panel I: Hand swab and faecal samples from aquaculture workers (n = 30) were examined. a and b illustrate the number (n) and species of Enterobacteriaceae isolated from hand swabs and faecal samples, respectively. c and d illustrate the number (n) of resistant Enterobacteriaceae isolates from hand swabs and faecal samples, respectively. Panel II: Faecal samples from inhabitants (n = 45) were examined. e illustrates the number (n) and species of Enterobacteriaceae isolated. f shows the number (n) of resistant Enterobacteriaceae. The strains are grouped based on resistance against carbapenems (CRE) and cephalosporins (CEPH: FOX, CAZ, CTX, and CRO) as follow: CRE and all CEPH (isolates resistant to at least one of the tested carbapenems and all tested cephalosporins); CRE and 2 CEPH (isolates resistant to at least one of the tested carbapenems and two of the tested cephalosporins); CEPH (isolates resistant to 2 or more of the tested cephalosporins but not to carbapenems). Each phenotype is marked with colour and its resistance genotypes are also provided; carbapenemase (bla , bla , and KPC OXA-48 bla ) and β-lactamase (bla , bla , bla , bla , and bla ) genes. (n) represents numbers of resistant strains NDM CTX-M-15 SHV OXA-1 TEM PER-1 the β-lactamase genes (bla , bla , bla , and for the presence and type of Inc. plasmids using PCR- CTX-M-15 SHV TEM bla ) (Panel I C and D). Based-Replicon-Typing (S2, Fig. 6). PER-1 Faecal samples were also collected from inhabitants of Therewerenodifferences in thetypeof Inc.plas- the aquaculture surrounding areas. Enterobacteriaceae mids, based on the source of the isolates (fish, fish- was isolated from 15 of the examined 45 samples with a pond water inlets, tap water, outlet water, and predominance of E. coli (Fig. 5, Panel II A). Of the 15 iso- workers) (S2). As demonstrated in Fig. 6a, of the 30 lates, 6 were resistant to carbapenems and all cephalospo- examined resistant strains, 29 showed the presence of rins, and 7 were resistant to cephalosporins alone (Fig. 5, the IncF and the IncHI groups of plasmids, 28 carried Panel II B). Consistent with the results of the aquaculture the IncX, 21 harboured the IncI complex group, 17 workers, the strains from the inhabitants were resistant to had the epidemic plasmid IncA/C, and 14 carried the carbapenems and all cephalosporins carried predomin- wide-host range IncP and the IncW groups. Other antly bla with other carbapenemase genes (bla Inc. plasmids found in minority of the isolates were KPC NDM, bla )orwith the β-lactamase genes (bla , as follows; IncN (n =9), IncN2 (n =7), IncR (n =5), OXA-48 CTX-M-15 bla , bla ,and bla )(Fig. 5, Panel II B). IncU (n =4), and IncT (n =4). SHV TEM PER-1 It is important to note that, the majority of the IncF plasmids present (22 / 29) were multi-replicon (Fig. 6a), Similar types of incompatible (Inc) plasmids were carried carrying the FII with FIA and or FIB replicons (S2)[30, by the Enterobacteriaceae resistant strains isolated from 31]. A divergence in FIB (FIB-KN and FIB-KQ) and FII different sources as determined by the PBRT-kits (FIIS and FIIK) replicons was observed. The FIB-KN was CRE isolates that were resistant to all tested antibiotics mainly found in the K. pneumoniae isolates, while the and showed similar resistance genotypes were examined FIB-KQ was present in K. pneumoniae and E. coli isolates. