Faecal colonization of E. coli and Klebsiella spp. producing extended-spectrum beta-lactamases and plasmid-mediated AmpC in Mozambican university students

Faecal colonization of E. coli and Klebsiella spp. producing extended-spectrum beta-lactamases... Background: In recent years, the world has seen a surge in Enterobacteriaceae resistant to broad-spectrum beta- lactam antibiotics due to the production of extended-spectrum beta-lactamases (ESBLs) or plasmid-mediated AmpC (pAmpC) enzymes. Data on the epidemiology of cephalosporin-resistant Enterobacteriaceae in Sub-Saharan Africa are still limited. Methods: Two hundred seventy-five non-repetitive stool samples were collected from Mozambican university students of both sexes. Samples were cultured on MacConkey agar with and without ceftriaxone (1 mg/L) for selection of third-generation cephalosporin-resistant isolates, which were subjected to antimicrobial susceptibility testing by disc diffusion, characterization of resistance genes by PCR and ERIC-PCR analysis for strain clonality. Results: Among the 275 students, 55 (20%) carried a total of 56 E. coli (n =35) and Klebsiella spp. (n = 21) isolates resistant to ceftriaxone and phenotypically positive for ESBL- and/or pAmpC-production. Forty-three percent of the isolates (24/56) contained only ESBL genes, 11% (6/56) only pAmpC genes, and 36% (20/56) both ESBL and pAmpC genes. The remaining six isolates were negative for the CTX-M/pAmpC genes included in the test panel. E. coli and Klebsiella spp. combined demonstrated 70% resistance to tetracycline and co-trimoxazole, 63% to ceftazidime and 34% to ciprofloxacin. In total, 89% of ESBL/pAmpC-positive isolates were defined as multi-resistant by being resistant to three or more antibiotic classes. ERIC-PCR fingerprinting demonstrated low similarity among isolates. None of the participants reported recent hospitalization and just 12.5% had taken antibiotics 3 months prior to the study. Conclusion: This study demonstrated 20% colonization with multi-resistant E. coli and Klebsiella spp. among Mozambican students with a diversity of ESBL and pAmpC genes. Colonization was not related to prior hospitalization or antimicrobial consumption. Keywords: E. coli, Klebsiella, ESBL, pAmpC, Colonization, Students, Mozambique * Correspondence: gunnar.skov.simonsen@unn.no Antimicrobial Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, South Africa Research Group for Host-Microbe Interaction, Institute of Medical Biology, Faculty of Health Sciences, UiT – Arctic University of Norway, Tromsø, Norway Full list of author information is available at the end of the article © The Author(s). 2018 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. Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 2 of 8 Background South Africa). A total of 56 E. coli (n = 35) and Klebsiella In recent years, the world has seen a surge in Enterobac- spp. (n = 21) isolates were identified by this procedure. teriaceae resistant to broad-spectrum beta-lactam antibi- Two isolates were recovered from the same sample. These otics attributed to the production of extended-spectrum putative ESBL- and/or pAmpC-producers constituted the beta-lactamases (ESBLs) and/or plasmid-mediated study sample and were subjected to antimicrobial suscep- AmpC (pAmpC) enzymes [1, 2]. ESBLs are, by the tibility testing by disc diffusion to the following antibiotics: classical definition, inhibited by clavulanic acid [3] cefoxitin, ceftazidime, ceftriaxone, imipenem, amikacin, whereas pAmpC enzymes are not [4]. The plasmid loca- gentamicin, ciprofloxacin and co-trimoxazole. The results tion of ESBL and pAmpC genes facilitates their spread were interpreted according to the CLSI breakpoints to via horizontal gene transfer [5, 6]. determine their susceptibility profile [18]. Multi-resistance Escherichia coli and Klebsiella pneumoniae are not was defined as resistance to three or more antibiotic only constituents of the commensal gut flora but also classes. E. coli ATCC 25922 (wild-type) and K. pneumoniae common opportunistic