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Prevalence and Characterization of Multi-Drug-Resistant Gram-Negative Bacilli Isolated From Lebanese Poultry: A Nationwide Study

Prevalence and Characterization of Multi-Drug-Resistant Gram-Negative Bacilli Isolated From... fmicb-09-00550 March 21, 2018 Time: 17:26 # 1 ORIGINAL RESEARCH published: 23 March 2018 doi: 10.3389/fmicb.2018.00550 Prevalence and Characterization of Multi-Drug-Resistant Gram-Negative Bacilli Isolated From Lebanese Poultry: A Nationwide Study 1,2 1 1 1 3 Iman Dandachi , Elie S. Sokhn , Elias A. Dahdouh , Eid Azar , Bassel El-Bazzal , 2 1 Jean-Marc Rolain and Ziad Daoud Clinical Microbiology Laboratory, Faculty of Medicine and Medical Sciences, University of Balamand, Beirut, Lebanon, 2 3 IRD, APHM, MEPHI, IHU-Méditerranée Infection, Aix-Marseille Université, Marseille, France, The Lebanese Ministry of Agriculture, Beirut, Lebanon Currently, antimicrobial resistance is one of the most prominent public health issues. In fact, there is increasing evidence that animals constitute a reservoir of antimicrobial Edited by: resistance. In collaboration with the Lebanese Ministry of Agriculture, the aim of this Miklos Fuzi, study was to determine the prevalence of intestinal carriage of multi-drug-resistant Semmelweis University, Hungary Gram-negative Bacilli in poultry farms at the national level. Between August and Reviewed by: Sebastian Guenther, December 2015, 981 fecal swabs were obtained from 49 poultry farms distributed University of Greifswald, Germany across Lebanon. The swabs were subcultured on MacConkey agar supplemented Djamel Drider, with cefotaxime (2 mg/ml). Isolated strains were identified using MALDI-TOF mass Lille University of Science and Technology, France spectrometry. Multilocus sequence typing analysis was performed for Escherichia coli. *Correspondence: Phenotypic detection of extended spectrum b-lactamases (ESBL) and AmpC production Ziad Daoud was performed using double disk synergy and the ampC disk test, respectively. ziad.daoud@balamand.edu.lb b-lactamase encoding genes bla , bla , bla , bla , bla , bla , bla , CTXM TEM SHV FOX MOX EBC ACC Specialty section: bla , and bla using PCR amplification. Out of 981 fecal swabs obtained, 203 DHA CMY This article was submitted to (20.6%) showed bacterial growth on the selective medium. Of the 235 strains isolated, Antimicrobials, Resistance and Chemotherapy, 217 were identified as E. coli (92%), eight as Klebsiella pneumoniae (3%), three as a section of the journal Proteus mirabilis (1%) and three as Enterobacter cloacae (1%). MLST analysis of E. coli Frontiers in Microbiology isolates showed the presence of ST156, ST5470, ST354, ST155, and ST3224. The Received: 02 December 2017 phenotypic tests revealed that 43.5, 28.5, and 20.5% of the strains were ampC, ESBL, Accepted: 12 March 2018 Published: 23 March 2018 and ampC/ESBL producers, respectively. The putative TEM gene was detected in 83% Citation: of the isolates, SHV in 20%, CTX-M in 53% and CMY ampC b-lactamase gene in 65%. Dandachi I, Sokhn ES, Dahdouh EA, Our study showed that chicken farms in Lebanon are reservoirs of ESBL and AmpC Azar E, El-Bazzal B, Rolain J-M and Daoud Z (2018) Prevalence producing Gram-negative bacilli. The level of antibiotic consumption in the Lebanese and Characterization veterinary medicine should be evaluated. Future studies should focus on the risk factors of Multi-Drug-Resistant associated with the acquisition of multi-drug-resistant organisms in farm animals in Gram-Negative Bacilli Isolated From Lebanese Poultry: A Nationwide Lebanon. Study. Front. Microbiol. 9:550. doi: 10.3389/fmicb.2018.00550 Keywords: ampC, ESBL, E. coli, poultry, carriage Frontiers in Microbiology | www.frontiersin.org 1 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 2 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry through direct contact or through the handling/consumption INTRODUCTION of undercooked/uncooked animal products (Dahms et al., Antibiotic resistance is currently a major topic of interest for 2014). researchers and physicians. In particular, the rise of multi- In Lebanon, several studies addressing MDROs in hospital drug resistance in Gram-negative bacteria is now a serious settings have been conducted. One study done at the American challenge encountered by healthcare professionals (Exner et al., University of Beirut Medical Center between 2008 and 2011 2017). Resistance in Gram-negative bacteria is mainly mediated reported that 1.07 and 2.45% of E. coli and Klebsiella via the production of extended spectrum b-lactamases (ESBL), pneumoniae clinical isolates, respectively, were ESBL producers ampC b-lactamases and carbapenemases (Schill et al., 2017). and ertapenem-resistant (Baroud et al., 2013). Another study Genes encoding these enzymes are often located on plasmids conducted in the north reported that over the period of carrying resistance genes to other commonly used antibiotics in 2009–2012, 9% and 28% of the bacteraemia episodes in clinical settings (Seiffert et al., 2013). Infections with these multi- febrile neutropenic patients were caused by carbapenem and drug-resistant organisms (MDROs) will thus pose therapeutic third-generation cephalosporin-resistant Gram-negative bacilli, challenges; the antibiotic pipeline is drying up, and no new respectively (Moghnieh et al., 2015). However, very few antimicrobial agents are anticipated in the near future to treat studies have addressed this issue in the environment. One infections caused by MDROs (Bettiol and Harbarth, 2015). study showed that Acinetobacter baumannii was detected In fact, it has been generally accepted that the main driver in 6.9% of water samples, 2.7% of milk samples, 8.0% of for the rapid evolution of bacterial resistance is the uncontrolled meat samples, 14.3% of cheese samples and 7.7% of animal usage of antibiotics in human medicine. It is suggested that samples (Rafei et al., 2015). Another study in which 115 this theory is also applicable to the veterinary sector (Kempf stool samples were collected from livestock animals from et al., 2015). The European Centre for Disease Prevention and different farms in north Lebanon reported the detection of Control/European Food Safety Authority/European Medicines four VIM-2 producing Pseudomonas aeruginosa, four OXA- Agency (ECDC/EFSA/EMA) joint report stated that in 2014, 23 producing A. baumannii and one OXA-23/OXA-58 co- the average antibiotic consumption in animals (152 mg/kg) producing A. baumannii (Al Bayssari et al., 2015a). Furthermore, was higher than in humans (124 mg/kg). Univariate analysis Al Bayssari et al. (2015b) reported the isolation of an OXA- showed a signification correlation between fluoroquinolone 48 harboring E. coli isolate from fowl in Lebanon. More consumption and resistance in Escherichia coli in the human recently, Diab et al. (2016) detected a relatively high prevalence and animal sectors, between polymyxins and tetracyclines and of CTX-M-15 producing E. coli in Lebanese cattle. In the E. coli in animals, and for 3rd/4th generation cephalosporins above-mentioned studies in Lebanese livestock, MLST analysis and E. coli in humans (ECDC/EFSA/EMA, 2017). Antibiotics are revealed the presence of sequence types common to both heavily administered for therapeutic and prophylaxis purposes humans and animals (Al Bayssari et al., 2015a; Rafei et al., in veterinary medicine. As growth promoters, this practice 2015; Diab et al., 2016), which suggests that Lebanese farms is no longer adapted in the European Union, whereas it are potent reservoirs of multi-drug-resistant organisms that persists in North America and other countries (Economou could be transmitted to humans. In the present study and in and Gousia, 2015). In their study, Chantziaras et al. (2014) collaboration with the Lebanese Ministry of Agriculture, our found a significant correlation between the use of antibiotics in aim was to determine the national epidemiology of multi-drug- livestock and the corresponding level of resistance toward these resistant Gram-negative bacilli in Lebanese chicken farms in antimicrobials in E. coli strains isolated from pigs, poultry and terms of intestinal carriage. cattle. During the last years, the prevalence of ESBLs, ampC, and carbapenemase producing Gram-negative bacteria has become MATERIALS AND METHODS extensively reported in food producing animals (Ghodousi et al., 2015; Gonzalez-Torralba et al., 2016; Haenni et al., 2016). In Ethics Statement their review paper, Schwarz et al. (2016) showed that studies describing the epidemiology of resistant organisms in livestock The Ministry of Agriculture in Lebanon granted approval to collect chicken samples from representative farms in the targeted mainly swine, cattle and poultry. The prevalence of resistance varied from one country to another (Alonso et al., country as per the national norms for animal sampling and manipulation. This sampling was in conformity with the 2017). Although the extent to which food of animal origin international regulations for animal safety. All of the involved contributes to the zoonotic transmission of multi-drug-resistant farms officially received authorization from the Ministry of organisms, i.e., ESBL and carbapenemase producers, has not Agriculture, and this was considered, after undergoing an yet been well established (Madec et al., 2017), it suggests that acceptance process, an official and legal document. Therefore, an sharing the same ESBL genes, plasmids and strains constitutes Institutional Review Board (IRB) approval was obtained for the possible evidence of zoonotic transmission of MDROs from animals to humans (Leverstein-van Hall et al., 2011; Dahms et al., present study. 2014). Furthermore, the increased risk of ESBL fecal carriage in individuals with a high degree of contact with broiler chickens Samples Collection is an indicator of transmission (Huijbers et al., 2014). Enteric- Between August and December 2015, 981 rectal swabs were resistant strains in livestock can be easily transferred to humans collected from 49 poultry farms distributed over the seven Frontiers in Microbiology | www.frontiersin.org 2 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 3 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry districts of Lebanon. Six to seven farms were visited in each a negative one. Furthermore, for the presumptive detection of district. The average number of samples taken from each farm carbapenemases, the carba NP test was performed as previously was 20 fecal swabs (Table 1). The 20 samples collected were described (Bakour et al., 2015). A bacterium was characterized randomly taken from each farm. Technical assistance, i.e., as being multi-drug-resistant when resistance to at least three fecal swabs, gloves, costumes, and a portable refrigerator, were classes of antibiotics was observed (Magiorakos et al., 2012). provided by the Ministry of Agriculture team. The collected swabs were directly placed in a portable refrigerator, and when Molecular Characterization of they arrived at the University Laboratory, they were stored at b-Lactamase Encoding Genes 80 C until use. The farms visited were selected by considering All of the isolates that showed a key hole effect or had cefoxitin their geographical location, presence or absence of a nearby resistance with non-susceptibility to cefepime were subjected community and the size of the farms (at least 3,000 chickens per to real-time PCR analysis for the detection of SHV, TEM and breeding site). Eighty percent of the samples were gathered from CTX-M encoding genes (Roschanski et al., 2014). Simplex PCRs broiler chickens, while 20% were taken from layers. The mean for the genes encoding AmpC b-lactamases FOX, MOX, ACC, average age of the broilers and layers was 31 days and 14 months, EBC, DHA, and CMY were conducted for all strains showing respectively. non-susceptibility to cefoxitin (Dallenne et al., 2010). Simplex PCR was also used to test the ADC ampC b-lactamase gene MALDI-TOF MS Identification in A. baumannii (Liu and Liu, 2015). DNA extraction was Rectal swabs were sub-cultured on a MacConkey agar performed according to the manufacturer’s instructions using supplemented with 2 mg/ml of cefotaxime for the preliminary EZ1 DNA extraction kits (Qiagen, Courtaboeuf, France) with the screening of antibiotic-resistant Gram-negative bacilli. After EZ1 Advanced XL biorobot. overnight incubation at 37 C, colonies showing different morphologies were picked up from each selective plate and Multilocus Sequence Typing tested separately with MALDI-TOF MS for identification using One E. coli strain from each cluster shown in the MSP the Microflex LT spectrometer (Bruker Daltonics, Bremen, dendrogram was chosen, and MLST typing was performed based Germany) (Seng et al., 2010; Singhal et al., 2015). The spectra on allelic profiles to determine their evolutionary relationship obtained for each strain were stored and downloaded into a (Peng and Zong, 2011). Seven housekeeping genes were used: MALDI Biotyper 