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 8 of 12 Fig. 6 Types of Incompatible (Inc) plasmids carried by the Enterobacteriaceae-resistant isolates. a illustrates the types of Inc. plasmids found in the examined CRE isolates (n = 30) that were resistant to all tested CEPH and showed similar genotypes. The red colour in the histogram of the IncF plasmid indicates number of isolates that harbour multi-replicon. b shows the differences between the CRE isolates producing-carbapenemase genes alone (CPE, n = 11) and those producing both carbapenamese and β-lactamase (CPE + βLPE, n = 19) genes, in the proportion and types of Inc. plasmids Moreover, the FIIS and FIIK divergences were found in E. were collected from inhabitants of the aquaculture sur- coli, K. pneumoniae,and ECC isolates (S2). rounding areas. The prevalence of Enterobacteriaceae iso- Some differences were noticed in the type of Inc. plas- lated from apparently healthy fresh Nile Tilapia fish was mids between strains producing carbapenemase genes 63%, which is higher than that reported by previous stud- alone (CPE) and those producing both carbapenemase ies performed on fish raised in earthen pond in Egypt and β-lactamase genes (CPE + βLPE) (Fig. 6b). Among (44–53%) [35–37]. Like in fish, the aquaculture water them, the IncI complex plasmid was found in the higher showed a high prevalence of Enterobacteriaceae, particu- proportion of the CPE + βLPE strains (15/19) than the larly the tap water (75%) as compared to fishpond water CPE strains (6/11). The IncI complex contains five groups inlets (50%) and outlet water (62%). Furthermore, of the IncI, IncL, IncM, IncK, and IncB/O combined, or at least 30 examined workers, 15 showed the presence of Entero- two of them. Another difference was the presence of the bacteriaceae in the hand and faecal samples, whereas, 5 IncW plasmid only in CPE + βLPE strains (14 /19). had Enterobacteriaceae only in the hand swabs. The spe- cies of Enterobacteriaceae isolated from fish, water sam- Discussion ples and the workers included E. coli, ECC,and K. For the past 60 years, β-lactam antibiotics have been pneumoniae,with E. coli being the predominant species. amongst the most successful drugs used for the treatment Although the species of Enterobacteriaceae found could of bacterial infections in humans, animals, and fish [32– be fish pathogens [35–40], it is not the most common 34]. In the current study, we investigated the occurrence found bacteria in fish [2]. This together with the high of β-lactamase (βLPE)- and carbapenemase (CPE)- produ- prevalence of Enterobacteriaceae found in water, suggests cing Enterobacteriaceae in fresh fish, fishpond water inlets, faecal contamination in the aquaculture environment. tap water, outlet water, and workers at four integrated A high proportion of the Enterobacteriaceae isolates agriculture-aquacultures. Additionally, faecal samples from the aquaculture showed resistance to the tested Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 9 of 12 antibiotics, as found in fish (64 / 66), water inlets (14 / shown to be commonly associated with bla on the TEM 15), tap water (33 / 33), outlet water (16 /16), and the same plasmid [52], and possibly with bla and the SHV workers (15 / 20). Regardless of the source of samples, a broad spectrum bla [15, 51]. Our findings then OXA-1 major number of these isolates were CRE, which encom- raised the question of how such CRE strains have pass predominantly strains resistant to carbapenems and reached the aquacultures. The most likely explanation is all tested cephalosporins, followed by strains resistant to a co-resistance to carbapenems by selective pressure carbapenems and some cephalosporins, while a few from overuse of other antibiotics, for which resistance strains were resistant to carbapenems alone. The three determinants are co-localised with carbapenemase on phenotypes of CRE isolates had different resistance ge- the same plasmid [53]. Another possible explanation notypes. However, CRE isolates resistant to all cephalo- might be unrestricted use of carbapenems to treat fish, sporins carried predominantly both carbapenemase and which is consistent with our results that strains resistant β-lactamase genes, CRE isolates resistant to some cepha- only to carbapenems were found mainly among fish iso- losporins or to carbapenems alone harboured only car- lates. Unfortunately, this cannot be clarified, as no data bapenemase