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Prevalence and molecular characteristics of Staphylococcus aureus, including methicillin resistant strains, isolated from bulk can milk and raw milk products in pastoral communities of South-West Uganda

Prevalence and molecular characteristics of Staphylococcus aureus, including methicillin... Background: Staphylococcus aureus strains are now regarded as zoonotic agents. In pastoral settings where human-animal interaction is intimate, multi-drug resistant microorganisms have become an emerging zoonotic issue of public health concern. The study of S. aureus prevalence, antimicrobial resistance and clonal lineages in humans, animals and food in African settings has great relevance, taking into consideration the high diversity of ethnicities, cultures and food habits that determine the lifestyle of the people. Little is known about milk carriage of methicillin resistant S. aureus strains (MRSA) and their virulence factors in Uganda. Here, we present the prevalence of MRSA in bulk can milk and raw milk products in pastoral communities of south-west Uganda. We also present PFGE profiles, spa-types, as well as frequency of enterotoxins genes. Methods: S. aureus was identified by the coagulase test, susceptibility testing by the Kirby-Bauer disc diffusion and E-test methods and MRSA by detection of the mecA gene and SCCmec types. The presence of Panton – Valentine Leucocidin (PVL) genes and staphylococcal enterotoxins was determined by PCR, while genotyping was by PFGE and spa typing. Results: S. aureus were isolated from 30/148 (20.3%) milk and 11/91(12%) sour milk samples. mecA gene carriage, hence MRSA, was detected in 23/41 (56.1%) of the isolates, with 21 of the 23 (91.3%) being SCCmec type V; while up to 30/41 (73.2%) of the isolates were resistant to tetracycline. Only five isolates carried the PVL virulence gene, while PFGE typing revealed ten clusters (ranging from two seven isolates each) that comprised 83% of the sample, and only eight isolates with unique pulsotypes. The largest PFGE profile (E) consisted of seven isolates while t7753, t1398, and t2112 were the most common spa-types. Thirty seven of the 41 strains (90.2%) showed at least one of the eight enterotoxin genes tested, with sem 29 (70.7%), sei 25 (61%) and seg 21 (51.2%) being the most frequently observed genes. Conclusion: This is the first study to demonstrate MRSA and enterotoxin genes in raw milk and its products in Uganda. The fact that over 90% of the isolates carried at least one gene encoding enterotoxins shows a high risk of spread of foodborne diseases through milk in this setting. * Correspondence: basiimwe@chs.mak.ac.ug Emerging Bacterial Pathogens Unit, IRCCS, Via Olgettina 58, Milan, Italy Universita Vita-Salute San Raffaele, Via Olgettina 58, Milan, Italy Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 2 of 8 Background being the heat-stable enterotoxins that cause the sporadic Staphylococcus aureus strains, especially those resistant food-poisoning syndrome or foodborne outbreaks [15]. to methicillin (methicillin resistant S. aureus, MRSA) Staphylococcal enterotoxins (SEs) cause toxic shock-like have been regarded as zoonotic agents and there is syndromes and have also been implicated in several growing genuine concern about the likely transmission allergic and autoimmune diseases, in addition to food poi- of MRSA between animals and humans from close inter- soning; therefore SEs function not only as potent gastro- action or from handling and/or consuming MRSA intestinal toxins but also as superantigens that stimulate infected animal products [1–5]. In settings where humans non-specific T-cell proliferation [16]. depend on animals and their products for food and liveli- Theaim of thepresent studywas to establishthe hood, such as in pastoral Africa, contact is intimate and prevalence and distribution of methicillin-susceptible multi-drug resistant microorganisms have become an S. aureus (MSSA) and MRSA in bulk can milk and emerging veterinary and zoonotic issue of public health raw milk products samples at household level. Fur- concern [6–8]. Additionally, S. aureus is known to be the thermore, we tested the susceptibility of the isolates third most reported cause of food-borne diseases in the to nine drugs and analyzed the genetic relatedness of world [4], culpable for production of staphylococcal theisolatesbyPFGE and spa typing. Additionally, enterotoxins that cause food poisoning. Moreover, con- resistance and virulence genes (SCCmec and PVL)as taminated milk and milk products have frequently been well as frequency of genes encoding three of the clas- implicated in staphylococcal food poisoning in different sical staphylococcal enterotoxins (SEs): SEA, SEB and industrialized countries [9]. The study of S. aureus preva- SEC; and five additional enterotoxins: SEG, SEH, SEI, lence, antimicrobial resistance and clonal lineages in SEL, and SEM were assayed. humans, animals and food in African settings has great relevance, taking into consideration the high diversity of Methods ethnicities, cultures and food habits that determine the Study area lifestyle of the people. The study was carried out in Sanga and Kanyaryeru sub- The treatment of bacterial infections in Uganda’s counties of Kiruhura district, South Western Uganda. health and veterinary care settings is largely empirical The area is a rangeland populated by agro-pastoralist with no laboratory confirmation to guide therapy, and households whose livelihoods are mainly dependent on antibiotics for both human and veterinary use are readily consumption and trade of cattle and their products, and available over the counter. This continuous use and the area is part of the cattle corridor and is considered misuse of the antibiotics has resulted into a surge in the milk basin of Uganda. multidrug microorganisms, now a growing problem on farms, in health care settings and the community Study design and sampling [7, 10, 11]. In Uganda, pastoralist communities are A total of 196 homesteads from the two sub-counties vulnerable to zoonotic infections, including MRSA, (100 from Kanyaryeru and 96 from Sanga) were ran- not only because of the weak veterinary/public health domly recruited and sampled. One pooled (bulk can) delivery systems but also due to their cherished way milk sample and a raw milk product (cow ghee (butter of life (culture) dependent on animals and their prod- from milk) or sour (fermented) milk) were collected ucts. There is limited data about prevalence and from each household on consent and when available at spread of S. aureus strains in rural pastoral farm the time of visit, from July to August 2013. In Kanyar- environments in Uganda. Previous studies have reported yeru sub-county, 81 milk samples, 60 ghee samples and on bacterial carriage of milk in peri and urban farmers in 53 sour milk samples were collected; while 67milk and around the capital, Kampala [7], while another samples, 57 ghee samples and 38 sour milk samples reported on burden of S. aureus infection on dairy farms were collected from Sanga sub-county. In all, 148 milk in the neighboring district of Kiboga [6]. However these samples, 117 ghee samples and 91 sour milk samples studies neither assessed resistance to methicillin nor were collected for the study. Samples were inoculated further characterized the isolates for resistance and viru- into Brain-Heart Infusion (BHI) broth in 15 ml propyl- lence markers as well as genetic relatedness and spread of ene tubes and stored on ice in any case for not more the strains in the study environments. than 12 h before transportation to the Department of In Uganda, 92% of the milk is marketed unregulated Microbiology at Mbarara University of Science and and in raw form [12]. This practice increases the risk of Technology for culture and identification. spread of staphylococcal food poisoning due to poor stor- age and transportation conditions. S. aureus is among the Culture, isolation and identification leading causes of foodborne bacterial intoxications world- Initial bacterial culture and isolation was done according wide [13, 14], with the most notable virulence factors to methods described by Cheesbrough [17], with minor Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 3 of 8 modifications. Briefly, 50 μl of culture