pathogens often implicated in ATCC 700603 (ESBL positive) were used as negative urinary tract and bloodstream infections [7, 8]. They and positive quality control strains for antimicrobial frequently harbor ESBL- and pAmpC-encoding genes. susceptibility testing and ESBL screening, respectively. Broad-spectrum beta-lactamase production is associated with increased morbidity and mortality in both high- and low/middle-income countries [9, 10]. Phenotypic and genotypic characterization of beta- Data on the epidemiology of ESBL- and pAmpC-- lactamases producing Enterobacteriaceae in Africa are still limited. ESBL production was confirmed by double disc synergy The majority of publications report on the prevalence of using ROSCO discs (Rosco Diagnostic, Taastrup, ESBL-producing Enterobacteriaceae in clinical samples, Denmark) containing ceftazidime, ceftriaxone and and there are only few studies on colonization [11, 12]. amoxicillin/clavulanic acid. pAmpC production was Furthermore, there is a predominance of reports from determined using ROSCO discs containing cefotaxime, Northern and Western Africa [13, 14] and only indi- cefotaxime+boronic acid, ceftazidime and ceftazidime vidual studies from Eastern and Southern Africa [15– +boronic acid [18]. Isolates with reduced susceptibility 17] (excluding South-Africa). The aim of this study to carbapenems were subjected to the Carba NP test as was to determine the prevalence of fecal colonization described previously [19]. E. coli ATCC 25922 and K. with ESBL- and pAmpC-producing E. coli and Klebsiella pneumoniae ATCC 700603 were used as negative and spp. among healthy university students in Maputo, positive quality control strains, respectively. Mozambique, and to analyze the resistance phenotype, For DNA extraction, 18–24 h colonies grown on ESBL- and pAmpC resistance gene content and clonal Muller Hinton agar were inoculated into Luria-Bertani relatedness of isolates. (LB) broth and incubated at 37 °C with shaking [20]. After 20 h of incubation, DNA was extracted using the Methods ZR Fungal/Bacterial DNA MiniPrep kit (Zymo Research, Study sample Lithuania). PCR amplifications for detection of bla , TEM A random sample of 275 university students of both bla , bla , bla , bla , bla and bla SHV CTX CMY DHA FOX MOX sexes living in three residences at the Eduardo (Table 1) were performed in a ThermalCycler T100™ Mondlane University in Maputo, Mozambique, provided (Bio-Rad, USA) with a final volume of 50 μL (25 μLof non-repetitive stool specimens within a six-week period, Master mix, 15 μL of water, 4 μL of each primer with a from January to February 2016 upon informed, voluntary final concentration of 0.8 μM (Inqaba Biotechnology consent. Students with diarrheal disease were excluded Industries, South Africa) and 2 μL of template DNA), from the study. Participants provided information about with an initial denaturation temperature of 98 °C for antibiotic use within 3 months and hospitalization within 10 s, extension at 72 °C for 15 s and a final extension at 6 months prior to the study. Stool samples were kept on 72 °C for 1 min. The annealing temperatures were as ice and transported to the laboratory for immediate follows: bla 60 °C, bla 56 °C, bla 57 °C, TEM SHV CTX culture twice daily. bla 57 °C, and bla , bla and bla 50 °C. CMY DHA FOX MOX PCR products were loaded on a 1.5% (w/v) agarose gel Identification and susceptibility testing and visualized by UV transillumination (Bio-Rad Chemi- All samples were cultured on MacConkey agar with and Doc™MP System) after staining in 0.1 mg/mL Gel Red without ceftriaxone (1 mg/L) for selection of third-genera- for 15 min. A random sample of 19 CTX-M PCR tion cephalosporin-resistant isolates. Lactose-positive amplicons were sent to Inqaba Biotec (South Africa) for isolates growing on the 1 mg/L ceftriaxone agar were DNA sequencing. TEM and SHV amplicons were not identified to the species level using API20E (bioMérieux, sequenced. Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 3 of 8 Table 1 Primers used for amplification of ESBL genes Results Setting and samples Target Primers Sequence (5′ to 3′) Annealing enzyme temperature (ref) Among the 275 samples collected in the study, 159 TEM-1 TEMMF AAA ATT CTT GAA GAC G 60 °C [29] (58%) were retrieved from male students and 116 (42%) from females in an age range from 19 to 32 years old. TEMMR TTA CCA ATG CTT AAT CA The students lived in separate blocks and/or floors for SHV SHVMF TTA ACT CCC TGT TAG CCA 56 °C [29] male and female students. Each floor had one kitchen SHVMR GAT TTG CTG ATT TCG CCC where students could prepare their own food. There CTX-1 CTXMF GGT TAA AAA ATC ACT GCG TC 57 °C [27] were students from different courses including, but not CTXMR TTG GTG ACG ATT TTA GCC GC limited to Engineering, Medicine, Political Science, CMY CMYMF GAT TCC TTG GAC TCT TCA G 57 °C [28] Biology and Sociology. All participants declared a history of non-hospitalization 6 months prior to the study and CMYMR TAA AAC CAG GTT CCC AGA TAG C 87.5% had not consumed any antibiotics for at least FOX FOXMF CAC CAC GAG AAT AAC CAT 57 °C [28] 3 months. FOXMR ATG TGG ACG CCT TGA ACT DHA DHAMF AAC TTT CAC AGG TGT GCT GGG T 57 °C [28] Frequency of E. coli and Klebsiella spp. ESBL colonization DHAMR CCG TAC GCA TAC TGG CTT TGC In 55 of 275 non-repetitive stool samples 35 E. coli and MOX MOXMF GCT GCT CAA GGA GCA CAG GAT 50 °C [28] 21 Klebsiella spp. grew on MacConkey agar containing 1 mg/L ceftriaxone, with one sample providing two MOXMR CAC ATT GAC ATA GGT GTG GTG C isolates. The overall prevalence of ceftriaxone-resistant E. coli and Klebsiella spp. colonization was thus 20% Genomic DNA isolation and ERIC-PCR analysis (55/275). There was no statistically significant difference Genomic DNA was isolated and purified using the in the rate of colonization between females (n = 28, 24%) GeneJet Genomic DNA Purification Kit (Thermo and males (n = 28, 17%). All samples displayed growth Scientific, USA). Antibiotic-susceptible E. coli ATCC on the ceftriaxone-free growth control agar. 25922 and beta-lactam resistant SHV-18 K. pneumo- niae ATCC 700603 were used as quality controls. The Phenotypic analyses of antimicrobial susceptibility total PCR reaction volume was 10 μL, which con- All the 56 E. coli (n = 35) and Klebsiella spp. (n = 21) tained 100 ng of template DNA, 50 pmol of each isolates were phenotypically confirmed as ESBL and/or primer, 2.8 μL nuclease-free water and 5 μLof pAmpC producers and showed high rates of resistance DreamTaq PCR Mastermix (2X) (Thermo Scientific). to tetracycline and co-trimoxazole (70%), ceftazidime The primers ERIC 1 and ERIC 2 [21]wereused. PCR (63%), cefoxitin (41%), and ciprofloxacin (34%). Two conditions were as follows: 94 °C for 3 min, 30 cycles isolates displayed carbapenem zone diameters below the of 30 s of denaturation at 94 °C, 1 min of annealing screening breakpoint for carbapenemase production, but at 50 °C, 8 min of extension at 65 °C, and a final were categorized as susceptible by the clinical break- elongation of 16 min at 65 °C, in an Applied Biosys- point. They were subsequently negative in the Carba NP tems 2720 thermal cycler. The ERIC-PCR products test and there were thus no carbapenemase producers were loaded onto 1% (w/v) agarose gels and subjected (Table 2). Eighty-eight percent of the isolates were to electrophoresis at 80 V using 1 × TAE buffer. Amp- defined as multi-resistant. Antimicrobial susceptibility lification products were visualized by UV transillu- results are presented in the Additional file 1: Table S1. mination (Syngene, UK) after staining in 0.1 mg/mL ethidium bromide for 15 min. Genotypic variation ESBL gene identification by PCR and sequencing was analyzed using the Gel Compare II version 6.0 The PCR results for CTX-M and pAmpC genes are software package (Applied Maths, Belgium) by summarized in Table 3. Among 56 isolates, 41% were Jacquard index and Unweighted Pair Group Method positive for at least two genes, 32% were positive for at with Arithmetic Mean (UPGMA) cluster analysis to least three, and 23% were positive for