genes. According to CLSI 2020, CRE is available on quantities and types of antibiotics used in isolates that produce one or more carbapenemase genes aquaculture in Egypt. Furthermore, the resistant strains usually test resistant to one or more of cephalosporins, might access the aquaculture from agricultural sources, however, some CPE isolates can still test susceptible to where animal manure is known to be used as fertilizers cephalosporins [41]. This can be a possible explanation [6, 54, 55]. CRE has been isolated from dairy farms in for our finding of CRE isolates which carry one or more Egypt [56]. Another possible source of the resistant of the carbapenemase genes (bla , bla , bla ), strains could be tap water, which showed the presence KPC OXA-48 NDM but susceptible to the tested cephalosporins (Figs. 2, 3). of high amounts of CRE and cephalosporins-resistant In this regard, a study from South Africa also found Enterobacteriaceae, indicating faecal contamination. The some CPE-CRE isolates which were susceptible to ceph- main source of tap water in Egypt [57] is from surface alosporins [42]. water like rivers, lakes or canals. In many Egyptian gov- It is important to note that among the 26 CRE fish iso- ernorates, sewage is usually discharged untreated into lates that showed resistance only to carbapenems, 9 iso- the surface water [58, 59]. Since sewage act as a pool of lates did not carry any of the tested carbapenemase genes, antibiotic residues as well as resistant bacteria from dif- suggesting that they might harbour other carbapenemases ferent sources [58, 59], this can result in contamination which were not screened in the present study. of tap water. This is supported by our results that Enterobacteriaceae isolates resistant to cephalosporins showed the occurrence of Enterobacteriaceae in faecal alone were also found but in a lower proportion than samples from inhabitants of the aquaculture surrounding the CRE. Slight differences were noticed between the areas, which have similar resistance phenotype and four examined aquacultures, in the numbers of CRE and genotype as the isolates from the aquaculture workers cephalosporins-resistant Enterobacteriaceae, which were with a predominance of bla and bla . In this KPC CTX-M-15 not statistically different. Cephalosporins-resistant En- regard, previous studies reported preponderance of En- terobacteriaceae isolates have previously been found in terobacteriaceae carrying bla and bla among KPC CTX-M-15 fish and aquatic environments from different countries, humans in Middle East [60] and North Africa [61, 62]. including Egypt [40, 43–48]. However, a few recent stud- Characterisation of the Inc. plasmid types harboured ies showed the presence of CRE strains among fish [49, by the CRE isolates that were resistant to all tested ceph- 50], indicating an increase in the prevalence of CRE in alosporins, revealed a similar profile among the isolates aquaculture. Irrespective of the species of bacteria or from fish, water, and workers. The major plasmid groups source of samples, the carbapenemase-producing isolates found were as follow; IncF, IncHI, IncX, IncI-complex, showed a predominance of bla over the other two IncA/C, IncP, and IncW, which agrees with findings that KPC genes bla and bla , which agrees with other IncF, IncHI, IncX, IncI-complex, and IncA/C are com- OXA-48 NDM study that detected the presence of bla in seafood monly associated with CRE isolated from humans and KPC isolates [51]. Furthermore, all the isolates that were posi- animals [19–21]. Crucially, the IncA/C is considered as tive for β-lactamase genes harboured bla in an epidemic plasmid, as it has been detected in different CTX-M-15 combination with one or more of the other β-lactamase countries and has a wide-host range of different sources genes (bla , bla , bla and bla ). This is [63]. IncP and IncW have been categorised among rarely OXA-1 