broth was inocu- Pulsed-field gel electrophoresis (PFGE) and spa typing lated on 5% sheep blood agar medium and incubated for All isolates were subjected to molecular epidemiological 18 - 20 h at 37 °C. Preliminary identification of the analysis by PFGE after SmaI digestion using the CHEF bacteria was carried out based on colony characteristics Genomic DNA Plug kit (Bio-Rad) according to a stan- such as shape, size, color and hemolysis patterns. dardized protocol [21]. PFGE was run using a CHEF Isolated suspected of being S. aureus were shipped in DRIII system (Bio-Rad). The InfoQuest FP (v5) software BHI with 20% glycerol to the Emerging Bacterial Pathogens (Bio-Rad Laboratories) was used to analyze PFGE pro- Unit (EBPU) at San Raffaele Scientific Institute in Milan, files, according to interpretation criteria described by Italy, for identification and molecular characterization. At Tenover et al. [22]. Clustering analysis was achieved the EBPU, all isolates were sub-cultured on mannitol salt using Dice similarity coefficients and the unweighted agar (Oxoid Ltd., Hampshire, England), then on blood agar pair group method with averages (UPGMA) at 1.5% (Becton Dickinson, Heidelberg, Germany), and subjected optimization and 1.5% position tolerance. Addition- to the tube coagulase test in rabbit plasma with EDTA ally, isolates were typed based on sequencing of the (Remel, KS, USA). Only isolates that passed the coagu- hypervariable region of the S. aureus protein A gene (spa); lase test were considered for further characterization. spa types were assigned MLST sequence types (ST) in- Isolates were also screened for susceptibility to eight ferred on the Ridom spaServer (http://spaserver.ridom.de) antibacterial agents (cefoxitin, tetracycline, gentamy- curated by the SeqNet.org initiative [23]. cin, ciprofloxacin, rifampicin, erythromycin, clindamy- cin, Sulfamethoxale/Trimethoprim) on Mueller-Hinton Staphylococcal enterotoxins genes detection by PCR agar (Oxoid Ltd., Hampshire, England) according to DNA amplification was performed using the following Kirby-Bauer disc diffusion method and two (oxacillin and conditions: initial denaturation for 15 min at 95 C vancomycin) by the E-test (Oxoid Ltd., Hampshire, followed by 35 cycles of denaturation (94 C for 1 min), England). Screen tests were performed in 0.5 McFarland annealing, and extension (72 C for 1 min), and a final according to EUCAST guidelines (http://www.eucast.org/ extension step (72 C for 10 min) after the completion clinical_breakpoints/). of the cycles. The primer sequences, annealing tempera- tures and expected amplicon sizes are as described in Jarraud et al. [24] and Diep et al. [25]. DNA extracted DNA extraction, SCCmec typing and PVL genes detection from the standard strains S. aureus Mu50 [26] was used Bacterial DNA was prepared from isolated colonies as positive control for enterotoxins SEA, SEC, SEG, SEI, suspended in 500 ml Triton X-100 lysis buffer with SEL and SEM; while DNA from S.aureus COL [27] 1% Triton and 50 mg/ml lysostaphin, incubated at controlled for enterotoxins SEB and SEH. 37 °C for 1 h, followed by phenol-chloroform extrac- tion. Staphylococcal Cassette Chromosome harboring Results mecA (SCCmec) gene multiplex PCR typing was done. Prevalence of MSSA and MRSA This was performed using primers and protocols for Of the samples that were collected, only 56 of 148 milk SCCmec types and subtypes I, II, III, IVa, IVb, IVc, samples, 25 of 91 sour milk samples and one of 117 cow IVd, and V and the mecA gene according to Zhang et ghee samples gave growth that was suspected to be S. al.[18]. We also assayed for the recently described aureus based on colony characteristics such as size, mecA homologue, mecC shown to be common in shape, color and hemolysis patterns on blood agar. animal-derived strains, using primers and conditions These samples were then sub-cultured and re-identified as described in Paterson et al. [19]. The MRSA strain at the EBPU and only 30 of 56 isolates from milk and 11 COL (SCCmec type I, mec gene complex B and ccr gene of the 25 isolates from sour milk were coagulase posi- complex 1) was also used as positive control. We further tive, while the single isolate from ghee failed the coagu- used control strains for SCCmec II (Mu50), III (ANS46), lase test. The prevalence of S. aureus in the bulk can IVa (USA300-FPR3757), IVb (strain from Ospedale San milk thus was 30/148 (20.3%) while that in the sour milk Raffale (OSR) MRSA 864), IVc (strain from OSR collec- was 11/91 (12.1%). Isolation of S. aureus was unevenly tion, MRSA 111), IVd (strain from OSR collection, MRSA distributed between the two study areas, with 25 isolates 1040) and V (S0385). Unfortunately a control for SCCmec from Kanyaryeru and 16 isolates from Sanga. type IV from subtype “e” to subtype “k” was not available to us. Additionally, amplification of the Panton – Valen- Drug susceptibility, mecA, SCCmec and PVL gene tine Leucocidin (PVL) toxin genes, lukS-PV and lukF-PV, Of the 356 samples cultured (148 fresh milk, 91 sour was performed using the S. aureus strain ATCC49775 as milk and 117 ghee), S. aureus was isolated from 30 positive control, and primers and protocols as described (53.6%) of milk, 11 (44%) of sour milk and none were by Lina et al. [20]. recovered from ghee samples. Carriage of the mecA gene Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 4 of 8 (hence MRSA) in this study, identified as part of the isolates (24.4%); while in the other enterotoxins, sem Staphylococcal Cassette Chromosome harboring mecA (29/41; 70.7%), sei (25/41; 61%) and seg (21/41; 51.2%) (SCCmec) after multiplex PCR, was 50% (15/30) and were the most frequently observed genes. The propor- 72.7% (8/11) of the milk and sour milk derived isolates tion of all the other enterotoxins can be seen in Table 3. respectively. Overall, only five isolates (5/41, 12.2%) car- Only four isolates (two from raw milk and two from ried the PVL virulence gene. A majority of the isolates sour milk) did not carry any of the eight enterotoxin with the mecA gene (21/23; 91.3%) were type V. Nosne genes that were assayed. of the isolates tested carried the mecA homologue, mecC. When screened with cefoxitin, only two of the 23 Discussion isolates carrying the mecA gene had zones of inhibition Pastoralists within Uganda’s cattle corridor are afflicted ≤22 mm, hence a discrepancy between the phenotypic by a variety of ecosystem-based human/livestock health and genotypic results. The two isolates resistant to problems, especially those zoonotic in nature, with cefoxitin also had MICs of oxacillin above the break- potential to spill over to neighboring communities and point of 2 mg/L, hence resistant. There was a high affecting the tourism industry. Unfortunately, not much resistance to tetracycline (73.2%), while all isolates were has been done to estimate the burden of many such fully susceptible to ciprofloxacin and vancomycin. The potentially zoonotic infections in these marginalized susceptibility pattern of the other drugs tested is shown communities. S. aureus, including MRSA, has recently in Table 1. been found to be a zoonotic problem in different settings [2, 3, 28]. We set out to study the prevalence of PFGE restriction profiles and spa types methicillin resistance, virulence factors, and genetic There were ten clusters (ranging from two to seven iso- relatedness of S. aureus strains isolated from bulk can lates each) that comprised 83% of the sample, while only milk and milk products from pastoral households eight isolates had unique pulsotypes (Table 2). The around L. Mburo national Park. largest PFGE profile (E) consisted of seven isolates, all S. aureus was isolated in 20.3% of bulk can milk sam- with unknown spa types. The most common spa types ples and 12.1% of sour milk samples. Generally, there is were t7753 and t1398 (four isolates each), while others a paucity of data on S. aureus from milk and raw milk were t2112 (three isolates), t3992 and t127 (two isolates products in the region. In urban and peri-urban each). The PFGE profiles and spa types of the isolates Kampala, a recent study of 97 milk samples yielded 85 are shown in Table 2. pure cultures, but only 1 of 58 (2%) Gram-positive bac- teria was identified as S. aureus while 20 of 58 (34%) Frequency of staphylococcal enterotoxins (SEs) were coagulase