3.0 system to create a single main spectrum for adk, fumC, gyrB, icd, mdh, purA, and recA. Analysis of the genes’ each bacterial isolate. Thereafter, a dendrogram was constructed allelic profiles was performed on the MLST to determine the using MALDI Biotyper 3.0 software. sequence type (ST) to which each isolate belongs. Antibiotic Susceptibility Testing Statistical Analysis Using the Kirby–Bauer disk diffusion method, antibiotic The prevalence, identification, and resistance profiles of isolated susceptibility testing was performed. The results were interpreted strains are all presented as the number (percentage). according to EUCAST guidelines 2017 (European Committee on Antimicrobial Susceptibility Testing, 2017). Sixteen antimicrobial agents were used including ampicillin, aztreonam, cefotaxime, ceftazidime, cefoxitin, cefepime, amoxicillin-clavulanic acid, RESULTS piperacillin-tazobactam, meropenem, imipenem, ertapenem, colistin, tigecycline, ciprofloxacin, gentamicin and trimethoprim- Bacterial Identification sulfamethoxazole (Bio-Rad, Marnes-la-Coquette, France). Out of 982 collected fecal swabs, 203 (20.6%) showed growth Phenotypic detection of ESBL was performed using the double- on selective medium. In total, 235 strains were isolated. All 235 disk synergy test by placing an amoxicillin–clavulanic acid disk isolated Gram-negative bacilli were identified by MALDI TOF in the center between aztreonam, cefepime and ceftazidime. The mass spectrometry with a score value 1.9. The distribution at observation of a “key hole effect” was considered a positive test. the species level was as follows: 217 were identified as E. coli On the other hand, ampC b-lactamase detection was performed (92%), eight as K. pneumoniae (3%), three as Proteus mirabilis using the ampC disk test (Black et al., 2005). In brief, a lawn (1%), three as Enterobacter cloacae (1%), two as E. albertii, one of cefoxitin-susceptible E. coli ATCC 25922 was inoculated on as E. fergusonii and one as A. baumannii. The MSP dendrogram the surface of a Mueller Hinton agar plate. A 30-mg cefoxitin of the 217 E. coli isolates revealed five clusters at a distance level disk was placed on the inoculated surface. A sterile filter paper of 500 (arbitrarily selected) (Figure 1). Cluster 1 was mainly disk was moistened by adding 20 ml of a 1:1 mixture of saline formed by isolates from the Akkar District. Cluster 2 contained and 100  Tris-EDTA (catalog code T-9285; Sigma-Aldrich two isolates: one from Saida and the other from Baalbek. Cluster Corporation, St. Louis, MO, United States). Several colonies 3 was composed of three strains isolated from Jabal Lebnen of the test isolate were then applied to the disk. The disk was District. Cluster 4 was mainly composed of isolates from the then positioned with its inoculated face in contact with the agar North Lebanon district, and Cluster 5 contained only one strain surface. After overnight incubation, a flattening or indentation of from Saida. the zone of inhibition around the cefoxitin disk was considered a positive result, while an absence of distortion was considered http://mlst.warwick.ac.uk/mlst/dbs/Ecoli Frontiers in Microbiology | www.frontiersin.org 3 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 4 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry TABLE 1 | Distribution of MDROs per farm and district. Collection date Farm size Age Type # of collected samples # of positive samples # of isolated strains Fl 18000 35 d B 27 11 11 F2 11300 35 d B 27 5 6 F3 20000 45 d B 27 2 2 North Leb F4 27-Aug 23000 4 m L 20 9 18 F5 4000 35 d B 20 14 23 F6 20000 25 d B 20 13 14 F7 15000 35 d B 20 8 9 F8 5000 25 d B 20 5 5 Akkar F9 31-Aug 4000 25 d B 20 5 5 F10 6000 25 d B 20 9 11 F11 4600 4 m L 20 11 14 F12 15000 40 d B 20 11 14 F13 6000 45 d B 20 1 1 F14 10700 36 d B 20 4 4 Bekaa F15 15-Sep 5000 45 d B 20 6 7 F16 3000 18 m L 20 3 3 F17 6000 36 d B 20 1 1 F18 6000 43 d B 20 6 7 F19 6000 43 d B 20 3 3 Baalbek F20 21-Sep 5000 14 m L 20 3 3 F21 6500 27 d B 20 3 3 F22 6700 12 m L 21 1 1 F23 11800 26 d B 20 4 4 Nabatieh F24 21-Oct 10000 27 d B 20 2 2 F25 10000 25 d B 20 1 1 F26 5000 25 d B 20 1 1 F27 10000 27 d B 20 8 8 F28 5000 28 d B 20 4 4 Jabal Leb F29 9-Nov 5000 25 d B 20 7 6 F30 10000 27 d B 20 2 2 F31 10000 28 d B 20 4 5 F32 18000 25 d B 20 5 5 F33 6000 25 d B 20 3 3 F34 6000 25 d B 20 6 6 Saida F35 7-Dec 3300 32 d B 20 10 10 F36 10000 25 d B 20 5 6 F37 10000 30 d B 20 1 1 F38 10000 28 d B 20 6 6 F, farm; Aug, August; Sept, September; Oct, October; Nov, November; Dec, December; d, days; m, month; B, broiler; L, layer. resistant to cefoxitin but tested negative with the ampC disk Phenotypic Profiles of Resistance test. Moreover, 32% of the isolated strains were co-resistant to The disk diffusion susceptibility testing results are summarized gentamicin, ciprofloxacin, and trimethoprim-sulfamethoxazole, in Table 2. All of the isolates were susceptible to tigecycline, whereas 40% were resistant to at least two non-b-lactam colistin and carbapenems. Phenotypic identification using the antibiotics, 19.5% were resistant to only one non-b-lactam, double disk synergy test, ampC disk test and carba NP test and 8% were susceptible to all of the non-b-lactam antibiotics revealed that 102 (43.5%) of the isolated strains were ampC b- tested. lactamase producers, 67 (28.5%) were ESBL producers, and 48 (20.5%) were co-producers of ESBL and ampC b-lactamases. Both ESBL and ESBL/ampC production were detected in E. coli, Prevalence of MDR-GNB K. pneumoniae, E. fergusonii, and E. cloacae (Table 2), whereas The distribution of samples showing positive growth on the only AmpC production was detected in E. coli, K. pneumoniae, selective medium was as follows: 54 samples in the North P. mirabilis, E. albertii, and A. baumannii. In addition, 18 District, 38 in the Akkar District, 37 in Saida, 26 in Bekaa, E. coli strains (7.5%) did not show a key hole effect and were 24 in Jabal Lebnen, 16 in Baalbek and eight in Nabatieh. Frontiers in Microbiology | www.frontiersin.org 4 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 5 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry FIGURE 1 | MSP dendrogram of Escherichia coli isolates. The number of positive samples from broilers exceeded the MLST Typing one obtained from Layers (176 vs. 27, respectively). Isolated The MLST typing of the strains, each chosen from the major strains (235) originated from 38 out of the 49 visited district-related isolates grouped in each cluster, revealed that they farms, i.e., 77.5% of the farms were positive for at least belong to five different STs: ST156 for Cluster 1, ST5470 for one multi-drug-resistant Gram-negative bacilli. As shown in Cluster 2, ST354 for Cluster 3, ST155 for Cluster 4 and ST3224 Figure 2, the highest prevalence was detected in the north- for Cluster 5. west of the country, with 74 and 44 isolated strains for the North and Akkar Districts, respectively, whereas the lowest prevalence was detected in the north–east and south–east of DISCUSSION Lebanon. Many years ago, hospitals and health care settings were regarded PCR Screening of CTX-M, SHV, TEM, and as the sole source of antimicrobial resistance. However, recent AmpC b-Lactamase Genes evidence has shown that food producing animals constitute a One hundred and twelve isolates suspected to be ESBL producers potent reservoir of multi-drug-resistant organisms (Belmahdi were subjected to a real-time PCR assay for the detection of et al., 2016; Bachiri et al., 2017). This was mainly linked to SHV, TEM, and CTX-M encoding genes. Of the 112 strains the over-use of antimicrobial agents in veterinary medicine for selected, 93 (83%) harbored the TEM gene, 59 (53%) the treatment, growth promotion and prophylaxis (Economou and CTX-M gene and 22 (20%) the SHV gene. Overall, 49% (55) Gousia, 2015). Although the zoonotic transmission of multi- of the ESBL suspected isolates harbored only one gene, 46% drug-resistant organisms from animals to humans remains (52) harbored at least two genes with the highest concordance controversial (Olsen et al., 2014), several studies have shown being between the TEM and CTX-M genes, and 4% (five) a direct link between direct contact with farm animals and showed the co-existence of all three genes together (Table 3). In the acquisition of bacterial resistance (Huijbers et al., 2014). parallel, 152 strains including 4 K. pneumoniae, 3 P. mirabilis, One study conducted by Olaitan et al. (2015) demonstrated the 2 E. albertii, and 143 E. coli were positive for bla ; whereas zoonotic transmission of a colistin-resistant E. coli strain from a CMY fifteen E. coli strains were negative fall ampC b-lactamase pig to its owner. This owner usually fed his pig without wearing genes tested. Furthermore, in A. baumannii the ADC gene was any protective equipment. The two colistin-resistant isolates (in detected. the pig and its owner) belonged to the same sequence type and Frontiers in Microbiology | www.frontiersin.org 5 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 6 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry presented with the same virulence and PFGE pattern (Olaitan et al., 2015). In Lebanon, very few studies have looked at the prevalence of MDROs in farm animals (Al Bayssari et al., 2015a). Our study is the first epidemiological study in Lebanon quantifying the prevalence of multi-drug-resistant Gram-negative bacilli in chicken farms in terms of intestinal carriage at the national level. The prevalence is similar to the one previously reported from cattle (84%) in Lebanon (Diab et al., 2016). The flock’s size did not influence the prevalence of resistance in each farm (Table 1). On a global level, the prevalence found in our study is approximate to the one reported in Romania (69%) (Maciuca et al., 2015) and Ecuador (60%) (Ortega-Paredes et al., 2016) but is higher than the ones described in Germany (44%) (Kola et al., 2012), Japan (23%) (Kawamura et al., 2014), and Vietnam (3.2%) (Nguyen et al., 2015). Differences in the screening methodologies, sample size used and the level of antibiotic consumption in each country could explain these variations (Rhouma et al., 2016). Escherichia coli was the most common multi-drug-resistant organism isolated; MALDI-TOF MSP dendrogram and MLST analysis revealed the presence of five clusters from which the representative strains belonged to different STs. Within each cluster, strains isolated from farms of the same district were grouped together; this is especially true for the Akkar and North Lebanon strains. This observation reveals that strains of the same region are closely related. Although PFGE is the standard method for the detection of clones, due to the large number of strains isolated in this study, PFGE typing was not performed; rather, we referred to the MSP dendrogram as a possible rapid tool for strain differentiation according to their geographical and/or phenotypic distribution in epidemiological studies as certain previous studies have suggested (Berrazeg et al., 2013; Khennouchi et al., 2015). With the exception of ST155, none of the sequence types identified in this study were among those frequently reported in chicken such as ST10, ST23, ST48, ST58, ST115, ST117, ST350, and ST648 (Olsen et al., 2014). However, looking at the Warwick E. coli MLST database, we found that the STs detected in our study were previously reported from livestock, cats and dogs, and humans. ST155 has been commonly reported in poultry (Pires-dos-Santos et al., 2013), and it appears to be associated with a zoonotic risk, which has been suggested by some studies (Lazarus et al., 2015). This emphasizes the hypothesis that MDROs in food-producing animals can be transmitted to humans and may be causative agents of infections with therapeutic challenges when high resistance is encountered. It should also be mentioned that clones in animals and humans are not always shared; some studies have shown that E. coli strains in food-producing animals differ from those reported in humans (Randall et al., 2012; Wu et al., 2013). This suggests that only some bacterial clones might be transmitted to the human population. As our study showed, ESBL producers dominate the Lebanese poultry sector. The prevalence of ampC producers is also elevated (43.5%). ESBL and ampC-producing Gram-negative bacilli were previously reported in clinical and community settings in Lebanon (Dandachi et al., 2016). Molecular characterization revealed that 50% of isolated strains co-harbored at least two Frontiers in Microbiology | www.frontiersin.org 6 March 2018 | Volume 9 | Article 550 TABLE 2 | Resistance profiles and phenotypes of multi-drug-resistant organisms isolated in this study. Species AMP AZT CTX CAZ FOX FEP AMC TZP SXT CIP GENT % of ESBL % of % of producers AmpC ESBL/AmpC producers co-producers Escherichia coli 217 .100/ 49 .23/ 195 .90/ 120 .55/ 104 .48/ 31 .14/ 77 .35/ 28 .13/ 150 .59/ 134 .62/ 152 .70/ 27 44 21 (n D 217) Klebsiella pneumonia 8 .100/ 2 .25/ 8 .100/ 3 .38/ 2 .25/ 2 .25/ 2 .25/ 2 .25/ 6 .75/ 7 .88/ 7 .88/ 50 37.5 12.5 (n D 8) Proteus mirabilis 3 .100/ 0 .0/ 2 .67/ 0 .0/ 3 .100/ 0 .0/ 3 .100/ 0 .0/ 3 .100/ 3 .100/ 1 .33/ 100 (n D 3) Enterobacter cloacae 3 .100/ 1 .33/ 3 .100/ 2 .67/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 1 .33/ 1 .33/ 3 .100/ 100 (n D 3) Escherichia albertii 2 .100/ 0 .0/ 1 .50/ 1 .50/ 2 .100/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 100 (n D 2) Escherichia fergusonii 1 .100/ 0 .0/ 1 .100/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 1 .100/ 0 .0/ 100 (n D l) Acinetobacter 1 .100/ 0 .0/ 1 .100/ 1 .100/ 1 .100/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 100 baumannii (n D l) Resistance profiles are presented as a number (percentage). N, number; %, percentage; AMP, ampicillin; AZT, aztreonam; CTX, cefotaxime; CAZ, ceftazidime; FOX, cefoxitin; FEP, cefepime; AMC, amoxicillin-clavulanic acid; TZP, piperacillin-tazobactam; SXT, trimethoprim-sulfamethoxazole; CIP, ciprofloxacin; GENT, gentamicin. fmicb-09-00550 March 21, 2018 Time: 17:26 # 7 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry FIGURE 2 | Prevalence of MDROs in Lebanese poultry farms. Prevalence is expressed as the “number of isolates (percentage).” Frontiers in Microbiology | www.frontiersin.org 7 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 8 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry TABLE 3 | Characteristics of the different phenotypes/genotypes of ESBL and ESBL/AmpC producers found in this study. Species Phenotype b -lactamase genes Co-resistance to non b -lactams Escherichia coli ESBL bla TEM bla CTX-M SXT-CIP-GNT bla TEM bla CTX-M SXT-CIP bla TEM bla CTX-M CIP-GNT bla TEM bla CTX-M SXT-GNT bla SHV bla TEM SXT-CIP-GNT bla SHV bla TEM CIP bla SHV bla TEM SXT-GNT bla SHV bla TEM SXT-CIP bla SHV bla TEM SXT bla CTX-M SXT-CIP-GNT bla CTX-M SXT-CIP bla CTX-M N.R bla TEM SXT-CIP-GNT bla TEM SXT-GNT bla TEM SXT-CIP bla TEM CIP-GNT bla TEM GNT bla TEM N.R bla SHV bla TEM bla CTX-M SXT-CIP-GNT bla SHV GNT AmpC/ESBL bla TEM SXT-CIP-GNT bla TEM SXT-GNT bla TEM CIP-GNT bla TEM SXT bla TEM N.R bla TEM bla CTX-M SXT-CIP-GNT bla TEM bla CTX-M SXT bla TEM bla CTX-M CIP-GNT bla TEM bla CTX-M SXT-CIP bla TEM bla CTX-M SXT-GNT bla TEM bla CTX-M N.R bla SHV bla TEM GNT bla SHV bla TEM CIP-GNT bla CTX-M SXT-CIP-GNT bla CTX-M N.R bla SHV bla CTX-M CIP-GNT bla SHV bla TEM bla CTX-M SXT-CIP-GNT Klebsiella pneumoniae ESBL bla SHV bla TEM SXT-CIP-GNT bla SHV bla TEM bla CTX-M SXT-CIP-GNT bla TEM bla CTX-M CIP-GNT AmpC/ESBL bla SHV bla TEM bla CTX-M SXT-CIP-GNT bla TEM bla CTX-M SXT-CIP-GNT bla SHV bla TEM SXT-GNT Escherichia fergusonii ESBL bla TEM bla CTX-M CIP Enterobacter cloacae ESBL bla CTX-M GNT SXT, trimethoprim-sulfamethoxazole; GNT, gentamicin; CIP, ciprofloxacin; N.R, no resistance. b-lactamase genes with the most common being CTX-M and AmpC production, these showed that there are some strains TEM. Moreover, the only AmpC b-lactamase encoding gene was that were negative with the ampC disk test but positive for the CMY ampC b-lactamase. This gene was previously reported an ampC b-lactamase gene and vice-versa. Phenotypically false in poultry (Dierikx et al., 2013; El-Shazly et al., 2017) as well negatives shows the importance of the molecular testing in the as in food producing animals (Sato et al., 2014; Aguilar-Montes detection of AmpC production. On the other hand, in the 15 de Oca et al., 2015) and healthy pets (Donati et al., 2014; Liu E. coli strains that were negative for plasmidic ampC b-lactamase et al., 2016). As per the phenotypic and genotypic detection of genes; one explanation for this might be due to an overexpression Frontiers in Microbiology | www.frontiersin.org 8 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 9 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry of the chromosomal ampC gene mediated by a mutation the number of strains (McGregor and Spratt, 2005; Zou et al., in the promoter/attenuator region as described in previous 2010). studies (Escudero et al., 2010; Haenni et al., 2014). Regarding non-b-lactam co-resistance in ESBL and/or ampC producers, antimicrobial resistance toward gentamicin was relatively high CONCLUSION in this study. In fact, 66% of ESBL and/or ampC producing Gram-negative bacilli were gentamicin resistant. This could Our study illustrates the current epidemiology of multi-drug- possibly be linked to the frequent use of this antibiotic in resistant Gram-negative bacilli in Lebanese chicken farms. ESBL Lebanese farms as several studies have reported (El-Rami et al., and ampC producers cross-resistant to antibiotics used in human 2012; Diab et al., 2016). One study conducted by Abdelnoor medicine are highly prevalent across the territory. Our study et al. (2013) found a significant association between gentamicin suggests that poultry farms are potent reservoirs of antimicrobial resistance in E. coli isolates and the use of this antimicrobial resistance in Lebanon. Although very few studies have reported agent as a food additive in poultry in Lebanon. Another study the detection of carbapenemase producers in Lebanese Livestock launched a questionnaire-based survey on the most common (Al Bayssari et al., 2015a,b), it will likely only be a matter of time antibiotics used in Lebanese livestock and found that gentamicin before these organisms become prevalent in Lebanese animal and streptomycin are the most common and heavily used farms. This is especially true if no strict rules are implemented antimicrobial agents (Kassaify et al., 2013). Another thing to to control the overuse and misuse of antibiotics for treatment, mention is that in this study, no carbapenemase producers growth promotion and prophylaxis in Lebanese agriculture. We were detected. There might be two possible explanations for believe that the prescription of antibiotics often used in human this: the first one is that carbapenemase producers are really medicine should be reduced or even banned in the veterinary scarce in Lebanese chicken farms; the second one is that these sector. isolates were missed due to the medium used for the screening of multi-drug-resistant organisms. As has been reported, OXA- 48 carbapenemase producers are frequently found in hospitals AUTHOR CONTRIBUTIONS and nursing homes and in fowls in Lebanon (Al Bayssari et al., 2015b). OXA-48 carbapenemases do not always confer ID, ES, and ED conducted the phenotypic and molecular work. resistance to third-generation cephalosporins unless there is BE-B was responsible for the collection of the samples. EA, J-MR, another mechanism of resistance that co-exists in the same and ZD reviewed and edited the manuscript. bacterial cell (Poirel et al., 2012). Therefore, Oxacillinase producers could have been missed or under-estimated in our study. FUNDING Our study has two main limitations. The first one is that the primers used for blaTEM and blaSHV screening were universal, This study was funded by the Lebanese Council for Research and and thus, the possibility of having non-ESBL variants cannot the French Government under the “Investissements d’Avenir” be ruled out. However, as the strains presented with a typical (Investments for the Future) program managed by the Agence ESBL phenotype, i.e., the key hole effect and resistance to Nationale de la Recherche (ANR, fr: National Agency for penicillin, monobactams and third-generation cephalosporins Research) (reference: Méditerranée Infection 10-IAHU-03). with susceptibility to carbapenems, the TEM-positive strains were considered as ESBL producers and were included in the description of the MDR-GNB prevalence in this study. The ACKNOWLEDGMENTS second limitation is the low number of isolates subjected to MLST typing. MLST and PFGE analysis remain the gold standard for We would like to thank Dr. Hervé Chaudet for his assistance in clone/cluster detection in epidemiological studies regardless of the construction of the MSP dendrogram. Al Bayssari, C., Olaitan, A. O., Dabboussi, F., Hamze, M., and Rolain, J. M. REFERENCES (2015b). Emergence of OXA-48-producing Escherichia coli clone ST38 in Abdelnoor, A. M., Chokr, S., Fayad, L., and Al-Akl, N. (2013). Review study on fowl. Antimicrob. Agents Chemother. 59, 745–746. doi: 10.1128/AAC.03 external-hospital bacteria as a source of infection and antimicrobial resistance 552-14 in lebanon. Int. Arab. J. Antimicrob. Agents 3, 1–6. Alonso, C. A., Zarazaga, M., Ben Sallem, R., Jouini, A., Ben Slama, K., and Aguilar-Montes de Oca, S., Talavera-Rojas, M., Soriano-Vargas, E., Barba-Leon, J., Torres, C. (2017). Antibiotic resistance in Escherichia coli in husbandry animals: and Vazquez-Navarrete, J. (2015). Determination of extended spectrum beta- the African perspective. Lett. Appl. Microbiol. 64, 318–334. doi: 10.1111/lam. lactamases/AmpC beta-lactamases and plasmid-mediated quinolone resistance 12724 in Escherichia coli isolates obtained from bovine carcasses in mexico. Trop. Bachiri, T., Bakour, S., Ladjouzi, R., Thongpan, L., Rolain, J. M., and Touati, A. Anim. Health Prod. 47, 975–981. doi: 10.1007/s11250- 015- 0818- 3 (2017). High rates of CTX-M-15-producing Escherichia coli and Klebsiella Al Bayssari, C., Dabboussi, F., Hamze, M., and Rolain, J. M. (2015a). Emergence pneumoniae in wild boars and barbary macaques in algeria. J. Glob. Antimicrob. of carbapenemase-producing Pseudomonas aeruginosa and Acinetobacter Resist. 8, 35–40. doi: 10.1016/j.jgar.2016.10.005 baumannii in livestock animals in lebanon. J. Antimicrob. Chemother. 70, Bakour, S., Garcia, V., Loucif, L., Brunel, J. M., Gharout-Sait, A., Touati, A., et al. 950–951. doi: 10.1093/jac/dku469 (2015). Rapid identification of carbapenemase-producing Enterobacteriaceae, Frontiers in Microbiology | www.frontiersin.org 9 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 10 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry Pseudomonas aeruginosa and Acinetobacter baumannii using a modified Carba Available at: http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/ NP test. New Microbes New Infect. 7, 89–93. doi: 10.1016/j.nmni.2015.07.001 Breakpoint_tables/v_7.1_Breakpoint_Tables.pdf Baroud, M., Dandache, I., Araj, G. F., Wakim, R., Kanj, S., Kanafani, Z., et al. (2013). Exner, M., Bhattacharya, S., Christiansen, B., Gebel, J., Goroncy-Bermes, P., Underlying mechanisms of carbapenem resistance in extended-spectrum beta- Hartemann, P., et al. (2017). Antibiotic resistance: What is so special lactamase-producing Klebsiella pneumoniae and Escherichia coli isolates at a about multidrug-resistant gram-negative bacteria? GMS Hyg. Infect. Control tertiary care centre in lebanon: role of OXA-48 and NDM-1 carbapenemases. 12:Doc05. doi: 10.3205/dgkh000290 Int. J. Antimicrob. Agents 41, 75–79. doi: 10.1016/j.ijantimicag.2012.08.010 Ghodousi, A., Bonura, C., Di Noto, A. M., and Mammina, C. (2015). Belmahdi, M., Bakour, S., Al Bayssari, C., Touati, A., and Rolain, J. M. (2016). Extended-spectrum ß-lactamase, AmpC-producing, and fluoroquinolone- Molecular characterisation of extended-spectrum beta-lactamase- and plasmid resistant Escherichia coli in retail broiler chicken meat, Italy. Foodborne Pathog. AmpC-producing Escherichia coli strains isolated from broilers in bejaia, Dis. 12, 619–625. doi: 10.1089/fpd.2015.1936 algeria. J. Glob. Antimicrob. Resist. 6, 108–112. doi: 10.1016/j.jgar.2016.04.006 Gonzalez-Torralba, A., Oteo, J., Asenjo, A., Bautista, V., Fuentes, E., and Alos, J. I. Berrazeg, M., Diene, S. M., Drissi, M., Kempf, M., Richet, H., Landraud, L., (2016). Survey of carbapenemase-producing Enterobacteriaceae in companion et al. (2013). Biotyping of multidrug-resistant Klebsiella pneumoniae clinical dogs in madrid, spain. Antimicrob. Agents Chemother. 60, 2499–2501. isolates from france and algeria using MALDI-TOF MS. PLoS One 8:e61428. doi: 10.1128/AAC.02383- 15 doi: 10.1371/journal.pone.0061428 Haenni, M., Chatre, P., and Madec, J. Y. (2014). Emergence of Escherichia Bettiol, E., and Harbarth, S. (2015). Development of new antibiotics: taking off coli producing extended-spectrum AmpC beta-lactamases (ESAC) in animals. finally? Swiss Med. Wkly. 145:w14167. doi: 10.4414/smw.2015.14167 Front. Microbiol. 5:53. doi: 10.3389/fmicb.2014.00053 Black, J. A., Moland, E. S., and Thomson, K. S. (2005). AmpC disk test for Haenni, M., Saras, E., Ponsin, C., Dahmen, S., Petitjean, M., Hocquet, D., detection of plasmid-mediated AmpC disk test for detection of plasmid- et al. (2016). High prevalence of international ESBL CTX-M-15-producing mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal Enterobacter cloacae ST114 clone in animals. J. Antimicrob. Chemother. 71, AmpC beta-lactamases. J. Clin. Microbiol. 43, 3110–3113. doi: 10.1128/JCM.43. 1497–1500. doi: 10.1093/jac/dkw006 7.3110- 3113.2005 Huijbers, P. M., Graat, E. A., Haenen, A. P., van Santen, M. G., van Essen- Chantziaras, I., Boyen, F., Callens, B., and Dewulf, J. (2014). Correlation between Zandbergen, A., Mevius, D. J., et al. (2014). Extended-spectrum and AmpC veterinary antimicrobial use and antimicrobial resistance in food-producing beta-lactamase-producing Escherichia coli in broilers and people living and/or animals: a report on seven countries. J. Antimicrob. Chemother. 