at least four genes. produce a dendrogram. Forty-three percent (24/56) of the isolates contained only CTX-M, 11% (6/56) only pAmpC, and 36% (20/56) both CTX-M and pAmpC sequences. Six isolates (11%) Statistical analyses were negative for CTX-M and pAmpC genes but con- Groups were compared by the Fischer Exact Test tained TEM and/or SHV sequences (data not shown). using the Epi Info StatCalc software version 7.2.2.6 However, as these amplicons were not sequenced it can- (CDC, Atlanta, USA) with statistical significance not be concluded that they encode broad-spectrum defined as p <0.05. beta-lactamases and the basis for ceftriaxone resistance Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 4 of 8 Table 2 Antimicrobial resistance (%) of ESBL-producing E. coli products are the minor amplification products of low and Klebsiella spp intensity [22]. All isolates were typeable using this Antibiotic E. coli (n = 35) Klebsiella spp. (n = 21) fingerprinting technique and band profiles were repro- ducibly obtained under similar experimental conditions Ceftriaxone 100 100 on repeat amplification. Ceftazidime 71 43 The ERIC-PCR profiles allowed differentiation of the Gentamicin 14 43 35 E. coli isolates into 24 ERIC-PCR types which were Cefoxitin 46 33 grouped into 15 clusters (A-O), each of which were Tetracycline 66 76 futher sub-divided into multiple sub-clusters (Fig. 1). Co-trimoxazole 63 76 CTX-M and pAmpC genes were amplified from isolates in different clusters. Isolates with similar profiles demon- Ciprofloxacin 37 29 strated varying beta-lactamase gene content. Imipenem 0 0 ERIC-PCR results for Klebsiella spp. in these isolates was consequently not determined. A Twenty-one Klebsiella spp. isolates were subjected to total of 67 CTX-M/pAmpC genes were found in the 35 ERIC-PCR analysis in comparison to the SHV-18 con- E. coli isolates and 41 in the 21 Klebsiella spp. isolates. taining K. pneumoniae ATCC 700603. Distinct profiles CTX-M was the most common gene detected in both E. were obtained for all isolates tested using ERIC-PCR coli (n = 25) and Klebsiella spp. (n = 16) followed by fingerprinting (Fig. 2). Banding patterns comprised DHA (n =15 in E. coli and n =9 in Klebsiella spp.). between 2 and 16 individual bands. Fragments of differ- DNA sequencing of 19 randomly selected CTX-M ent molecular weights were observed in the ERIC-PCR amplicons from E. coli (n = 13) and Klebsiella spp. (n =6) fingerprints, ranging from 0.5–20 kb (Fig. 2). revealed a predominance of bla (n =14) and occa- The ERIC-PCR profiles allowed differentiation of the 21 CTX-M-15 sional detection of bla (n =3), bla (n =1) isolates into 17 ERIC-PCR types which were grouped into CTX-M-55 CTX-M-3 and bla (n = 1). A full list of CTX-M and pAmpC 12 clusters (A-L), each of which were further sub-divided CTX-M-186 gene content in individual strains is presented in the into multiple sub-clusters (Fig. 2). CTX-M and pAmpC Additional file 1: Table S1. genes were identified in isolates from different clusters, predominantly clusters B-G. Isolates with similar profiles ERIC-PCR results for E. coli demonstrated varying beta-lactamase gene content. Distinct ERIC-PCR profiles were obtained for the 35 E. coli isolates (Fig. 1). The absence or presence of a band Discussion was noted in determining variation among isolates, and Antibiotic-resistant bacteria are an escalating cause of banding patterns comprised between 2 and 14 individual infections in Mozambique and worldwide, but informa- bands. Fragments of different molecular weights were tion regarding the molecular epidemiology of ESBL- and observed in the ERIC-PCR fingerprints, ranging from pAmpC-producing Enterobacteriaceae in the country 0.5–20 kb (Fig. 1). Amplification of different intensities remains scarce. To our knowledge, this is the first study was observed and visual analysis of the ERIC profiles reporting on gastrointestinal colonization with ESBL- included primary, secondary and tertiary amplification and pAmpC-producing Enterobacteriaceae among (Fig. 1). Primary amplification