negative Staphylococci [7]. Another Results for the detection of eight genes encoding the study carried out in dairy farms in rural Kiboga (another enterotoxins SEA, SEB, SEC, SEG, SEH, SEI, SEL, and cattle corridor district) in Uganda, however, found that SEM are shown in Table 2, in which 37 of 41 strains the most prevalent bacterial pathogens associated with (90.2%) were positive for at least one enterotoxin gene. sub-clinical mastitis in dairy cattle were Staphylococci The distribution of enterotoxin genes in MRSA and such as Coagulase negative Staphylococci and S. aureus MSSA strains as well as in milk and sour milk samples in 64.42 and 16.56% of the cases respectively [6], com- was unequal, with MSSA and milk-derived isolates parable to our finding of 19.6% S. aureus in fresh bulk showing more strains with multiple enterotoxins. There can milk samples in the current study. These findings were 17/30 (56.7%) milk-derived strains carrying at least support the expectation that S. aureus from infected three enterotoxins genes while only 3/11(27.3%) of iso- udders may contaminate bulk milk and, subsequently, lates from sour milk carried more than three. Among raw milk products as has been observed elsewhere [29]. the genes that code for three of the classic enterotoxins SCCmec PCR showed that 23/41 (56.1%) of the assayed (SEA-SEC), sec was the most frequent with 10 isolates carried the mecA gene implicated in resistance Table 1 Drug susceptibility pattern of the study isolates to eight of the 10 antimicrobial agents tested Source S. aureus Drug susceptibility pattern TE RD SXT CIP E CN DA VA R S R S RS RS RS R S RS RS Milk 30 20 10 1 29 1 29 0 30 1 29 1 29 2 28 0 30 Sour milk 11 10 1 0 11 1 10 0 11 0 11 0 11 0 11 0 11 Total 41 30 11 1 40 2 39 0 41 1 40 1 40 2 39 0 41 TE tetracycline, RD rifampicin, SXT trimethoprim-sulphamethoxale, CIP ciprofloxacin, E erythromycin, CN gentamycin, DA clindamycin, VA vancomycin Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 5 of 8 Table 2 Molecular characteristics of the 41 isolates of S. aureus Table 3 Genotypic profile of S. aureus strains, according to sea, in the study seb, sec, seg, seh, sei, sel, and sem genes Isolate ID Pulsotype spa type MLST PVL mecA SCCmec Genotypic profile Raw milk Sour milk Total (%) (n = 30) (n = 11) T130 m A1 t1398 −− − sea 1 1 2 (4.9) T132 m A1 t2112 − +V seb 4 2 6 (14.6) T111 m A2 t380 + −− Sec 7 3 10 (24.4) T119 m A3 t7753 −− − Seg 16 5 21 (51.2) T147y A4 t2112 − +V She 13 2 15 (36.6) T067 m B1 t127 ST-1 − +V Sei 17 8 25 (61) T089y B1 t127 ST-1 − +V Sel 8 2 10 (24.4) T120y C1 t1236 ST-97 −− − Sem 21 8 29 (70.7) T139 m C1 t7753 −− − sea + seb + sec 3 0 3 (7.3) T115 m C2 t10103 −− − seg + seh + sei + sel + sem 9 1 10 (24.4) T089 m C3 t7753 −− − At least 1 SE gene 28 9 37 (90.2) T099y C4 t14299 − +V No SE gene detected 2 2 4 (9.8) T041y D1 Unknown − +V T042 m D2 Unknown −− − to methicillin. Elsewhere in Africa, however, low level T127 m D3 Unknown − +V MRSA carriage in raw milk has been reported in Algeria T031 m E1 Unknown − +V where only two of five isolates were screened by the disc T117 m E2 Unknown −− − diffusion test, but there was no testing for the mecA T136 m E3 Unknown − +V gene [30]. In the current study, only two isolates had zones of clearance less than 22 mm around cefoxitin T105 m E4 Unknown −− − discs as recommended by EUCAST, to be considered T143 m E4 Unknown −− − MRSA. The two isolates also had MIC of >2 mg/L on T094 m E5 Unknown −− − oxacillin strips, hence resistant. The discrepancy T148 m E5 Unknown − +V between the phenotypic and genotypic results seen in T019 m F1 t645 ST-121 + + V this study may be due to a lack of expression of mecA. T057 m F1 Unknown − +V Routine oxacillin tests often fail to detect very heteroge- neous MRSA populations, which consequently are consid- T103 m G1 t1398 − +V ered methicillin-susceptible S. aureus (MSSA) because of T145 m G1 t1398 − +V their usual susceptibility to most non-β-lactam antistaphy- T030 m G2 t1398 −− − lococcal antibiotics. Therefore, several parameters have T098y H1 t186 ST-88 −− − been recommended to improve results and these include T120 m H2 Unknown − +V increasing the inoculum, growth at a low temperature, an T037 m I1 Unknown − +V oxacillin screen test with NaCl, or protracted incubation [31]. More recent methods for detection of MRSA include T067y I2 Unknown − +V the oxacillin E-test (AB BIODISK, Solna, Sweden) for T059y L1 t2112 + + V determination of MICs, and the automated Vitek 2 system T091 m L2 t1236 ST-97 −− − (bioMe’rieux, La Balme les Grottes, France). Phenotypic T032 m Unique Unknown + + IV results will therefore vary between laboratories depending T037y Unique Unknown − +V on the system used. Detection of the mecA gene, there- T049 m Unique Unknown −− − fore, would be a good way of standardizing identification of MRSA across laboratories. The results are similar to T055 m Unique Unknown −− − those in another study comparing methods for the detec- T056 m Unique t3992 ST-97 − +V tion of MRSA on isolates from foods of animal origin in T055y Unique t3992 ST-97 −− − Italy by Corrente et al. [31]. In that study, none of the six T144 m Unique t7753 − + IVc MRSA strains identified by mecA PCR were detected by T157y Unique t3772 + + V the cefoxitin test (sensitivity of 0%), and they concluded PVL = presence (+) or absence (−)of the PVL gene; mecA = presence (+) or that analysis for MRSA in isolates from food of animal ori- absence (−)ofthe mecA gene; SCCmec = SCCmec-types according to gin is better done with the mecA gene-specific PCR rather Zhang et al. [18] than conventional phenotypic assays [31]. More studies in Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 6 of 8 human derived strains also render support to low sensitiv- t2748, t2971, t2974, t3013, t3014, t3053, t3146, or t3208) ity of the cefoxitin test. For example, a recent study of low according to the recent work [37] were not found among level methicillin resistance in mecA positive S. aureus our isolates. However, we detected other spa types that strains showed zones of clearance of 28 mm diameter by have been reported to be associated with LA-MRSA cefoxitin testing [32]. In the current study, 17 of the 23 strains other than ST398 (Table 2). mecA positive strains also had zones of clearance of This, to the best of our knowledge, is the first report ≤28 mm (Table 1). The observation of a high proportion of SEs in milk from Uganda. While literature elsewhere of MRSA in the study sample is very worrisome because suggests that the classical SEA is the most frequently studies have shown that MRSA readily jumps from ani- observed in enterotoxigenic strains of S. aureus [38], it mals to exposed veterinarians, farm works and other farm was found in only two (4.9%) of the strains in the animals [2, 5, 28]. This, to the best of our knowledge, is current study. This is probably because of the fact the first report of MRSA isolated from milk and raw milk that different foods and strains carry different entero- products under communally grazing pastoral households toxins, as observed elsewhere [39]. On the other in Uganda. In this setting where animal – human inter- hand, the more recently discovered seg gene was action is intimate due to the pastoralists’ cherished way of observed in 21 (51.2%) of 41 isolates, and in 85.7% of life, it creates an urgent public health concern. the cases, it was associated with sei,which was Genotype analysis was done by SCCmec typing and present in 25 (61%) of the isolates, similar to reports PFGE. Of the 23 mecA positive (MRSA) isolates, 21 in other studies in Brazil [40] and France [41]. These harboured the SCCmec type V. The other SCCmec types genes are known to be frequently found together found can be seen in Table 2. It has recently been noted because they are within the same cluster, in a 3.2 kb that community-associated MRSA (CA-MRSA) isolates, DNA fragment as reported elsewhere [42]. In East which carry SCCmec type IV or V, are now prevalent Africa, most milk is produced by small holders and and exceed methicillin-susceptible S. aureus (MSSA) in the bulk of it (86% in Kenya and 92% in Uganda) is skin and soft tissue infections, and such isolates were traded through unregulated channels as unpasteurized reported to cause severe, often necrotizing, soft tissue milk or milk products [12]. Raw