SHV TEM, PER-1 consistent with findings that showed a predominance of reported plasmids [21, 64], however, we found them in a bla among β-lactam-resistant Enterobacteria- relatively high number of the isolates from different CTX-M-15 ceae isolated from wild fish in Algeria [44], from frozen sources. Since these two plasmids have a broad-host Mackerel in Saudi Arabia [45], and water collected from range and can carry multiple resistance genes [64, 65], fish farms in Egypt [46]. Moreover, the bla was their presence in our isolates indicates a role in the CTX-M Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 10 of 12 spread of resistance genes among bacteria of different gen- Supplementary information Supplementary information accompanies this paper at https://doi.org/10. era and sources. Other plasmids like IncN, IncN2, IncL, 1186/s13756-020-00736-3. IncU, IncT, and IncR were also present, but in minority of the current isolates. This is consistent with [19], who Additional file 1. stated that these plasmids are not often reported. One of the interesting results is a predominance of the Abbreviations multi-replicon IncF among the tested CRE isolates, with βLPE: β-lactamase-producing Enterobacteriaceae; CAZ: Ceftazidime; divergence in FII (FIIS, FIIK) and FIB (FIB-KQ, FIB-KN), CDC: Centre for Disease Control and Prevention; CEPH: Cephalosporins; but not in FA (S2), suggesting that such plasmids are rap- CLSI: Clinical and Laboratory Standards Institute; CPE: Carbapenemase- producing Enterobacteriaceae; CRE: Carbapenem-resistant Enterobacteriaceae; idly emerging with the subsequent increase in dissemin- CRO: Ceftriaxone; CTX: Cefotaxime; ECC: Enterobacter cloacae complex; ation of resistance genes. IncF plasmid is unique, as it can EMB: Eosin methylene blue agar; ESBL: Extended-spectrum β-lactam; carry many replicons, one replicon is strongly conserved ETP: Ertapenem; FOX: Cefoxitin; Inc. plasmids: Incompatible plasmids; IPM: Imipenem; K. pneumoniae: Klebsiella pneumoniae; MEM: Meropenem for plasmid replication, while the others are free to diverge as a mechanism of evolution [19, 30]. Additionally, the IncF plasmid can co-integrate with other plasmids found Acknowledgements We are grateful to the aquaculture workers and people living in the areas in the current study, like IncI1, IncA/C, and IncN forming surrounding the fish farms for participating in the current study. The a mega-plasmid with multi-resistance regions [19, 31, 66, cooperation of the aquaculture owners is highly appreciated. 67]. Taken together, the current CRE isolates carry a wide range of Inc. plasmids which are known to be associated Authors’ contributions with multi-drug resistance determinants. The profile of Dalia Hamza participated in the design and coordination of the study, performed the experiments, and assisted in the editing of the manuscript. these plasmids was similar between isolates from fish, Sohad Dorgham participated in the design of the study, collection of water, and humans working in contact with fish, suggest- samples and performed the experiments. Elshaima Ismael performed the ing a possible transfer among bacteria from different statistical analysis and preparation of tables. Sherein Ismail Abd El-Moez par- ticipated in the design of the study and collection of samples. Rehab Elhelw sources. To our knowledge, no data is available regarding and Mahmoud Elhariri performed the experiments and preparation of the the prevalent type of Inc. plasmids in Egypt. manuscript. Eman Hamza participated in the design of the study, assisted in the statistical analysis and preparation of the figures, and edited the manu- script. All the authors read and approved the final manuscript. Conclusion The present study reveals the occurrence of CRE which are resistant to all tested cephalosporins among fish, Funding This research did not receive any grant from funding agencies in the