69, 827–834. working on broiler farms: prevalence, risk factors and molecular characteristics. doi: 10.1093/jac/dkt443 J. Antimicrob. Chemother. 69, 2669–2675. doi: 10.1093/jac/dku178 Dahms, C., Hubner, N. O., Wilke, F., and Kramer, A. (2014). Mini-review: Kassaify, Z., Abi-Khalil, P., and Sleiman, F. (2013). Quantification of epidemiology and zoonotic potential of multiresistant bacteria and clostridium antibiotic residues and determination of antimicrobial resistance profiles difficile in livestock and food. GMS Hyg. Infect. Control 9:Doc21. doi: 10.3205/ of microorganisms isolated from bovine milk in lebanon. Food Nutr. Sci. 4, dgkh000241 1–9. doi: 10.4236/fns.2013.47A001 Dallenne, C., Da Costa, A., Decre, D., Favier, C., and Arlet, G. (2010). Development Kawamura, K., Goto, K., Nakane, K., and Arakawa, Y. (2014). Molecular of a set of multiplex PCR assays for the detection of genes encoding important epidemiology of extended-spectrum beta-lactamases and Escherichia coli beta-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother. 65, 490–495. isolated from retail foods including chicken meat in Japan. Foodborne Pathog. doi: 10.1093/jac/dkp498 Dis. 11, 104–110. doi: 10.1089/fpd.2013.1608 Dandachi, I., Salem Sokhn, E., Najem, E., Azar, E., and Daoud, Z. (2016). Carriage Kempf, I., Jouy, E., Granier, S. A., Chauvin, C., Sanders, P., Salvat, G., et al. (2015). of beta-lactamase-producing Enterobacteriaceae among nursing home residents Comment on “impact of antibiotic use in the swine industry”, by Mary D. in north lebanon. Int. J. Infect. Dis. 45, 24–31. doi: 10.1016/j.ijid.2016.02.007 Barton [Curr. Opin. Microbiol. 19 (June 2014) 9–15]. Curr. Opin. Microbiol. Diab, M., Hamze, M., Madec, J. Y., and Haenni, M. (2016). High prevalence of 26, 137–138. doi: 10.1016/j.mib.2015.06.013 non-ST131 CTX-M-15-producing Escherichia coli in healthy cattle in lebanon. Khennouchi, N. C., Loucif, L., Boutefnouchet, N., Allag, H., and Rolain, J. M. Microb. Drug Resist. 23, 261–266. doi: 10.1089/mdr.2016.0019 (2015). MALDI-TOF MS as a tool to detect a nosocomial outbreak of Dierikx, C. M., van der Goot, J. A., Smith, H. E., Kant, A., and Mevius, D. J. (2013). extended-spectrum-beta-lactamase- and ArmA methyltransferase-producing Presence of ESBL/AmpC-producing Escherichia coli in the broiler production Enterobacter cloacae clinical isolates in algeria. Antimicrob. Agents Chemother. pyramid: a descriptive study. PLoS One 8:e79005. doi: 10.1371/journal.pone. 59, 6477–6483. doi: 10.1128/AAC.00615- 15 0079005 Kola, A., Kohler, C., Pfeifer, Y., Schwab, F., Kuhn, K., Schulz, K., et al. Donati, V., Feltrin, F., Hendriksen, R. S., Svendsen, C. A., Cordaro, G., Garcia- (2012). High prevalence of extended-spectrum-beta-lactamase-producing Fernandez, A., et al. (2014). Extended-spectrum-beta-lactamases, AmpC beta- Enterobacteriaceae in organic and conventional retail chicken meat, Germany. lactamases and plasmid mediated quinolone resistance in klebsiella spp. from J. Antimicrob. Chemother. 67, 2631–2634. doi: 10.1093/jac/dks295 companion animals in italy. PLoS One 9:e90564. doi: 10.1371/journal.pone. Lazarus, B., Paterson, D. L., Mollinger, J. L., and Rogers, B. A. (2015). Do human 0090564 extraintestinal Escherichia coli infections resistant to expanded-spectrum ECDC/EFSA/EMA (2017). ECDC/EFSA/EMA second joint report on the cephalosporins originate from food-producing animals? A systematic review. integrated analysis of the consumption of antimicrobial agents and occurrence Clin. Infect. Dis. 60, 439–452. doi: 10.1093/cid/ciu785 of antimicrobial resistance in bacteria from humans and food-producing Leverstein-van Hall, M. A., Dierikx, C. M., Cohen Stuart, J., Voets, G. M., van den animals. Sci. Rep. 15:e04872. doi: 10.2903/j.efsa.2017.4872/epdf Munckhof, M. P., van Essen-Zandbergen, A., et al. (2011). Dutch patients, retail Economou, V., and Gousia, P. (2015). Agriculture and food animals as a source of chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin. antimicrobial-resistant bacteria. Infect. Drug Resist. 8, 49–61. doi: 10.2147/IDR. Microbiol. Infect. 17, 873–880. doi: 10.1111/j.1469- 0691.2011.03497.x S55778 Liu, X., Thungrat, K., and Boothe, D. M. (2016). Occurrence of OXA-48 El-Rami, F. E., Sleiman, F. T., and Abdelnoor, A. M. (2012). Identification and carbapenemase and other beta-lactamase genes in ESBL-producing multidrug antibacterial resistance of bacteria isolated from poultry. Pol. J. Microbiol. 61, resistant Escherichia coli from dogs and cats in the United States, 2009-2013. 323–326. Front. Microbiol. 7:1057. doi: 10.3389/fmicb.2016.01057 El-Shazly, D. A., Nasef, S. A., Mahmoud, F. F., and Jonas, D. (2017). Expanded Liu, Y., and Liu, X. (2015). Detection of AmpC beta-lactamases in Acinetobacter spectrum beta-lactamase producing Escherichia coli isolated from chickens with baumannii in the Xuzhou region and analysis of drug resistance. Exp. Ther. colibacillosis in Egypt. Poult. Sci. 96, 2375–2384. doi: 10.3382/ps/pew493 Med. 10, 933–936. doi: 10.3892/etm.2015.2612 Escudero, E., Vinue, L., Teshager, T., Torres, C., and Moreno, M. A. (2010). Maciuca, I. E., Williams, N. J., Tuchilus, C., Dorneanu, O., Guguianu, E., Carp- Resistance mechanisms and farm-level distribution of fecal Escherichia coli Carare, C., et al. (2015). High prevalence of Escherichia coli-producing CTX-M- isolates resistant to extended-spectrum cephalosporins in pigs in spain. Res. Vet. 15 extended-spectrum beta-lactamases in poultry and human clinical isolates in Sci. 88, 83–87. doi: 10.1016/j.rvsc.2009.05.021 romania. Microb. Drug Resist. 21, 651–662. doi: 10.1089/mdr.2014.0248 European Committee on Antimicrobial Susceptibility Testing (2017). Breakpoint Madec, J. Y., Haenni, M., Nordmann, P., and Poirel, L. (2017). Extended-spectrum Tables for Interpretation of MICs and Zone Diameters, Version 7.1. beta-lactamase/AmpC- and carbapenemase-producing Enterobacteriaceae in Frontiers in Microbiology | www.frontiersin.org 10 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 11 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry animals: a threat for humans? Clin. Microbiol. Infect. 23, 826–833. doi: 10.1016/ Rhouma, M., Beaudry, F., and Letellier, A. (2016). Resistance to colistin: What is the j.cmi.2017.01.013 fate for this antibiotic in pig production? Int. J. Antimicrob. Agents 48, 119–126. Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., doi: 10.1016/j.ijantimicag.2016.04.008 Giske, C. G., et al. (2012). Multidrug-resistant, extensively drug-resistant Roschanski, N., Fischer, J., Guerra, B., and Roesler, U. (2014). Development and pandrug-resistant bacteria: an international expert proposal for interim of a multiplex real-time PCR for the rapid detection of the predominant standard definitions for acquired resistance. Clin. Microbiol. Infect. 18, 268–281. beta-lactamase genes CTX-M, SHV, TEM and CIT-type AmpCs in doi: 10.1111/j.1469- 0691.2011.03570.x Enterobacteriaceae. PLoS One 9:e100956. doi: 10.1371/journal.pone.0100956 McGregor, K. F., and Spratt, B. G. (2005). Identity and prevalence of multilocus Sato, T., Okubo, T., Usui, M., Yokota, S., Izumiyama, S., and Tamura, Y. (2014). sequence typing-defined clones of group A streptococci within a hospital Association of veterinary third-generation cephalosporin use with the risk of setting. J. Clin. Microbiol. 43, 1963–1967. doi: 10.1128/JCM.43.4.1963- 1967. emergence of extended-spectrum-cephalosporin resistance in Escherichia coli 2005 from dairy cattle in Japan. PLoS One 9:e96101. doi: 10.1371/journal.pone. Moghnieh, R., Estaitieh, N., Mugharbil, A., Jisr, T., Abdallah, D. I., Ziade, F., 0096101 et al. (2015). Third generation cephalosporin resistant Enterobacteriaceae Schill, F., Abdulmawjood, A., Klein, G., and Reich, F. (2017). Prevalence and and multidrug resistant gram-negative bacteria causing bacteremia in febrile characterization of extended-spectrum beta-lactamase (ESBL) and AmpC beta- neutropenia adult cancer patients in lebanon, broad spectrum antibiotics use lactamase producing Enterobacteriaceae in fresh pork meat at processing level as a major risk factor, and correlation with poor prognosis. Front. Cell Infect. in germany. Int. J. Food Microbiol. 257, 58–66. doi: 10.1016/j.ijfoodmicro.2017. Microbiol. 5:11. doi: 10.3389/fcimb.2015.00011 06.010 Nguyen, V. T., Carrique-Mas, J. J., Ngo, T. H., Ho, H. M., Ha, T. T., Campbell, J. I., Schwarz, S., Enne, V. I., and van Duijkeren, E. (2016). 40 years of veterinary et al. (2015). Prevalence and risk factors for carriage of antimicrobial-resistant papers in JAC – what have we learnt? J. Antimicrob. Chemother. 71, 2681–2690. Escherichia coli on household and small-scale chicken farms in the mekong doi: 10.1093/jac/dkw363 delta of Vietnam. J. Antimicrob. Chemother. 70, 2144–2152. doi: 10.1093/jac/ Seiffert, S. N., Hilty, M., Perreten, V., and Endimiani, A. (2013). Extended- dkv053 spectrum cephalosporin-resistant gram-negative organisms in livestock: an Olaitan, A. O., Thongmalayvong, B., Akkhavong, K., Somphavong, S., emerging problem for human health? Drug Resist. Updat. 16, 22–45. doi: 10. Paboriboune, P., Khounsy, S., et al. (2015). Clonal transmission of a 1016/j.drup.2012.12.001 colistin-resistant Escherichia coli from a domesticated pig to a human in Seng, P., Rolain, J. M., Fournier, P. E., La Scola, B., Drancourt, M., and Raoult, D. laos. J. Antimicrob. Chemother. 70, 3402–3404. (2010). MALDI-TOF-mass spectrometry applications in clinical microbiology. Olsen, R. H., Bisgaard, M., Lohren, U., Robineau, B., and Christensen, H. (2014). Future Microbiol. 5, 1733–1754. doi: 10.2217/fmb.10.127 Extended-spectrum beta-lactamase-producing Escherichia coli isolated from Singhal, N., Kumar, M., Kanaujia, P. K., and Virdi, J. S. (2015). MALDI-TOF poultry: a review of current problems, illustrated with some laboratory findings. mass spectrometry: an emerging technology for microbial identification and Avian Pathol. 43, 199–208. doi: 10.1080/03079457.2014.907866 diagnosis. Front. Microbiol. 6:791. doi: 10.3389/fmicb.2015.00791 Ortega-Paredes, D., Barba, P., and Zurita, J. (2016). Colistin-resistant Escherichia Wu, G., Day, M. J., Mafura, M. T., Nunez-Garcia, J., Fenner, J. J., Sharma, M., et al. coli clinical isolate harbouring the mcr-1 gene in Ecuador. Epidemiol. Infect. 144, (2013). Comparative analysis of ESBL-positive Escherichia coli isolates from 2967–2970. doi: 10.1017/S0950268816001369 animals and humans from the UK, the Netherlands and Germany. PLoS One Peng, C., and Zong, Z. (2011). Sequence type 38 Escherichia coli carrying 8:e75392. doi: 10.1371/journal.pone.0075392 bla . J. Med. Microbiol. 60(Pt 5), 694–695. doi: 10.1099/jmm.0. Zou, W., Lin, W. J., Foley, S. L., Chen, C. H., Nayak, R., and Chen, J. J. CTX-M-14 028316-0 (2010). Evaluation of pulsed-field gel electrophoresis profiles for identification Pires-dos-Santos, T., Bisgaard, M., and Christensen, H. (2013). Genetic diversity of Salmonella serotypes. J. Clin. Microbiol. 48, 3122–3126. doi: 10.1128/JCM. and virulence profiles of Escherichia coli causing salpingitis and peritonitis 00645- 10 in broiler breeders. Vet. Microbiol. 162, 873–880. doi: 10.1016/j.vetmic.2012. 11.008 Conflict of Interest Statement: The authors declare that the research was Poirel, L., Potron, A., and Nordmann, P. (2012). OXA-48-like carbapenemases: the conducted in the absence of any commercial or financial relationships that could phantom menace. J. Antimicrob. Chemother. 67, 1597–1606. doi: 10.1093/jac/ be construed as a potential conflict of interest. dks121 Rafei, R., Hamze, M., Pailhories, H., Eveillard, M., Marsollier, L., Joly-Guillou, Copyright © 2018 Dandachi, Sokhn, Dahdouh, Azar, El-Bazzal, Rolain and Daoud. M. L., et al. (2015). Extrahuman epidemiology of Acinetobacter baumannii This is an open-access article distributed under the terms of the Creative Commons in lebanon. Appl. Environ. Microbiol. 81, 2359–2367. doi: 10.1128/AEM. Attribution License (CC BY). The use, distribution or reproduction in other forums 03824-14 is permitted, provided the original author(s) and the copyright owner are credited Randall, L., Wu, G., Phillips, N., Coldham, N., Mevius, D., and Teale, C. (2012). and that the original publication in this journal is cited, in accordance with accepted Virulence genes in bla Escherichia coli isolates from chickens and humans. academic practice. No use, distribution or reproduction is permitted which does not CTX-M Res. Vet. Sci. 93, 23–27. doi: 10.1016/j.rvsc.2011.06.016 comply with these terms. Frontiers in Microbiology | www.frontiersin.org 11 March 2018 | Volume 9 | Article 550 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Microbiology Pubmed Central

Prevalence and Characterization of Multi-Drug-Resistant Gram-Negative Bacilli Isolated From Lebanese Poultry: A Nationwide Study

Frontiers in Microbiology , Volume 9 – Mar 23, 2018

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Copyright © 2018 Dandachi, Sokhn, Dahdouh, Azar, El-Bazzal, Rolain and Daoud.