and sour milk are infections and pneumonia [33, 34]. Their presence in consumed in these settings for such beliefs as milk creates risk of spread of these genotypes in the enhanced nutritional quality and better taste. The fact communities. Additionally, assaying for the virulence that over 90% of the isolates in the current study car- marker PVL showed that only four isolates were positive ried at least one gene encoding for enterotoxins for this gene. A previous study of 50 S. aureus isolates shows a high risk of spread of foodborne diseases in from 14 dairy cow herds in Italy identified the PVL this setting, with milk as a vehicle for dispersion. gene in more than 50% of the isolates [35], while There is a need for farmer education and health pro- another in Brazil reported that none of the 84 milk motion messages in the communities about the need derived S. aureus isolates harbored the PVL gene [36]. for pasteurization of milk as this will kill S. aureus, According to criteria proposed by Tenover et al., [22], therefore reducing enterotoxin production. strains that differ by three bands or fewer by PFGE are likely to be closely related. The authors empha- sized that these criteria apply to epidemiologically Conclusion related isolates. By these criteria, isolates in the Our results show a high proportion of MRSA in raw current study group into ten clusters of two to seven milk and its products in the cattle corridor of isolates each, and only eight unique strains. Since Uganda, threatening treatment options for S. aureus these were not outbreak strains, spa typing was used in the near future in this setting, especially if the to determine the clonal lieneages of the isolates. The strains cross the species barrier into humans. Isolates most common lineages were t7753 and t1398 with carrying SCCmec type V are wide spread in this set- four isolates each. In all, there were 13 different line- ting. The fact that over 90% of the isolates in the ages, but most of the isolates (17/41, 41.5%) could current study carried at least one gene encoding for not be assigned to any lineage and were designated as enterotoxins shows a high risk of spread of foodborne unknown (Table 2). Only one lineage in the current diseases. A limitation of this study is that the entero- study, t645, had been isolated from one mastitic cow toxin genes were not studied for expression, therefore and four milkmen in peri-urban Kampala [7]. The we cannot confirm their significance in cases of food most common spa types associated with clone ST398 poisoning. Additionally, the initial processing and (t011, t034, t108, t567, t571, t588, t753, t753, t779, culture technique for the samples was not good t898, t899, t943, t1184, t1197, t1254, t1255, t1451, enough whereby only 82 of the 356 samples yielded t1456, t1457, t2123, t2287, t2329, t2330, t2383, t2582, suspected S. aureus. Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 7 of 8 Acknowledgments 8. Kamau NPM, Dolci S, Jans C, Wangoh J, Lacroix C, et al. Phenotypic and The authors wish to thank the households that participated in the study. genotypic antibiotic resistance patterns of Staphylococcus aureus from raw and We also thank Dr. F. Agnoletti and Dr. I. Drigo from the Istituto Zooprofilattico spontaneously fermented camel milk. Eur J Nutr Food Safety. 2013;3:87–98. Sperimentale delle Venezie, Italy, who kindly provided us with control 9. De Buyser ML, Dufour B, Maire M, Lafarge V. Implication of milk and milk strain LGA251. products in food-borne diseases in France and in different industrialised countries. Int J Food Microbiol. 2001;67:1–17. Funding 10. Kateete DP, Namazzi S, Okee M, Okeng A, Baluku H, et al. High prevalence of methicillin resistant Staphylococcus aureus in the surgical units of Mulago BBA was supported by a Marie Curie Fellowship (MARIE CURIE – COFUND hospital in Kampala, Uganda. 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Int J Food Microbiol. 2002; analyzing and interpreting PFGE data and critical revision of the manuscript; 78:31–41. AT participated in performing PFGE; DMC supervised the study and critically 15. Kerouanton A, Hennekinne JA, Letertre C, Petit L, Chesneau O, et al. revised the manuscript. All authors read and approved the final manuscript. Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France. Int J Food Microbiol. 2007;115:369–75. Competing interests 16. Balaban N, Rasooly A. Staphylococcal enterotoxins. Int J Food Microbiol. The authors declare that they have no competing interests. 2000;61:1–10. 17. Cheesbrough M District laboratory practice in tropical countries, part 2. 2nd Consent for publication edition.: 62-70, 132-143, 157-234. 2005. Not applicable. 18. Zhang K, McClure JA, Elsayed S, Louie T, Conly JM. 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Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary Publisher’sNote skin infections and pneumonia. Clin Infect Dis. 1999;29:1128–32. Springer Nature remains neutral with regard to jurisdictional claims in 21. Baldan R, Tassan Din C, Semeraro G, Costa C, Cichero P, et al. Severe published maps and institutional affiliations. community-onset infections in healthy individuals caused by community-acquired MRSA in an Italian teaching hospital, 2006-2008. Author details J Hosp Infect. 2009;72:271–3. Emerging Bacterial Pathogens Unit, IRCCS, Via Olgettina 58, Milan, Italy. 2 3 22. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, et al. Universita Vita-Salute San Raffaele, Via Olgettina 58, Milan, Italy. Department Interpreting chromosomal DNA restriction patterns produced by of Medical Microbiology, Makerere University College of Health Sciences, P O pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Box 7072, Kampala, Uganda. Microbiol. 1995;33:2233–9. 23. Mellmann A, Friedrich AW, Rosenkotter N, Rothganger J, Karch H, et al. Received: 14 February 2017 Accepted: 5 June 2017 Automated DNA sequence-based early warning system for the detection of methicillin-resistant Staphylococcus aureus outbreaks. PLoS Med. 2006;3:e33. 24. Jarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, et al. Relationships References between Staphylococcus aureus genetic background, virulence factors, agr 1. van Loo IH, Diederen BM, Savelkoul PH, Woudenberg JH, Roosendaal R, et groups (alleles), and human disease. Infect Immun. 2002;70:631–41. al. Methicillin-resistant Staphylococcus aureus in meat products, the 25. Diep BA, Carleton HA, Chang RF, Sensabaugh GF, Perdreau-Remington F. Netherlands. Emerg Infect Dis. 2007;13:1753–5. Roles of 34 virulence genes in the evolution of hospital- and community- 2. Juhasz-Kaszanyitzky E, Janosi S, Somogyi P, Dan A, van der Graaf-van Bloois associated strains of methicillin-resistant Staphylococcus aureus. J Infect Dis. L, et al. MRSA transmission between cows and humans. Emerg Infect Dis. 2006;193:1495–503. 2007;13:630–2. 26. Kuroda M, Ohta T, Uchiyama I, Baba T, Yuzawa H, et al. Whole genome 3. Garcia-Alvarez L, Holden MT, Lindsay H, Webb CR, Brown DF, et al. sequencing of meticillin-resistant Staphylococcus aureus. Lancet. 2001;357:1225–40. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in 27. Gill SR, Fouts DE, Archer GL, Mongodin EF, Deboy RT, et al. Insights on human and bovine populations in the UK and Denmark: a descriptive study. evolution of virulence and resistance from the complete genome analysis Lancet Infect Dis. 2011;11:595–603. of an early methicillin-resistant Staphylococcus aureus strain and a biofilm- 4. 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Egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J Immunol. 2001;166:669–77. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries � Our selector tool helps you to find the most relevant journal � We provide round the clock customer support � Convenient online submission � Thorough peer review � Inclusion in PubMed and all major indexing services � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Infectious Diseases Springer Journals

Prevalence and molecular characteristics of Staphylococcus aureus, including methicillin resistant strains, isolated from bulk can milk and raw milk products in pastoral communities of South-West Uganda

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Springer Journals
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Copyright © 2017 by The Author(s).