public, water, and workers at aquacultures integrated with agri- commercial, or not-for-profit sectors. culture systems in Egypt. The fish included in the study were apparently healthy, indicating they could act as res- Availability of data and materials ervoirs for potentially pathogenic bacteria in humans. All data generated or analysed during this study are included in this Such resistant strains might have occurred in the aqua- published article. culture due to unregulated use of antibiotics to treat fish, or through agricultural sources, faecal contaminated tap Ethics approval and consent to participate water, or the workers. The resistant strains carry a var- Protocols for collection of samples as well as the experiment plan and all methods were performed in accordance with the guidelines and regulations iety of resistance Inc. plasmids which are similar be- of Cairo University Council and were approved by the Scientific Research tween the strains from different sources. Most of these Committee and Bioethics Board of Cairo University, Faculty of Veterinary plasmids have a broad-host range and pose a great risk Medicine, Cairo, Egypt. Ethical clearance to use human subjects was obtained from the designated health facility (National Research Centre, Giza, for the possible spread of resistance. Therefore, we rec- Egypt) and written informed consent was obtained from each person upon ommend that studies on antimicrobial resistance should information of the use of samples. consider thorough, systematic studies of animals, fish, humans, tap water, and sewage, as all these factors are Consent for publication linked together. The current study has some limitations, Not applicable. including examination of integrated aquaculture from only one governorate in Egypt and the limited number Competing interests of the tested carbapenemase genes (bla , bla , The authors have no competing interests. KPC NDM-1 and bla ). Therefore, further work is required to OXA-48 Author details elucidate the extent of antibiotic resistance in more inte- 1 Department of Zoonoses, Faculty of Veterinary Medicine, Cairo University, grated aquaculture from other governorates. Particularly, Giza square, PO Box 12211, Cairo, Egypt. Department of Microbiology and Immunology, National Research Centre, Giza, Egypt. Department of to screen for carbapenems and extended-spectrum- Veterinary Hygiene and Management, Faculty of Veterinary Medicine, Cairo cephalosporins, with including a wide panel of carbape- 4 University, Giza, Egypt. Department of Microbiology, Faculty of Veterinary nemase genes. Medicine, Cairo University, Giza, Egypt. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 11 of 12 Received: 31 October 2019 Accepted: 9 May 2020 25. CDC, Centre for Disease Control and Prevention. Patient safety atlas. 2018. https://www.cdc.gov/hai/surveillance/ar-patient-safety-atlas.html. Accessed 13 July 2018. 26. Elhariri M, Hamza D, Elhelw R, Dorgham SM. Extended-spectrum beta- lactamase -producing Pseudomonas aeruginosa in camel in Egypt: potential References human hazard. Ann Clin Microbiol Antimicrob. 2017;16:1–6. 1. Cabello FC, Godfrey HP, Buschmann AH, Dölz HJ. Aquaculture as yet 27. Sugumar M, Kumar KM, Manoharan A, Anbarasu A, Ramaiah S. Detection of another environmental gateway to the development and globalization of OXA-1 β-lactamase gene of Klebsiella pneumoniae from blood stream antimicrobial resistance. Lancet Infect Dis. 2016;16:127–33. infections by conventional PCR and in-Silico analysis to understand the 2. Schmidt AS, Bruun MS, Dalsgaard I, Larsen JL. Incidence, distribution, and mechanism of OXA mediated resistance. PLoS One. 2014. https://doi.org/10. spread of tetracycline resistance determinants and integron-associated 1371/journal.pone.0091800. antibiotic resistance genes among motile aeromonads from a fish farming 28. Hamza E, Dorgham SM, Hamza DA. Carbapenemase-producing Klebsiella environment. Appl Environ Microbiol. 