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fmicb-09-00550 March 21, 2018 Time: 17:26 # 1 ORIGINAL RESEARCH published: 23 March 2018 doi: 10.3389/fmicb.2018.00550 Prevalence and Characterization of Multi-Drug-Resistant Gram-Negative Bacilli Isolated From Lebanese Poultry: A Nationwide Study 1,2 1 1 1 3 Iman Dandachi , Elie S. Sokhn , Elias A. Dahdouh , Eid Azar , Bassel El-Bazzal , 2 1 Jean-Marc Rolain and Ziad Daoud Clinical Microbiology Laboratory, Faculty of Medicine and Medical Sciences, University of Balamand, Beirut, Lebanon, 2 3 IRD, APHM, MEPHI, IHU-Méditerranée Infection, Aix-Marseille Université, Marseille, France, The Lebanese Ministry of Agriculture, Beirut, Lebanon Currently, antimicrobial resistance is one of the most prominent public health issues. In fact, there is increasing evidence that animals constitute a reservoir of antimicrobial Edited by: resistance. In collaboration with the Lebanese Ministry of Agriculture, the aim of this Miklos Fuzi, study was to determine the prevalence of intestinal carriage of multi-drug-resistant Semmelweis University, Hungary Gram-negative Bacilli in poultry farms at the national level. Between August and Reviewed by: Sebastian Guenther, December 2015, 981 fecal swabs were obtained from 49 poultry farms distributed University of Greifswald, Germany across Lebanon. The swabs were subcultured on MacConkey agar supplemented Djamel Drider, with cefotaxime (2 mg/ml). Isolated strains were identified using MALDI-TOF mass Lille University of Science and Technology, France spectrometry. Multilocus sequence typing analysis was performed for Escherichia coli. *Correspondence: Phenotypic detection of extended spectrum b-lactamases (ESBL) and AmpC production Ziad Daoud was performed using double disk synergy and the ampC disk test, respectively. ziad.daoud@balamand.edu.lb b-lactamase encoding genes bla , bla , bla , bla , bla , bla , bla , CTXM TEM SHV FOX MOX EBC ACC Specialty section: bla , and bla using PCR amplification. Out of 981 fecal swabs obtained, 203 DHA CMY This article was submitted to (20.6%) showed bacterial growth on the selective medium. Of the 235 strains isolated, Antimicrobials, Resistance and Chemotherapy, 217 were identified as E. coli (92%), eight as Klebsiella pneumoniae (3%), three as a section of the journal Proteus mirabilis (1%) and three as Enterobacter cloacae (1%). MLST analysis of E. coli Frontiers in Microbiology isolates showed the presence of ST156, ST5470, ST354, ST155, and ST3224. The Received: 02 December 2017 phenotypic tests revealed that 43.5, 28.5, and 20.5% of the strains were ampC, ESBL, Accepted: 12 March 2018 Published: 23 March 2018 and ampC/ESBL producers, respectively. The putative TEM gene was detected in 83% Citation: of the isolates, SHV in 20%, CTX-M in 53% and CMY ampC b-lactamase gene in 65%. Dandachi I, Sokhn ES, Dahdouh EA, Our study showed that chicken farms in Lebanon are reservoirs of ESBL and AmpC Azar E, El-Bazzal B, Rolain J-M and Daoud Z (2018) Prevalence producing Gram-negative bacilli. The level of antibiotic consumption in the Lebanese and Characterization veterinary medicine should be evaluated. Future studies should focus on the risk factors of Multi-Drug-Resistant associated with the acquisition of multi-drug-resistant organisms in farm animals in Gram-Negative Bacilli Isolated From Lebanese Poultry: A Nationwide Lebanon. Study. Front. Microbiol. 9:550. doi: 10.3389/fmicb.2018.00550 Keywords: ampC, ESBL, E. coli, poultry, carriage Frontiers in Microbiology | www.frontiersin.org 1 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 2 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry through direct contact or through the handling/consumption INTRODUCTION of undercooked/uncooked animal products (Dahms et al., Antibiotic resistance is currently a major topic of interest for 2014). researchers and physicians. In particular, the rise of multi- In Lebanon, several studies addressing MDROs in hospital drug resistance in Gram-negative bacteria is now a serious settings have been conducted. One study done at the American challenge encountered by healthcare professionals (Exner et al., University of Beirut Medical Center between 2008 and 2011 2017). Resistance in Gram-negative bacteria is mainly mediated reported that 1.07 and 2.45% of E. coli and Klebsiella via the production of extended spectrum b-lactamases (ESBL), pneumoniae clinical isolates, respectively, were ESBL producers ampC b-lactamases and carbapenemases (Schill et al., 2017). and ertapenem-resistant (Baroud et al., 2013). Another study Genes encoding these enzymes are often located on plasmids conducted in the north reported that over the period of carrying resistance genes to other commonly used antibiotics in 2009–2012, 9% and 28% of the bacteraemia episodes in clinical settings (Seiffert et al., 2013). Infections with these multi- febrile neutropenic patients were caused by carbapenem and drug-resistant organisms (MDROs) will thus pose therapeutic third-generation cephalosporin-resistant Gram-negative bacilli, challenges; the antibiotic pipeline is drying up, and no new respectively (Moghnieh et al., 2015). However, very few antimicrobial agents are anticipated in the near future to treat studies have addressed this issue in the environment. One infections caused by MDROs (Bettiol and Harbarth, 2015). study showed that Acinetobacter baumannii was detected In fact, it has been generally accepted that the main driver in 6.9% of water samples, 2.7% of milk samples, 8.0% of for the rapid evolution of bacterial resistance is the uncontrolled meat samples, 14.3% of cheese samples and 7.7% of animal usage of antibiotics in human medicine. It is suggested that samples (Rafei et al., 2015). Another study in which 115 this theory is also applicable to the veterinary sector (Kempf stool samples were collected from livestock animals from et al., 2015). The European Centre for Disease Prevention and different farms in north Lebanon reported the detection of Control/European Food Safety Authority/European Medicines four VIM-2 producing Pseudomonas aeruginosa, four OXA- Agency (ECDC/EFSA/EMA) joint report stated that in 2014, 23 producing A. baumannii and one OXA-23/OXA-58 co- the average antibiotic consumption in animals (152 mg/kg) producing A. baumannii (Al Bayssari et al., 2015a). Furthermore, was higher than in humans (124 mg/kg). Univariate analysis Al Bayssari et al. (2015b) reported the isolation of an OXA- showed a signification correlation between fluoroquinolone 48 harboring E. coli isolate from fowl in Lebanon. More consumption and resistance in Escherichia coli in the human recently, Diab et al. (2016) detected a relatively high prevalence and animal sectors, between polymyxins and tetracyclines and of CTX-M-15 producing E. coli in Lebanese cattle. In the E. coli in animals, and for 3rd/4th generation cephalosporins above-mentioned studies in Lebanese livestock, MLST analysis and E. coli in humans (ECDC/EFSA/EMA, 2017). Antibiotics are revealed the presence of sequence types common to both heavily administered for therapeutic and prophylaxis purposes humans and animals (Al Bayssari et al., 2015a; Rafei et al., in veterinary medicine. As growth promoters, this practice 2015; Diab et al., 2016), which suggests that Lebanese farms is no longer adapted in the European Union, whereas it are potent reservoirs of multi-drug-resistant organisms that persists in North America and other countries (Economou could be transmitted to humans. In the present study and in and Gousia, 2015). In their study, Chantziaras et al. (2014) collaboration with the Lebanese Ministry of Agriculture, our found a significant correlation between the use of antibiotics in aim was to determine the national epidemiology of multi-drug- livestock and the corresponding level of resistance toward these resistant Gram-negative bacilli in Lebanese chicken farms in antimicrobials in E. coli strains isolated from pigs, poultry and terms of intestinal carriage. cattle. During the last years, the prevalence of ESBLs, ampC, and carbapenemase producing Gram-negative bacteria has become MATERIALS AND METHODS extensively reported in food producing animals (Ghodousi et al., 2015; Gonzalez-Torralba et al., 2016; Haenni et al., 2016). In Ethics Statement their review paper, Schwarz et al. (2016) showed that studies describing the epidemiology of resistant organisms in livestock The Ministry of Agriculture in Lebanon granted approval to collect chicken samples from representative farms in the targeted mainly swine, cattle and poultry. The prevalence of resistance varied from one country to another (Alonso et al., country as per the national norms for animal sampling and manipulation. This sampling was in conformity with the 2017). Although the extent to which food of animal origin international regulations for animal safety. All of the involved contributes to the zoonotic transmission of multi-drug-resistant farms officially received authorization from the Ministry of organisms, i.e., ESBL and carbapenemase producers, has not Agriculture, and this was considered, after undergoing an yet been well established (Madec et al., 2017), it suggests that acceptance process, an official and legal document. Therefore, an sharing the same ESBL genes, plasmids and strains constitutes Institutional Review Board (IRB) approval was obtained for the possible evidence of zoonotic transmission of MDROs from animals to humans (Leverstein-van Hall et al., 2011; Dahms et al., present study. 2014). Furthermore, the increased risk of ESBL fecal carriage in individuals with a high degree of contact with broiler chickens Samples Collection is an indicator of transmission (Huijbers et al., 2014). Enteric- Between August and December 2015, 981 rectal swabs were resistant strains in livestock can be easily transferred to humans collected from 49 poultry farms distributed over the seven Frontiers in Microbiology | www.frontiersin.org 2 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 3 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry districts of Lebanon. Six to seven farms were visited in each a negative one. Furthermore, for the presumptive detection of district. The average number of samples taken from each farm carbapenemases, the carba NP test was performed as previously was 20 fecal swabs (Table 1). The 20 samples collected were described (Bakour et al., 2015). A bacterium was characterized randomly taken from each farm. Technical assistance, i.e., as being multi-drug-resistant when resistance to at least three fecal swabs, gloves, costumes, and a portable refrigerator, were classes of antibiotics was observed (Magiorakos et al., 2012). provided by the Ministry of Agriculture team. The collected swabs were directly placed in a portable refrigerator, and when Molecular Characterization of they arrived at the University Laboratory, they were stored at b-Lactamase Encoding Genes 80 C until use. The farms visited were selected by considering All of the isolates that showed a key hole effect or had cefoxitin their geographical location, presence or absence of a nearby resistance with non-susceptibility to cefepime were subjected community and the size of the farms (at least 3,000 chickens per to real-time PCR analysis for the detection of SHV, TEM and breeding site). Eighty percent of the samples were gathered from CTX-M encoding genes (Roschanski et al., 2014). Simplex PCRs broiler chickens, while 20% were taken from layers. The mean for the genes encoding AmpC b-lactamases FOX, MOX, ACC, average age of the broilers and layers was 31 days and 14 months, EBC, DHA, and CMY were conducted for all strains showing respectively. non-susceptibility to cefoxitin (Dallenne et al., 2010). Simplex PCR was also used to test the ADC ampC b-lactamase gene MALDI-TOF MS Identification in A. baumannii (Liu and Liu, 2015). DNA extraction was Rectal swabs were sub-cultured on a MacConkey agar performed according to the manufacturer’s instructions using supplemented with 2 mg/ml of cefotaxime for the preliminary EZ1 DNA extraction kits (Qiagen, Courtaboeuf, France) with the screening of antibiotic-resistant Gram-negative bacilli. After EZ1 Advanced XL biorobot. overnight incubation at 37 C, colonies showing different morphologies were picked up from each selective plate and Multilocus Sequence Typing tested separately with MALDI-TOF MS for identification using One E. coli strain from each cluster shown in the MSP the Microflex LT spectrometer (Bruker Daltonics, Bremen, dendrogram was chosen, and MLST typing was performed based Germany) (Seng et al., 2010; Singhal et al., 2015). The spectra on allelic profiles to determine their evolutionary relationship obtained for each strain were stored and downloaded into a (Peng and Zong, 2011). Seven housekeeping genes were used: MALDI Biotyper 3.0 system to create a single main spectrum for adk, fumC, gyrB, icd, mdh, purA, and recA. Analysis