Subject
Medicine & Public Health; Infectious Diseases; Parasitology; Medical Microbiology; Tropical Medicine; Internal Medicine
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1471-2334
DOI
10.1186/s12879-017-2524-4
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28610560
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Abstract

Background: Staphylococcus aureus strains are now regarded as zoonotic agents. In pastoral settings where human-animal interaction is intimate, multi-drug resistant microorganisms have become an emerging zoonotic issue of public health concern. The study of S. aureus prevalence, antimicrobial resistance and clonal lineages in humans, animals and food in African settings has great relevance, taking into consideration the high diversity of ethnicities, cultures and food habits that determine the lifestyle of the people. Little is known about milk carriage of methicillin resistant S. aureus strains (MRSA) and their virulence factors in Uganda. Here, we present the prevalence of MRSA in bulk can milk and raw milk products in pastoral communities of south-west Uganda. We also present PFGE profiles, spa-types, as well as frequency of enterotoxins genes. Methods: S. aureus was identified by the coagulase test, susceptibility testing by the Kirby-Bauer disc diffusion and E-test methods and MRSA by detection of the mecA gene and SCCmec types. The presence of Panton – Valentine Leucocidin (PVL) genes and staphylococcal enterotoxins was determined by PCR, while genotyping was by PFGE and spa typing. Results: S. aureus were isolated from 30/148 (20.3%) milk and 11/91(12%) sour milk samples. mecA gene carriage, hence MRSA, was detected in 23/41 (56.1%) of the isolates, with 21 of the 23 (91.3%) being SCCmec type V; while up to 30/41 (73.2%) of the isolates were resistant to tetracycline. Only five isolates carried the PVL virulence gene, while PFGE typing revealed ten clusters (ranging from two seven isolates each) that comprised 83% of the sample, and only eight isolates with unique pulsotypes. The largest PFGE profile (E) consisted of seven isolates while t7753, t1398, and t2112 were the most common spa-types. Thirty seven of the 41 strains (90.2%) showed at least one of the eight enterotoxin genes tested, with sem 29 (70.7%), sei 25 (61%) and seg 21 (51.2%) being the most frequently observed genes. Conclusion: This is the first study to demonstrate MRSA and enterotoxin genes in raw milk and its products in Uganda. The fact that over 90% of the isolates carried at least one gene encoding enterotoxins shows a high risk of spread of foodborne diseases through milk in this setting. * Correspondence: basiimwe@chs.mak.ac.ug Emerging Bacterial Pathogens Unit, IRCCS, Via Olgettina 58, Milan, Italy Universita Vita-Salute San Raffaele, Via Olgettina 58, Milan, Italy Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 2 of 8 Background being the heat-stable enterotoxins that cause the sporadic Staphylococcus aureus strains, especially those resistant food-poisoning syndrome or foodborne outbreaks [15]. to methicillin (methicillin resistant S. aureus, MRSA) Staphylococcal enterotoxins (SEs) cause toxic shock-like have been regarded as zoonotic agents and there is syndromes and have also been implicated in several growing genuine concern about the likely transmission allergic and autoimmune diseases, in addition to food poi- of MRSA between animals and humans from close inter- soning; therefore SEs function not only as potent gastro- action or from handling and/or consuming MRSA intestinal toxins but also as superantigens that stimulate infected animal products [1–5]. In settings where humans non-specific T-cell proliferation [16]. depend on animals and their products for food and liveli- Theaim of thepresent studywas to establishthe hood, such as in pastoral Africa, contact is intimate and prevalence and distribution of methicillin-susceptible multi-drug resistant microorganisms have become an S. aureus (MSSA) and MRSA in bulk can milk and emerging veterinary and zoonotic issue of public health raw milk products samples at household level. Fur- concern [6–8]. Additionally, S. aureus is known to be the thermore, we tested the susceptibility of the isolates third most reported cause of food-borne diseases in the to nine drugs and analyzed the genetic relatedness of world [4], culpable for production of staphylococcal theisolatesbyPFGE and spa typing. Additionally, enterotoxins that cause food poisoning. Moreover, con- resistance and virulence genes (SCCmec and PVL)as taminated milk and milk products have frequently been well as frequency of genes encoding three of the clas- implicated in staphylococcal food poisoning in different sical staphylococcal enterotoxins (SEs): SEA, SEB and industrialized countries [9]. The study of S. aureus preva- SEC; and five additional enterotoxins: SEG, SEH, SEI, lence, antimicrobial resistance and clonal lineages in SEL, and SEM were assayed. humans, animals and food in African settings has great relevance, taking into consideration the high diversity of Methods ethnicities, cultures and food habits that determine the Study area lifestyle of the people. The study was carried out in Sanga and Kanyaryeru sub- The treatment of bacterial infections in Uganda’s counties of Kiruhura district, South Western Uganda. health and veterinary care settings is largely empirical The area is a rangeland populated by agro-pastoralist with no laboratory confirmation to guide therapy, and households whose livelihoods are mainly dependent on antibiotics for both human and veterinary use are readily consumption and trade of cattle and their products, and available over the counter. This continuous use and the area is part of the cattle corridor and is considered misuse of the antibiotics has resulted into a surge in the milk basin of Uganda. multidrug microorganisms, now a growing problem on farms, in health care settings and the community Study design and sampling [7, 10, 11]. In Uganda, pastoralist communities are A total of 196 homesteads from the two sub-counties vulnerable to zoonotic infections, including MRSA, (100 from Kanyaryeru and 96 from Sanga) were ran- not only because of the weak veterinary/public health domly recruited and sampled. One pooled (bulk can) delivery systems but also due to their cherished way milk sample and a raw milk product (cow ghee (butter of life (culture) dependent on animals and their prod- from milk) or sour (fermented) milk) were collected ucts. There is limited data about prevalence and from each household on consent and when available at spread of S. aureus strains in rural pastoral farm the time of visit, from July to August 2013. In Kanyar- environments in Uganda. Previous studies have reported yeru sub-county, 81 milk samples, 60 ghee samples and on bacterial carriage of milk in peri and urban farmers in 53 sour milk samples were collected; while 67milk and around the capital, Kampala [7], while another samples, 57 ghee samples and 38 sour milk samples reported on burden of S. aureus infection on dairy farms were collected from Sanga sub-county. In all, 148 milk in the neighboring district of Kiboga [6]. However these samples, 117 ghee samples and 91 sour milk samples studies neither assessed resistance to methicillin nor were collected for the study. Samples were inoculated further characterized the isolates