2001;67:5675–82. pneumoniae in broiler poultry farming in Egypt. J Global Antimicrob Resist. 3. Defoirdt T, Sorgeloos P, Bossier P. Alternative to antibiotics for the control of 2016;7:8–10. bacterial disease in the aquaculture. Curr Opin Microbiol. 2011;14:251–8. 29. Li B, Yi Y, Wang Q, Woo PCY, Jing H, Gao GF. Analysis of drug resistance 4. Hoa PT, Managaki S, Nakada N, Takada H, Shimiz A, Anh DH. Antibiotic determinants in Klebsiella pneumoniae isolates from a tertiary-care hospital contamination and occurrence of antibiotic-resistant bacteria in aquatic in Beijing, China. PLoS One. 2012. https://doi.org/10.1371/journal.pone. environments of northern Vietnam. Sci Total Environ. 2011;409:2894–901. 5. Little DC, Edwards P. Integrated livestock-fish farming systems. Rome: Food 30. Villa L, Garcia-Fernandez A, Fortini D, Carattoli A. Replicon typing of IncF and Agriculture Organization of the United Nations; 2003. http://www.fao. plasmids carrying virulence and resistance determinants. J Antimicrob org/3/a-y5098e. Chemother. 2010;65:2518–29. 6. Neela FA, Banu NA, Rahman A, Rahman MH, Alam MF. Occurrence of 31. Osborn AM, da Silva Tatley FM, Steyn LM, Pickup RW, Saunders JR. Mosaic antibiotic resistant bacteria in pond water associated with integrated plasmids and mosaic replicons: evolutionary lessons from the analysis of poultry-fish farming in Bangladesh. Sains Malaysiana. 2015;44:371–7. genetic diversity in IncFII-related replicons. Microbiol. 2000;146:2267–75. 7. Seiler C, Berendonk TU. Heavy metal driven co-selection of antibiotic 32. Economou V, Gousia P. Agriculture and food animals as a source of resistance in soil and water bodies impacted by agriculture and antimicrobial-resistant bacteria. Infect Drug Resist. 2015;8:49–61. aquaculture. Front Microbiol. 2012;3:399. 33. Prestinaci F, Pezzoti P, Pantosti A. Antimicrobial resistance: a global 8. Burridge L, Weis JS, Cabello F, Pizarro J, Bostick K. Chemical use in salmon multifaceted phenomenon. Pathog Glob Health. 2015;109:309–18. aquaculture: a review of current practices and possible environmental 34. Sarmah AK, Meyer MT, Boxall AB. A global perspective on the use, sales, effects. Aquaculture. 2010;306:7–23. exposure pathways, occurrence, fate and effects of veterinary antibiotics 9. Buschmann AH, Tomova A, Lopez A, Maldonado MA, Henriquez LA, Ivanova (VAs) in the environment. Chemosphere. 2006;65:725–59. L, et al. Salmon aquaculture and antimicrobial resistance in the marine environment. PLoS One. 2012;7:42724. 35. Abdel-Latif HMR, Sedeek EK. Diversity of Enterobacteriaceae retrieved from diseased cultured Oreochromis niloticus. Int J Fish Aqua Stud. 2017;5:29–34. 10. Heuer OE, Kruse H, Grave K, Collignon P, Karunasagar I, Angulo FJ. 2009. 36. Aly SM, Nouh WG, Salem-Bekhit MM. Bacteriological and histopathological Human health consequences of use of antimicrobial agents in studies on Enterobacteriaceae in Nile Tilapia, Oreochromis niloticus. J Pharm aquagculture. Clin Infect Dis. 2009;49:1248–53. Biomed Sci. 2012;2:94–104. 11. Rhodes G, Huys G, Swings J, Mcgann P, Hiney M, Smith P, et al. Distribution 37. Elsherief MF, Mousa MM, Abd El-Galil H, El-Bahy EF. Enterobacteriaceae of oxytetracycline resistance plasmids between aeromonads in hospital and associated with farm fish and retailed ones. Alex J Vet Sci. 2014;42:99–104. aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Appl Environ Microbiol. 2000;66: 38. Hassan AHM, Noor-El Deen AE, Galal HM, Dorgham SM, Bakry MA, Hakim AS. Further characterization of Enterobacteriaceae isolated from cultured 3883–90. 12. Sheu CC, Chang YT, Lin SY, ChengYH HPR. Infections caused by freshwater fish in Kafr El Shiek governorate: clinical, biochemical and carbapenem-resistant Enterobacteriaceae: An update on therapeutic histopathological study with emphasis on treatment trials. Glob Veter. 2012; options. Front Microbiol. 