products refer to those university students in Mozambique. products of high intensity, which appear extremely We systematically collected samples from healthy stu- bright on the gels. Secondary amplification products are dents to determine the prevalence of colonization by E. those products that are not as bright as the primary coli and Klebsiella spp. carrying ESBL and/or pAmpC, amplification products but more intense that the tertiary as well as their antibiotic susceptibility. In total, 20% of amplification products, while the tertiary amplification students (55/275) were colonized with ESBL and/or pAmpC positive E. coli and Klebsiella spp. Similar re- Table 3 Distribution of ESBL enzymes according to species sults were found in a study conducted in pre-school ESBL enzymes E. coli (n = 35) Klebsiella spp. (n = 21) Total children attending childcare facilities in the Lao People’s CTX-M 28 16 44 Democratic Republic where the ESBL prevalence was DHA 15 9 24 23% [23] while the prevalence amongst patients in inten- sive care in Korea was 28.2% with a higher frequency in MOX 10 7 17 E. coli (78%) compared with Klebsiella spp. (18%) [24]. A FOX 7 5 12 study recently conducted in Madagascar [18] in commu- CMY 7 4 11 nity settings demonstrated lower rates of colonization by TOTAL 104 57 161 ESBL-positive Gram-negative bacilli compared to this Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 5 of 8 Fig. 1 Dendrogram representing the genetic relatedness and cluster analysis of 35 E. coli strains isolated from stool samples of university students, based on ERIC-PCR fingerprinting patterns using Jacquard index and UPGMA algorithm. The scale at the top represents percentage similarity to E. coli ATCC 25922 study, despite the fact that more species were included facilities [27]. We found 36% co-existence of ESBL and in addition to E. coli and Klebsiella spp. The present pAmpC beta-lactamase genes which is high compared to study showed a higher prevalence of ESBL and pAmpC reports from clinical isolates in Turkey (13.9%) [28]. colonization compared to a study conducted in rural The CTX-M positive isolates carried different genotypes; communities in France where the frequency of ESBL bla (n =14), bla (n =3), bla CTX-M-15 CTX-M-55 CTX-M-186 colonization was 5.3% [25]. This large difference may be (n =1) and bla (n = 1). These results are similar to CTX-M-3 attributed to poorly controlled antibiotic consumption findings in a study conducted in Tanzania [29]that found and sub-optimal hygiene conditions in developing coun- bla genes in 95% of the carriers, but differ from a CTX-M-15 tries [26]. In Mozambique, antibiotic therapy is mostly study in Kenya [30] which demonstrated only 29% of the empirical because of scarce diagnostic microbiology isolates carrying bla and 4% carrying bla . CTX-M-15 CTX-M-3 Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 6 of 8 Fig. 2 Dendrogram representing the genetic relatedness and cluster analysis of 21 Klebsiella spp. strains isolated from stool samples of university students, based on ERIC-PCR fingerprinting patterns using Jacquard index and UPGMA algorithm. The scale at the top represents percentage similarity to Klebsiella pneumoniae ATCC 700603 No isolates harbored bla or bla in the The prevalence in different studies may vary depending CTX-M-55 CTX-M-186 Kenyan study. bla is endemic in many Asian on socio-economic status of individuals involved [32], CTX-M-55 countries, and the detection of this variant in Mozambique and this makes it difficult to estimate the prevalence in may reflect travel and trade with this part of the world [31]. the general population in Mozambique. One may In this study of healthy students, none of the partici- suppose that transmission between students is facilitated pants had a history of hospitalization 6 months prior to because of the conditions they live under with many the study, and 87.5% had not consumed any antibiotics at individuals sharing the same bathroom and kitchen. least 3 months prior to the study. This suggests that the However, the