of the genes’ each bacterial isolate. Thereafter, a dendrogram was constructed allelic profiles was performed on the MLST to determine the using MALDI Biotyper 3.0 software. sequence type (ST) to which each isolate belongs. Antibiotic Susceptibility Testing Statistical Analysis Using the Kirby–Bauer disk diffusion method, antibiotic The prevalence, identification, and resistance profiles of isolated susceptibility testing was performed. The results were interpreted strains are all presented as the number (percentage). according to EUCAST guidelines 2017 (European Committee on Antimicrobial Susceptibility Testing, 2017). Sixteen antimicrobial agents were used including ampicillin, aztreonam, cefotaxime, ceftazidime, cefoxitin, cefepime, amoxicillin-clavulanic acid, RESULTS piperacillin-tazobactam, meropenem, imipenem, ertapenem, colistin, tigecycline, ciprofloxacin, gentamicin and trimethoprim- Bacterial Identification sulfamethoxazole (Bio-Rad, Marnes-la-Coquette, France). Out of 982 collected fecal swabs, 203 (20.6%) showed growth Phenotypic detection of ESBL was performed using the double- on selective medium. In total, 235 strains were isolated. All 235 disk synergy test by placing an amoxicillin–clavulanic acid disk isolated Gram-negative bacilli were identified by MALDI TOF in the center between aztreonam, cefepime and ceftazidime. The mass spectrometry with a score value 1.9. The distribution at observation of a “key hole effect” was considered a positive test. the species level was as follows: 217 were identified as E. coli On the other hand, ampC b-lactamase detection was performed (92%), eight as K. pneumoniae (3%), three as Proteus mirabilis using the ampC disk test (Black et al., 2005). In brief, a lawn (1%), three as Enterobacter cloacae (1%), two as E. albertii, one of cefoxitin-susceptible E. coli ATCC 25922 was inoculated on as E. fergusonii and one as A. baumannii. The MSP dendrogram the surface of a Mueller Hinton agar plate. A 30-mg cefoxitin of the 217 E. coli isolates revealed five clusters at a distance level disk was placed on the inoculated surface. A sterile filter paper of 500 (arbitrarily selected) (Figure 1). Cluster 1 was mainly disk was moistened by adding 20 ml of a 1:1 mixture of saline formed by isolates from the Akkar District. Cluster 2 contained and 100  Tris-EDTA (catalog code T-9285; Sigma-Aldrich two isolates: one from Saida and the other from Baalbek. Cluster Corporation, St. Louis, MO, United States). Several colonies 3 was composed of three strains isolated from Jabal Lebnen of the test isolate were then applied to the disk. The disk was District. Cluster 4 was mainly composed of isolates from the then positioned with its inoculated face in contact with the agar North Lebanon district, and Cluster 5 contained only one strain surface. After overnight incubation, a flattening or indentation of from Saida. the zone of inhibition around the cefoxitin disk was considered a positive result, while an absence of distortion was considered http://mlst.warwick.ac.uk/mlst/dbs/Ecoli Frontiers in Microbiology | www.frontiersin.org 3 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 4 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry TABLE 1 | Distribution of MDROs per farm and district. Collection date Farm size Age Type # of collected samples # of positive samples # of isolated strains Fl 18000 35 d B 27 11 11 F2 11300 35 d B 27 5 6 F3 20000 45 d B 27 2 2 North Leb F4 27-Aug 23000 4 m L 20 9 18 F5 4000 35 d B 20 14 23 F6 20000 25 d B 20 13 14 F7 15000 35 d B 20 8 9 F8 5000 25 d B 20 5 5 Akkar F9 31-Aug 4000 25 d B 20 5 5 F10 6000 25 d B 20 9 11 F11 4600 4 m L 20 11 14 F12 15000 40 d B 20 11 14 F13 6000 45 d B 20 1 1 F14 10700 36 d B 20 4 4 Bekaa F15 15-Sep 5000 45 d B 20 6 7 F16 3000 18 m L 20 3 3 F17 6000 36 d B 20 1 1 F18 6000 43 d B 20 6 7 F19 6000 43 d B 20 3 3 Baalbek F20 21-Sep 5000 14 m L 20 3 3 F21 6500 27 d B 20 3 3 F22 6700 12 m L 21 1 1 F23 11800 26 d B 20 4 4 Nabatieh F24 21-Oct 10000 27 d B 20 2 2 F25 10000 25 d B 20 1 1 F26 5000 25 d B 20 1 1 F27 10000 27 d B 20 8 8 F28 5000 28 d B 20 4 4 Jabal Leb F29 9-Nov 5000 25 d B 20 7 6 F30 10000 27 d B 20 2 2 F31 10000 28 d B 20 4 5 F32 18000 25 d B 20 5 5 F33 6000 25 d B 20 3 3 F34 6000 25 d B 20 6 6 Saida F35 7-Dec 3300 32 d B 20 10 10 F36 10000 25 d B 20 5 6 F37 10000 30 d B 20 1 1 F38 10000 28 d B 20 6 6 F, farm; Aug, August; Sept, September; Oct, October; Nov, November; Dec, December; d, days; m, month; B, broiler; L, layer. resistant to cefoxitin but tested negative with the ampC disk Phenotypic Profiles of Resistance test. Moreover, 32% of the isolated strains were co-resistant to The disk diffusion susceptibility testing results are summarized gentamicin, ciprofloxacin, and trimethoprim-sulfamethoxazole, in Table 2. All of the isolates were susceptible to tigecycline, whereas 40% were resistant to at least two non-b-lactam colistin and carbapenems. Phenotypic identification using the antibiotics, 19.5% were resistant to only one non-b-lactam, double disk synergy test, ampC disk test and carba NP test and 8% were susceptible to all of the non-b-lactam antibiotics revealed that 102 (43.5%) of the isolated strains were ampC b- tested. lactamase producers, 67 (28.5%) were ESBL producers, and 48 (20.5%) were co-producers of ESBL and ampC b-lactamases. Both ESBL and ESBL/ampC production were detected in E. coli, Prevalence of MDR-GNB K. pneumoniae, E. fergusonii, and E. cloacae (Table 2), whereas The distribution of samples showing positive growth on the only AmpC production was detected in E. coli, K. pneumoniae, selective medium was as follows: 54 samples in the North P. mirabilis, E. albertii, and A. baumannii. In addition, 18 District, 38 in the Akkar District, 37 in Saida, 26 in Bekaa, E. coli strains (7.5%) did not show a key hole effect and were 24 in Jabal Lebnen, 16 in Baalbek and eight in Nabatieh. Frontiers in Microbiology | www.frontiersin.org 4 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 5 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry FIGURE 1 | MSP dendrogram of Escherichia coli isolates. The number of positive samples from broilers exceeded the MLST Typing one obtained from Layers (176 vs. 27, respectively). Isolated The MLST typing of the strains, each chosen from the major strains (235) originated from 38 out of the 49 visited district-related isolates grouped in each cluster, revealed that they farms, i.e., 77.5% of the farms were positive for at least belong to five different STs: ST156 for Cluster 1, ST5470 for one multi-drug-resistant Gram-negative bacilli. As shown in Cluster 2, ST354 for Cluster 3, ST155 for Cluster 4 and ST3224 Figure 2, the highest prevalence was detected in the north- for Cluster 5. west of the country, with 74 and 44 isolated strains for the North and Akkar Districts, respectively, whereas the lowest prevalence was detected in the north–east and south–east of DISCUSSION Lebanon. Many years ago, hospitals and health care settings were regarded PCR Screening of CTX-M, SHV, TEM, and as the sole source of antimicrobial resistance. However, recent AmpC b-Lactamase Genes evidence has shown that food producing animals constitute a One hundred and twelve isolates suspected to be ESBL producers potent reservoir of multi-drug-resistant organisms (Belmahdi were subjected to a real-time PCR assay for the detection of et al., 2016; Bachiri et al., 2017). This was mainly linked to SHV, TEM, and CTX-M encoding genes. Of the 112 strains the over-use of antimicrobial agents in veterinary medicine for selected, 93 (83%) harbored the TEM gene, 59 (53%) the treatment, growth promotion and prophylaxis (Economou and CTX-M gene and 22 (20%) the SHV gene. Overall, 49% (55) Gousia, 2015). Although the zoonotic transmission of multi- of the ESBL suspected isolates harbored only one gene, 46% drug-resistant organisms from animals to humans remains (52) harbored at least two genes with the highest concordance controversial (Olsen et al., 2014), several studies have shown being between the TEM and CTX-M genes, and 4% (five) a direct link between direct contact with farm animals and showed the co-existence of all three genes together (Table 3). In the acquisition of bacterial resistance (Huijbers et al., 2014). parallel, 152 strains including 4 K. pneumoniae, 3 P. mirabilis, One study conducted by Olaitan et al. (2015) demonstrated the 2 E. albertii, and 143 E. coli were positive for bla ; whereas zoonotic transmission of a colistin-resistant E. coli strain from a CMY fifteen E. coli strains were negative fall ampC b-lactamase pig to its owner. This owner usually fed his pig without wearing genes tested. Furthermore, in A. baumannii the ADC gene was any protective equipment. The two colistin-resistant isolates (in detected. the pig and its owner) belonged to the same sequence type and Frontiers in Microbiology | www.frontiersin.org 5 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 6 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry presented with the same virulence and PFGE pattern (Olaitan et al., 2015). In Lebanon, very few studies have looked at the prevalence of MDROs in farm animals (Al Bayssari et al., 2015a). Our study is the first epidemiological study in Lebanon quantifying the prevalence of multi-drug-resistant Gram-negative bacilli in chicken farms in terms of intestinal carriage at the national level. The prevalence is similar to the one previously reported from cattle (84%) in Lebanon (Diab et al., 2016). The flock’s size did not influence the prevalence of resistance in each farm (Table 1). On a global level, the prevalence found in our study is approximate to the one reported in Romania (69%) (Maciuca et al., 2015) and Ecuador (60%) (Ortega-Paredes et al., 2016) but is higher than the ones described in Germany (44%) (Kola et al., 2012), Japan (23%) (Kawamura et al., 2014), and Vietnam (3.2%) (Nguyen et al., 2015). Differences in the screening methodologies, sample size used and the level of antibiotic consumption in each country could explain these variations (Rhouma et al., 2016). Escherichia coli was the most common multi-drug-resistant organism isolated; MALDI-TOF MSP dendrogram and MLST analysis revealed the presence of five clusters from which the representative strains belonged to different STs. Within each cluster, strains isolated from farms of the same district were grouped together; this is especially true for the Akkar and North Lebanon strains. This observation reveals that strains of the same region are closely related. Although PFGE is the standard method for the detection of clones, due to the large number of strains isolated in this study, PFGE typing was not performed; rather, we referred to the MSP dendrogram as a possible rapid tool for strain differentiation according to their geographical and/or phenotypic distribution in epidemiological studies as certain previous studies have suggested (Berrazeg et al., 2013; Khennouchi et al., 2015). With the exception of ST155, none of the sequence types identified in this study were among those frequently reported in chicken such as ST10, ST23, ST48, ST58, ST115, ST117, ST350, and ST648 (Olsen et al., 2014). However, looking at the Warwick E. coli MLST database, we found that the STs detected in our study were previously reported from livestock, cats and dogs, and humans. ST155 has been commonly reported in poultry (Pires-dos-Santos et al., 2013), and it appears to be associated with a zoonotic risk, which has been suggested by some studies (Lazarus et al., 2015). This emphasizes the hypothesis that MDROs in food-producing animals can be transmitted to humans and may be causative agents of infections with therapeutic challenges when high resistance is encountered. It should also be mentioned that clones in animals and humans are not always shared; some studies have shown that E. coli strains in food-producing animals differ from those reported in humans (Randall et al., 2012; Wu et al., 2013). This suggests that only some bacterial clones might be transmitted to the human population. As our study showed, ESBL producers dominate the Lebanese poultry sector. The prevalence of ampC producers is also elevated (43.5%). ESBL and ampC-producing Gram-negative bacilli were previously reported in clinical and community settings in Lebanon (Dandachi et al., 2016). Molecular characterization revealed that 50% of isolated strains co-harbored at least two Frontiers in Microbiology | www.frontiersin.org 6 March 2018 | Volume 9 | Article 550 TABLE 2 | Resistance profiles and phenotypes of multi-drug-resistant organisms isolated in this study. Species AMP AZT CTX CAZ FOX FEP AMC TZP SXT CIP GENT % of ESBL % of % of producers AmpC ESBL/AmpC producers co-producers Escherichia coli 217 .100/ 49 .23/ 195 .90/ 120 .55/ 104 .48/ 31 .14/ 77 .35/ 28 .13/ 150 .59/ 134 .62/ 152 .70/ 27 44 21 (n D 217) Klebsiella pneumonia 8 .100/ 2 .25/ 8 .100/ 3 .38/ 2 .25/ 2 .25/ 2 .25/ 2 .25/ 6 .75/ 7 .88/ 7 .88/ 50 37.5 12.5 (n D 8) Proteus mirabilis 3 .100/ 0 .0/ 2 .67/ 0 .0/ 3 .100/ 0 .0/ 3 .100/ 0 .0/ 3 .100/ 3 .100/ 1 .33/ 100 (n D 3) Enterobacter cloacae 3 .100/ 1 .33/ 3 .100/ 2 .67/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 1 .33/ 1 .33/ 3 .100/ 100 (n D 3) Escherichia albertii 2 .100/ 0 .0/ 1 .50/ 1 .50/ 2 .100/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 100 (n D 2) Escherichia fergusonii 1 .100/ 0 .0/ 1 .100/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 1 .100/ 0 .0/ 100 (n D l) Acinetobacter 1 .100/ 0 .0/ 1 .100/ 1 .100/ 1 .100/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 0 .0/ 100 baumannii (n D l) Resistance profiles are presented as a number (percentage). N, number; %, percentage; AMP, ampicillin; AZT, aztreonam; CTX, cefotaxime; CAZ, ceftazidime; FOX, cefoxitin; FEP, cefepime; AMC, amoxicillin-clavulanic acid; TZP, piperacillin-tazobactam; SXT, trimethoprim-sulfamethoxazole; CIP, ciprofloxacin; GENT, gentamicin. fmicb-09-00550 March 21, 2018 Time: 17:26 # 7 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry FIGURE 2 | Prevalence of MDROs in Lebanese poultry farms. Prevalence is expressed as the “number of isolates (percentage).” Frontiers in Microbiology | www.frontiersin.org 7 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 8 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry TABLE 3 | Characteristics of the different phenotypes/genotypes of ESBL and ESBL/AmpC producers found in this study. Species Phenotype b -lactamase genes Co-resistance to non b -lactams Escherichia coli ESBL bla TEM bla CTX-M SXT-CIP-GNT bla TEM bla CTX-M SXT-CIP bla TEM bla CTX-M CIP-GNT bla TEM bla CTX-M SXT-GNT bla SHV bla TEM SXT-CIP-GNT bla SHV bla TEM CIP bla SHV bla TEM SXT-GNT bla SHV bla TEM SXT-CIP bla SHV bla TEM SXT bla CTX-M SXT-CIP-GNT bla CTX-M SXT-CIP bla CTX-M N.R bla TEM SXT-CIP-GNT bla TEM SXT-GNT bla TEM SXT-CIP bla TEM CIP-GNT bla TEM GNT bla TEM N.R bla SHV bla TEM bla CTX-M SXT-CIP-GNT bla SHV GNT AmpC/ESBL bla TEM SXT-CIP-GNT bla TEM SXT-GNT bla TEM CIP-GNT bla TEM SXT bla TEM N.R bla TEM bla CTX-M SXT-CIP-GNT bla TEM bla CTX-M SXT bla TEM bla CTX-M CIP-GNT bla TEM bla CTX-M SXT-CIP bla TEM bla CTX-M SXT-GNT bla TEM bla CTX-M N.R bla SHV bla TEM GNT bla SHV bla TEM CIP-GNT bla CTX-M SXT-CIP-GNT bla CTX-M N.R bla SHV bla CTX-M CIP-GNT bla SHV bla TEM bla CTX-M SXT-CIP-GNT Klebsiella pneumoniae ESBL bla SHV bla TEM SXT-CIP-GNT bla SHV bla TEM bla CTX-M SXT-CIP-GNT bla TEM bla CTX-M CIP-GNT AmpC/ESBL bla SHV bla TEM bla CTX-M SXT-CIP-GNT bla TEM bla CTX-M SXT-CIP-GNT bla SHV bla TEM SXT-GNT Escherichia fergusonii ESBL bla TEM bla CTX-M CIP Enterobacter cloacae ESBL bla CTX-M GNT SXT, trimethoprim-sulfamethoxazole; GNT, gentamicin; CIP, ciprofloxacin; N.R, no resistance. b-lactamase genes with the most common being CTX-M and AmpC production, these showed that there are some strains TEM. Moreover, the only AmpC b-lactamase encoding gene was that were negative with the ampC disk test but positive for the CMY ampC b-lactamase. This gene was previously reported an ampC b-lactamase gene and vice-versa. Phenotypically false in poultry (Dierikx et al., 2013; El-Shazly et al., 2017) as well negatives shows the importance of the molecular testing in the as in food producing animals (Sato et al., 2014; Aguilar-Montes detection of AmpC production. On the other hand, in the 15 de Oca et al., 2015) and healthy pets (Donati et al., 2014; Liu E. coli strains that were negative for plasmidic ampC b-lactamase et al., 2016). As per the phenotypic and genotypic detection of genes; one explanation for this might be due to an overexpression Frontiers in Microbiology | www.frontiersin.org 8 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 9 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry of the chromosomal ampC gene mediated by a mutation the number of strains (McGregor and Spratt, 2005; Zou et al., in the promoter/attenuator region as described in previous 2010). studies (Escudero et al., 2010; Haenni et al., 2014). Regarding non-b-lactam co-resistance in ESBL and/or ampC producers, antimicrobial resistance toward gentamicin was relatively high CONCLUSION in this study. In fact, 66% of ESBL and/or ampC producing Gram-negative bacilli were gentamicin resistant. This could Our study illustrates the current epidemiology of multi-drug- possibly be linked to the frequent use of this antibiotic in resistant Gram-negative bacilli in Lebanese chicken farms. ESBL Lebanese farms as several studies have reported (El-Rami et al., and ampC producers cross-resistant to antibiotics used in human 2012; Diab et al., 2016). One study conducted by Abdelnoor medicine are highly prevalent across the territory. Our study et al. (2013) found a significant association between gentamicin suggests that poultry farms are potent reservoirs of antimicrobial resistance in E. coli isolates and the use of this antimicrobial resistance in Lebanon. Although very few studies have reported agent as a food additive in poultry in Lebanon. Another study the detection of carbapenemase producers in Lebanese Livestock launched a questionnaire-based survey on the most common (Al Bayssari et al., 2015a,b), it will likely only be a matter of time antibiotics used in Lebanese livestock and found that gentamicin before these organisms become prevalent in Lebanese animal and streptomycin are the most common and heavily used farms. This is especially true if no strict rules are implemented antimicrobial agents (Kassaify et al., 2013). Another thing to to control the overuse and misuse of antibiotics for treatment, mention is that in this study, no carbapenemase producers growth promotion and prophylaxis in Lebanese agriculture. We were detected. There might be two possible explanations for believe that the prescription of antibiotics often used in human this: the first one is that carbapenemase producers are really medicine should be reduced or even banned in the veterinary scarce in Lebanese chicken farms; the second one is that these sector. isolates were missed due to the medium used for the screening of multi-drug-resistant organisms. As has been reported, OXA- 48 carbapenemase producers are frequently found in hospitals AUTHOR CONTRIBUTIONS and nursing homes and in fowls in Lebanon (Al Bayssari et al., 2015b). OXA-48 carbapenemases do not always confer ID, ES, and ED conducted the phenotypic and molecular work. resistance to third-generation cephalosporins unless there is BE-B was responsible for the collection of the samples. EA, J-MR, another mechanism of resistance that co-exists in the same and ZD reviewed and edited the manuscript. bacterial cell (Poirel et al., 2012). Therefore, Oxacillinase producers could have been missed or under-estimated in our study. FUNDING Our study has two main limitations. The first one is that the primers used for blaTEM and blaSHV screening were universal, This study was funded by the Lebanese Council for Research and and thus, the possibility of having non-ESBL variants cannot the French Government under the “Investissements d’Avenir” be ruled out. However, as the strains presented with a typical (Investments for the Future) program managed by the Agence ESBL phenotype, i.e., the key hole effect and resistance to Nationale de la Recherche (ANR, fr: National Agency for penicillin, monobactams and third-generation cephalosporins Research) (reference: Méditerranée Infection 10-IAHU-03). with susceptibility to carbapenems, the TEM-positive strains were considered as ESBL producers and were included in the description of the MDR-GNB prevalence in this study. The ACKNOWLEDGMENTS second limitation is the low number of isolates subjected to MLST typing. MLST and PFGE analysis remain the gold standard for We would like to thank Dr. Hervé Chaudet for his assistance in clone/cluster detection in epidemiological studies regardless of the construction of the MSP dendrogram. Al Bayssari, C., Olaitan, A. O., Dabboussi, F., Hamze, M., and Rolain, J. M. REFERENCES (2015b). Emergence of OXA-48-producing Escherichia coli clone ST38 in Abdelnoor, A. M., Chokr, S., Fayad, L., and Al-Akl, N. (2013). Review study on fowl. Antimicrob. Agents Chemother. 59, 745–746. doi: 10.1128/AAC.03 external-hospital bacteria as a source of infection and antimicrobial resistance 552-14 in lebanon. Int. Arab. J. Antimicrob. Agents 3, 1–6. Alonso, C. A., Zarazaga, M., Ben Sallem, R., Jouini, A., Ben Slama, K., and Aguilar-Montes de Oca, S., Talavera-Rojas, M., Soriano-Vargas, E., Barba-Leon, J., Torres, C. (2017). Antibiotic resistance in Escherichia coli in husbandry animals: and Vazquez-Navarrete, J. (2015). Determination of extended spectrum beta- the African perspective. Lett. Appl. Microbiol. 64, 318–334. doi: 10.1111/lam. lactamases/AmpC beta-lactamases and plasmid-mediated quinolone resistance 12724 in Escherichia coli isolates obtained from bovine carcasses in mexico. Trop. Bachiri, T., Bakour, S., Ladjouzi, R., Thongpan, L., Rolain, J. M., and Touati, A. Anim. Health Prod. 47, 975–981. doi: 10.1007/s11250- 015- 0818- 3 (2017). High rates of CTX-M-15-producing Escherichia coli and Klebsiella Al Bayssari, C., Dabboussi, F., Hamze, M., and Rolain, J. M. (2015a). Emergence pneumoniae in wild boars and barbary macaques in algeria. J. Glob. Antimicrob. of carbapenemase-producing Pseudomonas aeruginosa and Acinetobacter Resist. 8, 35–40. doi: 10.1016/j.jgar.2016.10.005 baumannii in livestock animals in lebanon. J. Antimicrob. Chemother. 70, Bakour, S., Garcia, V., Loucif, L., Brunel, J. M., Gharout-Sait, A., Touati, A., et al. 950–951. doi: 10.1093/jac/dku469 (2015). Rapid identification of carbapenemase-producing Enterobacteriaceae, Frontiers in Microbiology | www.frontiersin.org 9 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 10 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry Pseudomonas aeruginosa and Acinetobacter baumannii using a modified Carba Available at: http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/ NP test. New Microbes New Infect. 7, 89–93. doi: 10.1016/j.nmni.2015.07.001 Breakpoint_tables/v_7.1_Breakpoint_Tables.pdf Baroud, M., Dandache, I., Araj, G. F., Wakim, R., Kanj, S., Kanafani, Z., et al. (2013). Exner, M., Bhattacharya, S., Christiansen, B., Gebel, J., Goroncy-Bermes, P., Underlying mechanisms of carbapenem resistance in extended-spectrum beta- Hartemann, P., et al. (2017). Antibiotic resistance: What is so special lactamase-producing Klebsiella pneumoniae and Escherichia coli isolates at a about multidrug-resistant gram-negative bacteria? GMS Hyg. Infect. Control tertiary care centre in lebanon: role of OXA-48 and NDM-1 carbapenemases. 12:Doc05. doi: 10.3205/dgkh000290 Int. J. Antimicrob. Agents 41, 75–79. doi: 10.1016/j.ijantimicag.2012.08.010 Ghodousi, A., Bonura, C., Di Noto, A. M., and Mammina, C. (2015). Belmahdi, M., Bakour, S., Al Bayssari, C., Touati, A., and Rolain, J. M. (2016). Extended-spectrum ß-lactamase, AmpC-producing, and fluoroquinolone- Molecular characterisation of extended-spectrum beta-lactamase- and plasmid resistant Escherichia coli in retail broiler chicken meat, Italy. Foodborne Pathog. AmpC-producing Escherichia coli strains isolated from broilers in bejaia, Dis. 12, 619–625. doi: 10.1089/fpd.2015.1936 algeria. J. Glob. Antimicrob. Resist. 6, 108–112. doi: 10.1016/j.jgar.2016.04.006 Gonzalez-Torralba, A., Oteo, J., Asenjo, A., Bautista, V., Fuentes, E., and Alos, J. I. Berrazeg, M., Diene, S. M., Drissi, M., Kempf, M., Richet, H., Landraud, L., (2016). Survey of carbapenemase-producing Enterobacteriaceae in companion et al. (2013). Biotyping of multidrug-resistant Klebsiella pneumoniae clinical dogs in madrid, spain. Antimicrob. Agents Chemother. 60, 2499–2501. isolates from france and algeria using MALDI-TOF MS. PLoS One 8:e61428. doi: 10.1128/AAC.02383- 15 doi: 10.1371/journal.pone.0061428 Haenni, M., Chatre, P., and Madec, J. Y. (2014). Emergence of Escherichia Bettiol, E., and Harbarth, S. (2015). Development of new antibiotics: taking off coli producing extended-spectrum AmpC beta-lactamases (ESAC) in animals. finally? Swiss Med. Wkly. 145:w14167. doi: 10.4414/smw.2015.14167 Front. Microbiol. 5:53. doi: 10.3389/fmicb.2014.00053 Black, J. A., Moland, E. S., and Thomson, K. S. (2005). AmpC disk test for Haenni, M., Saras, E., Ponsin, C., Dahmen, S., Petitjean, M., Hocquet, D., detection of plasmid-mediated AmpC disk test for detection of plasmid- et al. (2016). High prevalence of international ESBL CTX-M-15-producing mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal Enterobacter cloacae ST114 clone in animals. J. Antimicrob. Chemother. 71, AmpC beta-lactamases. J. Clin. Microbiol. 43, 3110–3113. doi: 10.1128/JCM.43. 1497–1500. doi: 10.1093/jac/dkw006 7.3110- 3113.2005 Huijbers, P. M., Graat, E. A., Haenen, A. P., van Santen, M. G., van Essen- Chantziaras, I., Boyen, F., Callens, B., and Dewulf, J. (2014). Correlation between Zandbergen, A., Mevius, D. J., et al. (2014). Extended-spectrum and AmpC veterinary antimicrobial use and antimicrobial resistance in food-producing beta-lactamase-producing Escherichia coli in broilers and people living and/or animals: a report on seven countries. J. Antimicrob. Chemother. 