2019;10:80. 9:617–29. 13. Wilson H, Török ME. Extended spectrum β-lactamase producing and 39. Saswathi R, Sumithra P, Sivakami R. Identification of Enterobacteriaceae carbapenamese producing Enterobacteriaceae. Microb Genom. 2018;4:7. studies in carps during rearing a freshwater pond. Int J Curr Microbiol Appl 14. Bush K, Jacoby GA. Updated functional classification of beta-lactamases. Sci. 2015;4:661–9. Antimicrob Agents Chemother. 2010;54:969–76. 40. Abgottspon H, Nüesch-Inderbinen MT, Zurfluh K, Althaus D, Hächler H, 15. Dandachi I, Chaddad A, Hanna J, Matta J, Daoud Z. Understanding the Stephan R. Enterobacteriaceae with extended-Spectrum- and pAmpC-type epidemiology of multi-drug resistant gram-negative bacilli in the Middle -lactamase-encoding genes isolated from freshwater fish from two lakes in East using a one health approach. Front Microbiol. 2019;10:1941. Switzerland. Antimicrob Agents Chemother. 2014;58:2482–4. 41. CLSI, Clinical and Laboratory Standards Institute. Performance standards for 16. Exner M, Bhattacharya S, Christiansen B, Gebel J, Goroncy-Bermes P. th antimicrobial susceptibility testing (M100, 30 Edition); 2020. Hartemann p. Antibiotic resistance: What is so special about multidrug- resistant gram-negative bacteria?. GMS Hyg Infect Control. 2017;12:1–24. 42. Singh-Moodley A, Perovic O. Antimicrobial susceptibility testing in 17. Canton R, Ruiz-Garbajosa P. Co-resistance: an opportunity for the bacteria predicting the presence of carbapenemase genes in Enterobacteriaceae in and 2 resistance genes. Curr Opin Pharmacol. 2011;11:477–85. South Africa. BMC Infect Dis. 2016;16:536. 18. Austin S, Nordström K. Partition-mediated incompatibility of bacterial 43. Akinbowale OL, Peng H, Barton MD. Antimicrobial resistance in bacteria plasmids. Cell. 1990;60:351–4. isolated from aquaculture sources in Australia. J Appl Microbiol. 2006;100: 19. Rozwandowicz M, Brouwer MSM, Fischer J, Wagenaar JA, Gonzalez-Zorn B, 1103–13. Guerrat B. Plasmids carrying antimicrobial resistance genes in 44. Brahmi S, Tuoati A, Dunyach-Remy C, Sotto A, Pantel A, Lavigne JP. High Enterobacteriaceae. J Antimicrob Chemother. 2018;73:1121–37. prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae 20. Carattoli A. Resistance plasmid families in Enterobacteriaceae. Antimicrob in wild fish from the Mediterranean Sea in Algeria. Microb Drug Resist. 2018; Agents Chemother. 2009;53:2227–38. 24:290–8. 21. Carloni E, Andreoni F, Omiccioli E, Villa L, Magnani M, Carratoli A. 45. Elhadi N, Alsamman K. Incidence and antimicrobial susceptibility pattern of Comparative analysis of the standard PCR-based replicon typing (PBRT) with extended-spectrum-β-lactamase-producing Escherichia coli isolated from the commercial PBRT-Kit. Plasmid. 2017;90:10–4. retail imported mackerel fish. Afr J Biotech. 2015;14:1954–60. 22. Bezuidenhout CC, Mthembu N, Puckree T, Lin J. Microbiological evaluation 46. Ishida M, Ahmed AM, Mahfouz NB, Kimura T, El-Khodery SA, Moawad AA. of the Mhlathuze River, Kwazulu-Natal (RSA). Water SA. 2002;28:281–6. Molecular analysis of antimicrobial resistance in gram-negative bacteria 23. Collee JG, Fraser AG, Marmion BP, Simmons A. Practical medical isolated from fish farms in Egypt. J Vet Med Sci. 2010;72:727–34. microbiology. New York: Churchill Livingstone; 1996. 47. Sarter S, Nguyen K, Hung LT, Lazart J, Montet D. Antibiotic resistance in 24. CLSI, Clinical and Laboratory Standards Institute. Performance standards for gram-negative bacteria isolated from farmed catfish. Food Control. 2007;18: antimicrobial susceptibility testing (M100-S24);2016. 1391–6. Hamza et al. Antimicrobial Resistance and Infection Control (2020) 9:67 Page 12 of 12 48. Singh AS, Lekshmi M, Prakasan S, Nayak BB, Kumar S. Multiple antibiotic- resistant, Extended Spectrum-Lactamase (ESBL)-producing Enterobacteriaceae in fresh seafood. Microorganisms. 2017;5(53):1–10. 