for resistance and viru- into Brain-Heart Infusion (BHI) broth in 15 ml propyl- lence markers as well as genetic relatedness and spread of ene tubes and stored on ice in any case for not more the strains in the study environments. than 12 h before transportation to the Department of In Uganda, 92% of the milk is marketed unregulated Microbiology at Mbarara University of Science and and in raw form [12]. This practice increases the risk of Technology for culture and identification. spread of staphylococcal food poisoning due to poor stor- age and transportation conditions. S. aureus is among the Culture, isolation and identification leading causes of foodborne bacterial intoxications world- Initial bacterial culture and isolation was done according wide [13, 14], with the most notable virulence factors to methods described by Cheesbrough [17], with minor Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 3 of 8 modifications. Briefly, 50 μl of culture broth was inocu- Pulsed-field gel electrophoresis (PFGE) and spa typing lated on 5% sheep blood agar medium and incubated for All isolates were subjected to molecular epidemiological 18 - 20 h at 37 °C. Preliminary identification of the analysis by PFGE after SmaI digestion using the CHEF bacteria was carried out based on colony characteristics Genomic DNA Plug kit (Bio-Rad) according to a stan- such as shape, size, color and hemolysis patterns. dardized protocol [21]. PFGE was run using a CHEF Isolated suspected of being S. aureus were shipped in DRIII system (Bio-Rad). The InfoQuest FP (v5) software BHI with 20% glycerol to the Emerging Bacterial Pathogens (Bio-Rad Laboratories) was used to analyze PFGE pro- Unit (EBPU) at San Raffaele Scientific Institute in Milan, files, according to interpretation criteria described by Italy, for identification and molecular characterization. At Tenover et al. [22]. Clustering analysis was achieved the EBPU, all isolates were sub-cultured on mannitol salt using Dice similarity coefficients and the unweighted agar (Oxoid Ltd., Hampshire, England), then on blood agar pair group method with averages (UPGMA) at 1.5% (Becton Dickinson, Heidelberg, Germany), and subjected optimization and 1.5% position tolerance. Addition- to the tube coagulase test in rabbit plasma with EDTA ally, isolates were typed based on sequencing of the (Remel, KS, USA). Only isolates that passed the coagu- hypervariable region of the S. aureus protein A gene (spa); lase test were considered for further characterization. spa types were assigned MLST sequence types (ST) in- Isolates were also screened for susceptibility to eight ferred on the Ridom spaServer (http://spaserver.ridom.de) antibacterial agents (cefoxitin, tetracycline, gentamy- curated by the SeqNet.org initiative [23]. cin, ciprofloxacin, rifampicin, erythromycin, clindamy- cin, Sulfamethoxale/Trimethoprim) on Mueller-Hinton Staphylococcal enterotoxins genes detection by PCR agar (Oxoid Ltd., Hampshire, England) according to DNA amplification was performed using the following Kirby-Bauer disc diffusion method and two (oxacillin and conditions: initial denaturation for 15 min at 95 C vancomycin) by the E-test (Oxoid Ltd., Hampshire, followed by 35 cycles of denaturation (94 C for 1 min), England). Screen tests were performed in 0.5 McFarland annealing, and extension (72 C for 1 min), and a final according to EUCAST guidelines (http://www.eucast.org/ extension step (72 C for 10 min) after the completion clinical_breakpoints/). of the cycles. The primer sequences, annealing tempera- tures and expected amplicon sizes are as described in Jarraud et al. [24] and Diep et al. [25]. DNA extracted DNA extraction, SCCmec typing and PVL genes detection from the standard strains S. aureus Mu50 [26] was used Bacterial DNA was prepared from isolated colonies as positive control for enterotoxins SEA, SEC, SEG, SEI, suspended in 500 ml Triton X-100 lysis buffer with SEL and SEM; while DNA from S.aureus COL [27] 1% Triton and 50 mg/ml lysostaphin, incubated at controlled for enterotoxins SEB and SEH. 37 °C for 1 h, followed by phenol-chloroform extrac- tion. Staphylococcal Cassette Chromosome harboring Results mecA (SCCmec) gene multiplex PCR typing was done. Prevalence of MSSA and MRSA This was performed using primers and protocols for Of the samples that were collected, only 56 of 148 milk SCCmec types and subtypes I, II, III, IVa, IVb, IVc, samples, 25 of 91 sour milk samples and one of 117 cow IVd, and V and the mecA gene according to Zhang et ghee samples gave growth that was suspected to be S. al.[18]. We also assayed for the recently described aureus based on colony characteristics such as size, mecA homologue, mecC shown to be common in shape, color and hemolysis patterns on blood agar. animal-derived strains, using primers and conditions These samples were then sub-cultured and re-identified as described in Paterson et al. [19]. The MRSA strain at the EBPU and only 30 of 56 isolates from milk and 11 COL (SCCmec type I, mec gene complex B and ccr gene of the 25 isolates from sour milk were coagulase posi- complex 1) was also used as positive control. We further tive, while the single isolate from ghee failed the coagu- used control strains for SCCmec II (Mu50), III (ANS46), lase test. The prevalence of S. aureus in the bulk can IVa (USA300-FPR3757), IVb (strain from Ospedale San milk thus was 30/148 (20.3%) while that in the sour milk Raffale (OSR) MRSA 864), IVc (strain from OSR collec- was 11/91 (12.1%). Isolation of S. aureus was unevenly tion, MRSA 111), IVd (strain from OSR collection, MRSA distributed between the two study areas, with 25 isolates 1040) and V (S0385). Unfortunately a control for SCCmec from Kanyaryeru and 16 isolates from Sanga. type IV from subtype “e” to subtype “k” was not available to us. Additionally, amplification of the Panton – Valen- Drug susceptibility, mecA, SCCmec and PVL gene tine Leucocidin (PVL) toxin genes, lukS-PV and lukF-PV, Of the 356 samples cultured (148 fresh milk, 91 sour was performed using the S. aureus strain ATCC49775 as milk and 117 ghee), S. aureus was isolated from 30 positive control, and primers and protocols as described (53.6%) of milk, 11 (44%) of sour milk and none were by Lina et al. [20]. recovered from ghee samples. Carriage of the mecA gene Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 4 of 8 (hence MRSA) in this study, identified as part of the isolates (24.4%); while in the other enterotoxins, sem Staphylococcal Cassette Chromosome harboring mecA (29/41; 70.7%), sei (25/41; 61%) and seg (21/41; 51.2%) (SCCmec) after multiplex PCR, was 50% (15/30) and were the most frequently observed genes. The propor- 72.7% (8/11) of the milk and sour milk derived isolates tion of all the other enterotoxins can be seen in Table 3. respectively. Overall, only five isolates (5/41, 12.2%) car- Only four isolates (two from raw milk and two from ried the PVL virulence gene. A majority of the isolates sour milk) did not carry any of the eight enterotoxin with the mecA gene (21/23; 91.3%) were type V. Nosne genes that were assayed. of the isolates tested carried the mecA homologue, mecC. When screened with cefoxitin, only two of the 23 Discussion isolates carrying the mecA gene had zones of inhibition Pastoralists within Uganda’s cattle corridor are afflicted ≤22 mm, hence a discrepancy between the phenotypic by a variety