69, 827–834. working on broiler farms: prevalence, risk factors and molecular characteristics. doi: 10.1093/jac/dkt443 J. Antimicrob. Chemother. 69, 2669–2675. doi: 10.1093/jac/dku178 Dahms, C., Hubner, N. O., Wilke, F., and Kramer, A. (2014). Mini-review: Kassaify, Z., Abi-Khalil, P., and Sleiman, F. (2013). Quantification of epidemiology and zoonotic potential of multiresistant bacteria and clostridium antibiotic residues and determination of antimicrobial resistance profiles difficile in livestock and food. GMS Hyg. Infect. Control 9:Doc21. doi: 10.3205/ of microorganisms isolated from bovine milk in lebanon. Food Nutr. Sci. 4, dgkh000241 1–9. doi: 10.4236/fns.2013.47A001 Dallenne, C., Da Costa, A., Decre, D., Favier, C., and Arlet, G. (2010). Development Kawamura, K., Goto, K., Nakane, K., and Arakawa, Y. (2014). Molecular of a set of multiplex PCR assays for the detection of genes encoding important epidemiology of extended-spectrum beta-lactamases and Escherichia coli beta-lactamases in Enterobacteriaceae. J. Antimicrob. Chemother. 65, 490–495. isolated from retail foods including chicken meat in Japan. Foodborne Pathog. doi: 10.1093/jac/dkp498 Dis. 11, 104–110. doi: 10.1089/fpd.2013.1608 Dandachi, I., Salem Sokhn, E., Najem, E., Azar, E., and Daoud, Z. (2016). Carriage Kempf, I., Jouy, E., Granier, S. A., Chauvin, C., Sanders, P., Salvat, G., et al. (2015). of beta-lactamase-producing Enterobacteriaceae among nursing home residents Comment on “impact of antibiotic use in the swine industry”, by Mary D. in north lebanon. Int. J. Infect. Dis. 45, 24–31. doi: 10.1016/j.ijid.2016.02.007 Barton [Curr. Opin. Microbiol. 19 (June 2014) 9–15]. Curr. Opin. Microbiol. Diab, M., Hamze, M., Madec, J. Y., and Haenni, M. (2016). High prevalence of 26, 137–138. doi: 10.1016/j.mib.2015.06.013 non-ST131 CTX-M-15-producing Escherichia coli in healthy cattle in lebanon. Khennouchi, N. C., Loucif, L., Boutefnouchet, N., Allag, H., and Rolain, J. M. Microb. Drug Resist. 23, 261–266. doi: 10.1089/mdr.2016.0019 (2015). MALDI-TOF MS as a tool to detect a nosocomial outbreak of Dierikx, C. M., van der Goot, J. A., Smith, H. E., Kant, A., and Mevius, D. J. (2013). extended-spectrum-beta-lactamase- and ArmA methyltransferase-producing Presence of ESBL/AmpC-producing Escherichia coli in the broiler production Enterobacter cloacae clinical isolates in algeria. Antimicrob. Agents Chemother. pyramid: a descriptive study. PLoS One 8:e79005. doi: 10.1371/journal.pone. 59, 6477–6483. doi: 10.1128/AAC.00615- 15 0079005 Kola, A., Kohler, C., Pfeifer, Y., Schwab, F., Kuhn, K., Schulz, K., et al. Donati, V., Feltrin, F., Hendriksen, R. S., Svendsen, C. A., Cordaro, G., Garcia- (2012). High prevalence of extended-spectrum-beta-lactamase-producing Fernandez, A., et al. (2014). Extended-spectrum-beta-lactamases, AmpC beta- Enterobacteriaceae in organic and conventional retail chicken meat, Germany. lactamases and plasmid mediated quinolone resistance in klebsiella spp. from J. Antimicrob. Chemother. 67, 2631–2634. doi: 10.1093/jac/dks295 companion animals in italy. PLoS One 9:e90564. doi: 10.1371/journal.pone. Lazarus, B., Paterson, D. L., Mollinger, J. L., and Rogers, B. A. (2015). Do human 0090564 extraintestinal Escherichia coli infections resistant to expanded-spectrum ECDC/EFSA/EMA (2017). ECDC/EFSA/EMA second joint report on the cephalosporins originate from food-producing animals? A systematic review. integrated analysis of the consumption of antimicrobial agents and occurrence Clin. Infect. Dis. 60, 439–452. doi: 10.1093/cid/ciu785 of antimicrobial resistance in bacteria from humans and food-producing Leverstein-van Hall, M. A., Dierikx, C. M., Cohen Stuart, J., Voets, G. M., van den animals. Sci. Rep. 15:e04872. doi: 10.2903/j.efsa.2017.4872/epdf Munckhof, M. P., van Essen-Zandbergen, A., et al. (2011). Dutch patients, retail Economou, V., and Gousia, P. (2015). Agriculture and food animals as a source of chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin. antimicrobial-resistant bacteria. Infect. Drug Resist. 8, 49–61. doi: 10.2147/IDR. Microbiol. Infect. 17, 873–880. doi: 10.1111/j.1469- 0691.2011.03497.x S55778 Liu, X., Thungrat, K., and Boothe, D. M. (2016). Occurrence of OXA-48 El-Rami, F. E., Sleiman, F. T., and Abdelnoor, A. M. (2012). Identification and carbapenemase and other beta-lactamase genes in ESBL-producing multidrug antibacterial resistance of bacteria isolated from poultry. Pol. J. Microbiol. 61, resistant Escherichia coli from dogs and cats in the United States, 2009-2013. 323–326. Front. Microbiol. 7:1057. doi: 10.3389/fmicb.2016.01057 El-Shazly, D. A., Nasef, S. A., Mahmoud, F. F., and Jonas, D. (2017). Expanded Liu, Y., and Liu, X. (2015). Detection of AmpC beta-lactamases in Acinetobacter spectrum beta-lactamase producing Escherichia coli isolated from chickens with baumannii in the Xuzhou region and analysis of drug resistance. Exp. Ther. colibacillosis in Egypt. Poult. Sci. 96, 2375–2384. doi: 10.3382/ps/pew493 Med. 10, 933–936. doi: 10.3892/etm.2015.2612 Escudero, E., Vinue, L., Teshager, T., Torres, C., and Moreno, M. A. (2010). Maciuca, I. E., Williams, N. J., Tuchilus, C., Dorneanu, O., Guguianu, E., Carp- Resistance mechanisms and farm-level distribution of fecal Escherichia coli Carare, C., et al. (2015). High prevalence of Escherichia coli-producing CTX-M- isolates resistant to extended-spectrum cephalosporins in pigs in spain. Res. Vet. 15 extended-spectrum beta-lactamases in poultry and human clinical isolates in Sci. 88, 83–87. doi: 10.1016/j.rvsc.2009.05.021 romania. Microb. Drug Resist. 21, 651–662. doi: 10.1089/mdr.2014.0248 European Committee on Antimicrobial Susceptibility Testing (2017). Breakpoint Madec, J. Y., Haenni, M., Nordmann, P., and Poirel, L. (2017). Extended-spectrum Tables for Interpretation of MICs and Zone Diameters, Version 7.1. beta-lactamase/AmpC- and carbapenemase-producing Enterobacteriaceae in Frontiers in Microbiology | www.frontiersin.org 10 March 2018 | Volume 9 | Article 550 fmicb-09-00550 March 21, 2018 Time: 17:26 # 11 Dandachi et al. Antimicrobial Resistant Organisms in Lebanese Poultry animals: a threat for humans? Clin. Microbiol. Infect. 23, 826–833. doi: 10.1016/ Rhouma, M., Beaudry, F., and Letellier, A. (2016). Resistance to colistin: What is the j.cmi.2017.01.013 fate for this antibiotic in pig production? Int. J. Antimicrob. Agents 48, 119–126. Magiorakos, A. P., Srinivasan, A., Carey, R. B., Carmeli, Y., Falagas, M. E., doi: 10.1016/j.ijantimicag.2016.04.008 Giske, C. G., et al. (2012). Multidrug-resistant, extensively drug-resistant Roschanski, N., Fischer, J., Guerra, B., and Roesler, U. (2014). Development and pandrug-resistant bacteria: an international expert proposal for interim of a multiplex real-time PCR for the rapid detection of the predominant standard definitions for acquired resistance. Clin. Microbiol. Infect. 18, 268–281. beta-lactamase genes CTX-M, SHV, TEM and CIT-type AmpCs in doi: 10.1111/j.1469- 0691.2011.03570.x Enterobacteriaceae. PLoS One 9:e100956. doi: 10.1371/journal.pone.0100956 McGregor, K. F., and Spratt, B. G. (2005). Identity and prevalence of multilocus Sato, T., Okubo, T., Usui, M., Yokota, S., Izumiyama, S., and Tamura, Y. (2014). sequence typing-defined clones of group A streptococci within a hospital Association of veterinary third-generation cephalosporin use with the risk of setting. J. Clin. Microbiol. 43, 1963–1967. doi: 10.1128/JCM.43.4.1963- 1967. emergence of extended-spectrum-cephalosporin resistance in Escherichia coli 2005 from dairy cattle in Japan. PLoS One 9:e96101. doi: 10.1371/journal.pone. Moghnieh, R., Estaitieh, N., Mugharbil, A., Jisr, T., Abdallah, D. I., Ziade, F., 0096101 et al. (2015). Third generation cephalosporin resistant Enterobacteriaceae Schill, F., Abdulmawjood, A., Klein, G., and Reich, F. (2017). Prevalence and and multidrug resistant gram-negative bacteria causing bacteremia in febrile characterization of extended-spectrum beta-lactamase (ESBL) and AmpC beta- neutropenia adult cancer patients in lebanon, broad spectrum antibiotics use lactamase producing Enterobacteriaceae in fresh pork meat at processing level as a major risk factor, and correlation with poor prognosis. Front. Cell Infect. in germany. Int. J. Food Microbiol. 257, 58–66. doi: 10.1016/j.ijfoodmicro.2017. Microbiol. 5:11. doi: 10.3389/fcimb.2015.00011 06.010 Nguyen, V. T., Carrique-Mas, J. J., Ngo, T. H., Ho, H. M., Ha, T. T., Campbell, J. I., Schwarz, S., Enne, V. I., and van Duijkeren, E. (2016). 40 years of veterinary et al. (2015). Prevalence and risk factors for carriage of antimicrobial-resistant papers in JAC – what have we learnt? J. Antimicrob. Chemother. 71, 2681–2690. Escherichia coli on household and small-scale chicken farms in the mekong doi: 10.1093/jac/dkw363 delta of Vietnam. J. Antimicrob. Chemother. 70, 2144–2152. doi: 10.1093/jac/ Seiffert, S. N., Hilty, M., Perreten, V., and Endimiani, A. (2013). Extended- dkv053 spectrum cephalosporin-resistant gram-negative organisms in livestock: an Olaitan, A. O., Thongmalayvong, B., Akkhavong, K., Somphavong, S., emerging problem for human health? Drug Resist. Updat. 16, 22–45. doi: 10. Paboriboune, P., Khounsy, S., et al. (2015). Clonal transmission of a 1016/j.drup.2012.12.001 colistin-resistant Escherichia coli from a domesticated pig to a human in Seng, P., Rolain, J. M., Fournier, P. E., La Scola, B., Drancourt, M., and Raoult, D. laos. J. Antimicrob. Chemother. 70, 3402–3404. (2010). MALDI-TOF-mass spectrometry applications in clinical microbiology. Olsen, R. H., Bisgaard, M., Lohren, U., Robineau, B., and Christensen, H. (2014). Future Microbiol. 5, 1733–1754. doi: 10.2217/fmb.10.127 Extended-spectrum beta-lactamase-producing Escherichia coli isolated from Singhal, N., Kumar, M., Kanaujia, P. K., and Virdi, J. S. (2015). MALDI-TOF poultry: a review of current problems, illustrated with some laboratory findings. mass spectrometry: an emerging technology for microbial identification and Avian Pathol. 43, 199–208. doi: 10.1080/03079457.2014.907866 diagnosis. Front. Microbiol. 6:791. doi: 10.3389/fmicb.2015.00791 Ortega-Paredes, D., Barba, P., and Zurita, J. (2016). Colistin-resistant Escherichia Wu, G., Day, M. J., Mafura, M. T., Nunez-Garcia, J., Fenner, J. J., Sharma, M., et al. coli clinical isolate harbouring the mcr-1 gene in Ecuador. Epidemiol. Infect. 144, (2013). Comparative analysis of ESBL-positive Escherichia coli isolates from 2967–2970. doi: 10.1017/S0950268816001369 animals and humans from the UK, the Netherlands and Germany. PLoS One Peng, C., and Zong, Z. (2011). Sequence type 38 Escherichia coli carrying 8:e75392. doi: 10.1371/journal.pone.0075392 bla . J. Med. Microbiol. 60(Pt 5), 694–695. doi: 10.1099/jmm.0. Zou, W., Lin, W. J., Foley, S. L., Chen, C. H., Nayak, R., and Chen, J. J. CTX-M-14 028316-0 (2010). Evaluation of pulsed-field gel electrophoresis profiles for identification Pires-dos-Santos, T., Bisgaard, M., and Christensen, H. (2013). Genetic diversity of Salmonella serotypes. J. Clin. Microbiol. 48, 3122–3126. doi: 10.1128/JCM. and virulence profiles of Escherichia coli causing salpingitis and peritonitis 00645- 10 in broiler breeders. Vet. Microbiol. 162, 873–880. doi: 10.1016/j.vetmic.2012. 11.008 Conflict of Interest Statement: The authors declare that the research was Poirel, L., Potron, A., and Nordmann, P. (2012). OXA-48-like carbapenemases: the conducted in the absence of any commercial or financial relationships that could phantom menace. J. Antimicrob. Chemother. 67, 1597–1606. doi: 10.1093/jac/ be construed as a potential conflict of interest. dks121 Rafei, R., Hamze, M., Pailhories, H., Eveillard, M., Marsollier, L., Joly-Guillou, Copyright © 2018 Dandachi, Sokhn, Dahdouh, Azar, El-Bazzal, Rolain and Daoud. M. L., et al. (2015). Extrahuman epidemiology of Acinetobacter baumannii This is an open-access article distributed under the terms of the Creative Commons in lebanon. Appl. Environ. Microbiol. 81, 2359–2367. doi: 10.1128/AEM. Attribution License (CC BY). The use, distribution or reproduction in other forums 03824-14 is permitted, provided the original author(s) and the copyright owner are credited Randall, L., Wu, G., Phillips, N., Coldham, N., Mevius, D., and Teale, C. (2012). and that the original publication in this journal is cited, in accordance with accepted Virulence genes in bla Escherichia coli isolates from chickens and humans. academic practice. No use, distribution or reproduction is permitted which does not CTX-M Res. Vet. Sci. 93, 23–27. doi: 10.1016/j.rvsc.2011.06.016 comply with these terms. Frontiers in Microbiology | www.frontiersin.org 11 March 2018 | Volume 9 | Article 550

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