49. Janecko N, Martz SL, Avery BP, Daignault D, Desruisseau A, Boyd D. Carbapenem-resistant Enterobacter spp. in retail seafood imported from Southeast Asia to Canada. Emerg Infect Dis. 2016;22:1675–7. 50. Mangat CS, Boyd D, Janecko N, Martz SL, Desruisseau A, Carpenter M. Characterization of VCC-1, a novel ambler class a carbapenemase from Vibrio cholerae isolated from imported retail shrimp sold in Canada. Antimicrob Agents Chemother. 2016;60:1819–25. 51. Sabate M, Tarrago R, Navarro F, Miro E, Verges C. Barbe, et al. cloning and sequence of the gene encoding a novel cefotaxime-hydrolysing β- lactamase (CTX-M-9) from Escherichia coli in Spain. Antimicrob Agents Chemother. 2000;44:1970–3. 52. Karim A, Poirel L, Nagarajan S, Nordmann P. Plasmid-mediated extended- spectrum β-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiol Banner. 2006;201:237–41. 53. Barron SA, Diene SM, Rolain JM. Human microbiomes and antibiotic resistance. Hum Microb J. 2018;10:43–52. 54. Dang STT, Petersen A, Truong DV, Chu HTT, Dalsgaard A. Resistance in bacteria at integrated pig-fish farms in Vietnam. Appl Environ Microbiol. 2011;77:4494–8. 55. Petersen A, Andersen JS, Kaewmak T, Somsiri T, Dalsgaard A. Impact of integrated fish farming on antimicrobial resistance in a pond environment. Appl Environ Microbiol. 2002;68:6036–42. 56. Braun S, Ahmed MFE, El-Adway H, Hotzel H, Engelmann I, Weiss D, et al. Surveillance of extended Spectrum β-lactamase-producing Escherichia coli in dairy cattle farms in the Nile Delta, Egypt. Front Microbiol. 2016;7(1020):1–14. 57. El-Kowrany SI, El-Zamarany EA, El-Nouby KA, El-Mehy DA, Abo Ali EA, Othman AA. Water pollution in the middle Nile Delta, Egypt: an environmental study. J Adv Res. 2016;7:781–9. 58. Ezzat SM, Mahdy HM, Abo-State MA, Abd-Elshakour EH, El-Bahnasawy MA. Water quality assessment of River Nile at Rosetta branch Middle East. J Sci Res. 2012;12:413–23. 59. Amine AEK. Extended spectrum Beta-lactamase producing bacteria in sewage water, Alexandria, Egypt. Int J Biosc Bioch Bioinf. 2013;3:605–8. 60. Sonnevend A, Al-Dhaheri K, Mag T, Herpay M, Kolodziejek J, Nowotny N. CTX-M-15-producing multidrug-resistant enteroaggregative Escherichia coli in the United Arab Emirates. Clin Microbiol Infect. 2006;12:582–285. 61. Iabadene H, Messai Y, Ammari H, Alouache S, Verdet C, Bakour R, Arlet G. Prevalence of plasmid-mediated AmpC β-lactamases among Enterobacteriaceae in Algiers hospitals. Int J Antimicrob Agents. 2009;34: 340–2. 62. Mamlouk K, Boubaker IB, Gautier V, Vimont S, Picard B, Ben RS. Emergence and outbreaks of CTX-M β-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital. J Clin Microbiol. 2006;44: 4049–56. 63. Hancok S, Phan MD, Peters KM, Forde BM, Chong TM, Yin WF, et al. Identification of IncA/C plasmid replication and maintenance genes and development of a plasmid multi-locus sequence typing scheme. Antimicrob Agents Chemother. 2017;61(2):1–17. 64. Almeida AC. de Sa Cavalcanti FL, Vilela MA, gales AC, de Morais MA, Jr Camargo de Morais MM. Escherichia coli ST502 and Klebsiella pneumoniae ST11 sharing an IncW plasmid harbouring the bla gene in an intensive KPC-2 care unit patient. Int J Antimicrob Agents. 2012;40:374–6. 65. Yano H, Rogers LM, Knox MG, Heuer H, Smalla K, Brown CJ, et al. Host range diversification within the IncP1 plasmid group. Microbiol. 2013;159:2303–15. 66. Froehlich B, Parkhill J, Sanders M, Quail MA, Scott JR. The pCoo plasmid of enterotoxigenic Escherichia coli is a mosaic conitegrate. J Bacteriol. 2005; 187:6509–16. 67. Desmet S, Nepal S, van Dijl JM, van Ranst M, Chlebowicz MA, Rossen JW, et al. Antibiotic resistance plasmids cointegrated into a megaplasmid harbouring the bla carbapenemase gene. Antimicrob Agents OXA-427 Chemother. 2018;62(3):1–6. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Journal

Antimicrobial Resistance & Infection Control – Springer Journals

Published: May 19, 2020

There are no references for this article.