diversity of strains demonstrated using high antibiotic resistance rates are not related to antibiotic ERIC-PCR indicates that there is at present no wide- consumption or hospitalization, and may indicate that the spread clonal outbreak, although there could be horizon- E. coli and Klebsiella ESBL- and pAmpC-producers tal transfer of plasmids or other mobile genetic isolated in this study were acquired in the community. In elements. One may speculate that students are exposed the university residences there is a mixture of students to ESBL and/or pAmpC isolates from some external from different courses including medicine and health source like dissemination in the food supply. Very little sciences, but the study was not powered to analyse differ- is known about the occurrence of ESBL and pAmpC ences between student groups (data not shown). ESBL- strains in the food supply in Mozambique, but it is and/or pAmpC-colonized students working in health reported from other countries that ESBL and pAmpC E. institutions may constitute a reservoir for further spread coli and Klebsiella spp. may disseminate in food animals of multi-resistant microorganisms among patients. and environmental sources [33–37]. To control the rapid dissemination of resistant Entero- bacteriaceae among students and, consequently, the gen- Conclusions eral population, it is necessary to educate students about This study demonstrated a 20% prevalence of colonization the importance of personal hygiene. It is also necessary by multi-drug resistant, non-clonally-related ESBL- and/ to perform further studies to determine the prevalence or pAmpC positive E. coli and Klebsiella spp. isolates of colonization in different groups of the population. among male and female university students in Chirindze et al. 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Ewers C, Bethe A, Semmler T, Guenther S, Wieler LH. Extended-spectrum beta-lactamase-producing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective. Clin Microbiol Infect. 2012;18(7):646–55. 36. Zurfluh K, Nüesch-Inderbinen M, Morach M, Zihler Berner A, Hächler H, Stephan R. Extended-spectrum beta-lactamase-producing Enterobacteriaceae isolated from vegetables imported from the Dominican Republic, India, Thailand, and Vietnam. Appl Environ Microbiol. 2015;81(9):3115–20. 37. Stefani S, Giovanelli I, Anacarso I, Condò C, Messi P, de Niederhäusern S, Bondi M, Iseppi R, Sabia C. Prevalence and characterization of extended- spectrum beta-lactamase-producing Enterobacteriaceae in food-producing animals in Northern Italy. New Microbiol. 2014;37(4):551–5. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Infectious Diseases Springer Journals

Faecal colonization of E. coli and Klebsiella spp. producing extended-spectrum beta-lactamases and plasmid-mediated AmpC in Mozambican university students

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Medicine & Public Health; Infectious Diseases; Parasitology; Medical Microbiology; Tropical Medicine; Internal Medicine
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Abstract

Background: In recent years, the world has seen a surge in Enterobacteriaceae resistant to broad-spectrum beta- lactam antibiotics due to the production of extended-spectrum beta-lactamases (ESBLs) or plasmid-mediated AmpC (pAmpC) enzymes. Data on the epidemiology of cephalosporin-resistant Enterobacteriaceae in Sub-Saharan Africa are still limited. Methods: Two hundred seventy-five non-repetitive stool samples were collected from Mozambican university students of both sexes. Samples were cultured on MacConkey agar with and without ceftriaxone (1 mg/L) for selection of third-generation cephalosporin-resistant isolates, which were subjected to antimicrobial susceptibility testing by disc diffusion, characterization of resistance genes by PCR and ERIC-PCR analysis for strain clonality. Results: Among the 275 students, 55 (20%) carried a total of 56 E. coli (n =35) and Klebsiella spp. (n = 21) isolates resistant to ceftriaxone and phenotypically positive for