of ecosystem-based human/livestock health and genotypic results. The two isolates resistant to problems, especially those zoonotic in nature, with cefoxitin also had MICs of oxacillin above the break- potential to spill over to neighboring communities and point of 2 mg/L, hence resistant. There was a high affecting the tourism industry. Unfortunately, not much resistance to tetracycline (73.2%), while all isolates were has been done to estimate the burden of many such fully susceptible to ciprofloxacin and vancomycin. The potentially zoonotic infections in these marginalized susceptibility pattern of the other drugs tested is shown communities. S. aureus, including MRSA, has recently in Table 1. been found to be a zoonotic problem in different settings [2, 3, 28]. We set out to study the prevalence of PFGE restriction profiles and spa types methicillin resistance, virulence factors, and genetic There were ten clusters (ranging from two to seven iso- relatedness of S. aureus strains isolated from bulk can lates each) that comprised 83% of the sample, while only milk and milk products from pastoral households eight isolates had unique pulsotypes (Table 2). The around L. Mburo national Park. largest PFGE profile (E) consisted of seven isolates, all S. aureus was isolated in 20.3% of bulk can milk sam- with unknown spa types. The most common spa types ples and 12.1% of sour milk samples. Generally, there is were t7753 and t1398 (four isolates each), while others a paucity of data on S. aureus from milk and raw milk were t2112 (three isolates), t3992 and t127 (two isolates products in the region. In urban and peri-urban each). The PFGE profiles and spa types of the isolates Kampala, a recent study of 97 milk samples yielded 85 are shown in Table 2. pure cultures, but only 1 of 58 (2%) Gram-positive bac- teria was identified as S. aureus while 20 of 58 (34%) Frequency of staphylococcal enterotoxins (SEs) were coagulase negative Staphylococci [7]. Another Results for the detection of eight genes encoding the study carried out in dairy farms in rural Kiboga (another enterotoxins SEA, SEB, SEC, SEG, SEH, SEI, SEL, and cattle corridor district) in Uganda, however, found that SEM are shown in Table 2, in which 37 of 41 strains the most prevalent bacterial pathogens associated with (90.2%) were positive for at least one enterotoxin gene. sub-clinical mastitis in dairy cattle were Staphylococci The distribution of enterotoxin genes in MRSA and such as Coagulase negative Staphylococci and S. aureus MSSA strains as well as in milk and sour milk samples in 64.42 and 16.56% of the cases respectively [6], com- was unequal, with MSSA and milk-derived isolates parable to our finding of 19.6% S. aureus in fresh bulk showing more strains with multiple enterotoxins. There can milk samples in the current study. These findings were 17/30 (56.7%) milk-derived strains carrying at least support the expectation that S. aureus from infected three enterotoxins genes while only 3/11(27.3%) of iso- udders may contaminate bulk milk and, subsequently, lates from sour milk carried more than three. Among raw milk products as has been observed elsewhere [29]. the genes that code for three of the classic enterotoxins SCCmec PCR showed that 23/41 (56.1%) of the assayed (SEA-SEC), sec was the most frequent with 10 isolates carried the mecA gene implicated in resistance Table 1 Drug susceptibility pattern of the study isolates to eight of the 10 antimicrobial agents tested Source S. aureus Drug susceptibility pattern TE RD SXT CIP E CN DA VA R S R S RS RS RS R S RS RS Milk 30 20 10 1 29 1 29 0 30 1 29 1 29 2 28 0 30 Sour milk 11 10 1 0 11 1 10 0 11 0 11 0 11 0 11 0 11 Total 41 30 11 1 40 2 39 0 41 1 40 1 40 2 39 0 41 TE tetracycline, RD rifampicin, SXT trimethoprim-sulphamethoxale, CIP ciprofloxacin, E erythromycin, CN gentamycin, DA clindamycin, VA vancomycin Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 5 of 8 Table 2 Molecular characteristics of the 41 isolates of S. aureus Table 3 Genotypic profile of S. aureus strains, according to sea, in the study seb, sec, seg, seh, sei, sel, and sem genes Isolate ID Pulsotype spa type MLST PVL mecA SCCmec Genotypic profile Raw milk Sour milk Total (%) (n = 30) (n = 11) T130 m A1 t1398 −− − sea 1 1 2 (4.9) T132 m A1 t2112 − +V seb 4 2 6 (14.6) T111 m A2 t380 + −− Sec 7 3 10 (24.4) T119 m A3 t7753 −− − Seg 16 5 21 (51.2) T147y A4 t2112 − +V She 13 2 15 (36.6) T067 m B1 t127 ST-1 − +V Sei 17 8 25 (61) T089y B1 t127 ST-1 − +V Sel 8 2 10 (24.4) T120y C1 t1236 ST-97 −− − Sem 21 8 29 (70.7) T139 m C1 t7753 −− − sea + seb + sec 3 0 3 (7.3) T115 m C2 t10103 −− − seg + seh + sei + sel + sem 9 1 10 (24.4) T089 m C3 t7753 −− − At least 1 SE gene 28 9 37 (90.2) T099y C4 t14299 − +V No SE gene detected 2 2 4 (9.8) T041y D1 Unknown − +V T042 m D2 Unknown −− − to methicillin. Elsewhere in Africa, however, low level T127 m D3 Unknown − +V MRSA carriage in raw milk has been reported in Algeria T031 m E1 Unknown − +V where only two of five isolates were screened by the disc T117 m E2 Unknown −− − diffusion test, but there was no testing for the mecA T136 m E3 Unknown − +V gene [30]. In the current study, only two isolates had zones of clearance less than 22 mm around cefoxitin T105 m E4 Unknown −− − discs as recommended by EUCAST, to be considered T143 m E4 Unknown −− − MRSA. The two isolates also had MIC of >2 mg/L on T094 m E5 Unknown −− − oxacillin strips, hence resistant. The discrepancy T148 m E5 Unknown − +V between the phenotypic and genotypic results seen in T019 m F1 t645 ST-121 + + V this study may be due to a lack of expression of mecA. T057 m F1 Unknown − +V Routine oxacillin tests often fail to detect very heteroge- neous MRSA populations, which consequently are consid- T103 m G1 t1398 − +V ered methicillin-susceptible S. aureus (MSSA) because of T145 m G1 t1398 − +V their usual susceptibility to most non-β-lactam antistaphy- T030 m G2 t1398 −− − lococcal antibiotics. Therefore, several parameters have T098y H1 t186 ST-88 −− − been recommended to improve results and these include T120 m H2 Unknown − +V increasing the inoculum, growth at a low temperature, an T037 m I1 Unknown − +V oxacillin screen test with NaCl, or protracted incubation [31]. More recent methods for detection of MRSA include T067y I2 Unknown − +V the oxacillin E-test (AB BIODISK, Solna, Sweden) for T059y L1 t2112 + + V determination of MICs, and the automated Vitek 2 system T091 m L2 t1236 ST-97 −− − (bioMe’rieux, La Balme les Grottes, France). Phenotypic T032 m Unique Unknown + + IV results will therefore vary between laboratories depending T037y Unique Unknown − +V on the system used. Detection of the mecA gene, there- T049 m Unique Unknown −− − fore, would be a good way of standardizing identification of MRSA across laboratories. The results are similar to T055 m Unique Unknown −− − those in another study comparing methods for the detec- T056 m Unique t3992 ST-97 − +V tion of MRSA on isolates from foods of animal origin in T055y Unique t3992 ST-97 −− − Italy by Corrente et al. [31]. In that study, none of the six T144 m Unique t7753 − + IVc MRSA strains identified by mecA PCR were detected by T157y Unique t3772 + + V the cefoxitin test (sensitivity of 0%), and they concluded PVL = presence (+) or absence (−)of the PVL gene; mecA = presence (+) or that analysis for MRSA in isolates from food of animal