ESBL- and/or pAmpC-production. Forty-three percent of the isolates (24/56) contained only ESBL genes, 11% (6/56) only pAmpC genes, and 36% (20/56) both ESBL and pAmpC genes. The remaining six isolates were negative for the CTX-M/pAmpC genes included in the test panel. E. coli and Klebsiella spp. combined demonstrated 70% resistance to tetracycline and co-trimoxazole, 63% to ceftazidime and 34% to ciprofloxacin. In total, 89% of ESBL/pAmpC-positive isolates were defined as multi-resistant by being resistant to three or more antibiotic classes. ERIC-PCR fingerprinting demonstrated low similarity among isolates. None of the participants reported recent hospitalization and just 12.5% had taken antibiotics 3 months prior to the study. Conclusion: This study demonstrated 20% colonization with multi-resistant E. coli and Klebsiella spp. among Mozambican students with a diversity of ESBL and pAmpC genes. Colonization was not related to prior hospitalization or antimicrobial consumption. Keywords: E. coli, Klebsiella, ESBL, pAmpC, Colonization, Students, Mozambique * Correspondence: gunnar.skov.simonsen@unn.no Antimicrobial Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban, South Africa Research Group for Host-Microbe Interaction, Institute of Medical Biology, Faculty of Health Sciences, UiT – Arctic University of Norway, Tromsø, Norway Full list of author information is available at the end of the article © The Author(s). 2018 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. Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 2 of 8 Background South Africa). A total of 56 E. coli (n = 35) and Klebsiella In recent years, the world has seen a surge in Enterobac- spp. (n = 21) isolates were identified by this procedure. teriaceae resistant to broad-spectrum beta-lactam antibi- Two isolates were recovered from the same sample. These otics attributed to the production of extended-spectrum putative ESBL- and/or pAmpC-producers constituted the beta-lactamases (ESBLs) and/or plasmid-mediated study sample and were subjected to antimicrobial suscep- AmpC (pAmpC) enzymes [1, 2]. ESBLs are, by the tibility testing by disc diffusion to the following antibiotics: classical definition, inhibited by clavulanic acid [3] cefoxitin, ceftazidime, ceftriaxone, imipenem, amikacin, whereas pAmpC enzymes are not [4]. The plasmid loca- gentamicin, ciprofloxacin and co-trimoxazole. The results tion of ESBL and pAmpC genes facilitates their spread were interpreted according to the CLSI breakpoints to via horizontal gene transfer [5, 6]. determine their susceptibility profile [18]. Multi-resistance Escherichia coli and Klebsiella pneumoniae are not was defined as resistance to three or more antibiotic only constituents of the commensal gut flora but also classes. E. coli ATCC 25922 (wild-type) and K. pneumoniae common opportunistic pathogens often implicated in ATCC 700603 (ESBL positive) were used as negative urinary tract and bloodstream infections [7, 8]. They and positive quality control strains for antimicrobial frequently harbor ESBL- and pAmpC-encoding genes. susceptibility testing and ESBL screening, respectively. Broad-spectrum beta-lactamase production is associated with increased morbidity and mortality in both high- and low/middle-income countries [9, 10]. Phenotypic and genotypic characterization of beta- Data on the epidemiology of ESBL- and pAmpC-- lactamases producing Enterobacteriaceae in Africa are still limited. ESBL production was confirmed by double disc synergy The majority of publications report on the prevalence of using ROSCO discs (Rosco Diagnostic, Taastrup, ESBL-producing Enterobacteriaceae in clinical samples, Denmark) containing ceftazidime, ceftriaxone and and there are only few studies on colonization [11, 12]. amoxicillin/clavulanic acid. pAmpC production was Furthermore, there is a predominance of reports from determined using ROSCO discs containing cefotaxime, Northern and Western Africa [13, 14] and only indi- cefotaxime+boronic acid, ceftazidime and ceftazidime vidual studies from Eastern and