ori- absence (−)ofthe mecA gene; SCCmec = SCCmec-types according to gin is better done with the mecA gene-specific PCR rather Zhang et al. [18] than conventional phenotypic assays [31]. More studies in Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 6 of 8 human derived strains also render support to low sensitiv- t2748, t2971, t2974, t3013, t3014, t3053, t3146, or t3208) ity of the cefoxitin test. For example, a recent study of low according to the recent work [37] were not found among level methicillin resistance in mecA positive S. aureus our isolates. However, we detected other spa types that strains showed zones of clearance of 28 mm diameter by have been reported to be associated with LA-MRSA cefoxitin testing [32]. In the current study, 17 of the 23 strains other than ST398 (Table 2). mecA positive strains also had zones of clearance of This, to the best of our knowledge, is the first report ≤28 mm (Table 1). The observation of a high proportion of SEs in milk from Uganda. While literature elsewhere of MRSA in the study sample is very worrisome because suggests that the classical SEA is the most frequently studies have shown that MRSA readily jumps from ani- observed in enterotoxigenic strains of S. aureus [38], it mals to exposed veterinarians, farm works and other farm was found in only two (4.9%) of the strains in the animals [2, 5, 28]. This, to the best of our knowledge, is current study. This is probably because of the fact the first report of MRSA isolated from milk and raw milk that different foods and strains carry different entero- products under communally grazing pastoral households toxins, as observed elsewhere [39]. On the other in Uganda. In this setting where animal – human inter- hand, the more recently discovered seg gene was action is intimate due to the pastoralists’ cherished way of observed in 21 (51.2%) of 41 isolates, and in 85.7% of life, it creates an urgent public health concern. the cases, it was associated with sei,which was Genotype analysis was done by SCCmec typing and present in 25 (61%) of the isolates, similar to reports PFGE. Of the 23 mecA positive (MRSA) isolates, 21 in other studies in Brazil [40] and France [41]. These harboured the SCCmec type V. The other SCCmec types genes are known to be frequently found together found can be seen in Table 2. It has recently been noted because they are within the same cluster, in a 3.2 kb that community-associated MRSA (CA-MRSA) isolates, DNA fragment as reported elsewhere [42]. In East which carry SCCmec type IV or V, are now prevalent Africa, most milk is produced by small holders and and exceed methicillin-susceptible S. aureus (MSSA) in the bulk of it (86% in Kenya and 92% in Uganda) is skin and soft tissue infections, and such isolates were traded through unregulated channels as unpasteurized reported to cause severe, often necrotizing, soft tissue milk or milk products [12]. Raw and sour milk are infections and pneumonia [33, 34]. Their presence in consumed in these settings for such beliefs as milk creates risk of spread of these genotypes in the enhanced nutritional quality and better taste. The fact communities. Additionally, assaying for the virulence that over 90% of the isolates in the current study car- marker PVL showed that only four isolates were positive ried at least one gene encoding for enterotoxins for this gene. A previous study of 50 S. aureus isolates shows a high risk of spread of foodborne diseases in from 14 dairy cow herds in Italy identified the PVL this setting, with milk as a vehicle for dispersion. gene in more than 50% of the isolates [35], while There is a need for farmer education and health pro- another in Brazil reported that none of the 84 milk motion messages in the communities about the need derived S. aureus isolates harbored the PVL gene [36]. for pasteurization of milk as this will kill S. aureus, According to criteria proposed by Tenover et al., [22], therefore reducing enterotoxin production. strains that differ by three bands or fewer by PFGE are likely to be closely related. The authors empha- sized that these criteria apply to epidemiologically Conclusion related isolates. By these criteria, isolates in the Our results show a high proportion of MRSA in raw current study group into ten clusters of two to seven milk and its products in the cattle corridor of isolates each, and only eight unique strains. Since Uganda, threatening treatment options for S. aureus these were not outbreak strains, spa typing was used in the near future in this setting, especially if the to determine the clonal lieneages of the isolates. The strains cross the species barrier into humans. Isolates most common lineages were t7753 and t1398 with carrying SCCmec type V are wide spread in this set- four isolates each. In all, there were 13 different line- ting. The fact that over 90% of the isolates in the ages, but most of the isolates (17/41, 41.5%) could current study carried at least one gene encoding for not be assigned to any lineage and were designated as enterotoxins shows a high risk of spread of foodborne unknown (Table 2). Only one lineage in the current diseases. A limitation of this study is that the entero- study, t645, had been isolated from one mastitic cow toxin genes were not studied for expression, therefore and four milkmen in peri-urban Kampala [7]. The we cannot confirm their significance in cases of food most common spa types associated with clone ST398 poisoning. Additionally, the initial processing and (t011, t034, t108, t567, t571, t588, t753, t753, t779, culture technique for the samples was not good t898, t899, t943, t1184, t1197, t1254, t1255, t1451, enough whereby only 82 of the 356 samples yielded t1456, t1457, t2123, t2287, t2329, t2330, t2383, t2582, suspected S. aureus. Asiimwe et al. BMC Infectious Diseases (2017) 17:422 Page 7 of 8 Acknowledgments 8. Kamau NPM, Dolci S, Jans C, Wangoh J, Lacroix C, et al. Phenotypic and The authors wish to thank the households that participated in the study. genotypic antibiotic resistance patterns of Staphylococcus aureus from raw and We also thank Dr. F. Agnoletti and Dr. I. Drigo from the Istituto Zooprofilattico spontaneously fermented camel milk. Eur J Nutr Food Safety. 2013;3:87–98. Sperimentale delle Venezie, Italy, who kindly provided us with control 9. De Buyser ML, Dufour B, Maire M, Lafarge V. Implication of milk and milk strain LGA251. products in food-borne diseases in France and in different industrialised countries. Int J Food Microbiol. 2001;67:1–17. Funding 10. Kateete DP, Namazzi S, Okee M, Okeng A, Baluku H, et al. High prevalence of methicillin resistant Staphylococcus aureus in the surgical units of Mulago BBA was supported by a Marie Curie Fellowship (MARIE CURIE – COFUND hospital in Kampala, Uganda. 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Egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J Immunol. 2001;166:669–77. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries � Our selector tool helps you to find the most relevant journal � We provide round the clock customer support � Convenient online submission � Thorough peer review � Inclusion in PubMed and all major indexing services � Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit

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Published: Jun 13, 2017

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