Southern Africa [15– +boronic acid [18]. Isolates with reduced susceptibility 17] (excluding South-Africa). The aim of this study to carbapenems were subjected to the Carba NP test as was to determine the prevalence of fecal colonization described previously [19]. E. coli ATCC 25922 and K. with ESBL- and pAmpC-producing E. coli and Klebsiella pneumoniae ATCC 700603 were used as negative and spp. among healthy university students in Maputo, positive quality control strains, respectively. Mozambique, and to analyze the resistance phenotype, For DNA extraction, 18–24 h colonies grown on ESBL- and pAmpC resistance gene content and clonal Muller Hinton agar were inoculated into Luria-Bertani relatedness of isolates. (LB) broth and incubated at 37 °C with shaking [20]. After 20 h of incubation, DNA was extracted using the Methods ZR Fungal/Bacterial DNA MiniPrep kit (Zymo Research, Study sample Lithuania). PCR amplifications for detection of bla , TEM A random sample of 275 university students of both bla , bla , bla , bla , bla and bla SHV CTX CMY DHA FOX MOX sexes living in three residences at the Eduardo (Table 1) were performed in a ThermalCycler T100™ Mondlane University in Maputo, Mozambique, provided (Bio-Rad, USA) with a final volume of 50 μL (25 μLof non-repetitive stool specimens within a six-week period, Master mix, 15 μL of water, 4 μL of each primer with a from January to February 2016 upon informed, voluntary final concentration of 0.8 μM (Inqaba Biotechnology consent. Students with diarrheal disease were excluded Industries, South Africa) and 2 μL of template DNA), from the study. Participants provided information about with an initial denaturation temperature of 98 °C for antibiotic use within 3 months and hospitalization within 10 s, extension at 72 °C for 15 s and a final extension at 6 months prior to the study. Stool samples were kept on 72 °C for 1 min. The annealing temperatures were as ice and transported to the laboratory for immediate follows: bla 60 °C, bla 56 °C, bla 57 °C, TEM SHV CTX culture twice daily. bla 57 °C, and bla , bla and bla 50 °C. CMY DHA FOX MOX PCR products were loaded on a 1.5% (w/v) agarose gel Identification and susceptibility testing and visualized by UV transillumination (Bio-Rad Chemi- All samples were cultured on MacConkey agar with and Doc™MP System) after staining in 0.1 mg/mL Gel Red without ceftriaxone (1 mg/L) for selection of third-genera- for 15 min. A random sample of 19 CTX-M PCR tion cephalosporin-resistant isolates. Lactose-positive amplicons were sent to Inqaba Biotec (South Africa) for isolates growing on the 1 mg/L ceftriaxone agar were DNA sequencing. TEM and SHV amplicons were not identified to the species level using API20E (bioMérieux, sequenced. Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 3 of 8 Table 1 Primers used for amplification of ESBL genes Results Setting and samples Target Primers Sequence (5′ to 3′) Annealing enzyme temperature (ref) Among the 275 samples collected in the study, 159 TEM-1 TEMMF AAA ATT CTT GAA GAC G 60 °C [29] (58%) were retrieved from male students and 116 (42%) from females in an age range from 19 to 32 years old. TEMMR TTA CCA ATG CTT AAT CA The students lived in separate blocks and/or floors for SHV SHVMF TTA ACT CCC TGT TAG CCA 56 °C [29] male and female students. Each floor had one kitchen SHVMR GAT TTG CTG ATT TCG CCC where students could prepare their own food. There CTX-1 CTXMF GGT TAA AAA ATC ACT GCG TC 57 °C [27] were students from different courses including, but not CTXMR TTG GTG ACG ATT TTA GCC GC limited to Engineering, Medicine, Political Science, CMY CMYMF GAT TCC TTG GAC TCT TCA G 57 °C [28] Biology and Sociology. All participants declared a history of non-hospitalization 6 months prior to the study and CMYMR TAA AAC CAG GTT CCC AGA TAG C 87.5% had not consumed any antibiotics for at least FOX FOXMF CAC CAC GAG AAT AAC CAT 57 °C [28] 3 months. FOXMR ATG TGG ACG CCT TGA ACT DHA DHAMF AAC TTT CAC AGG TGT GCT GGG T 57 °C [28] Frequency of E. coli and Klebsiella spp. ESBL colonization DHAMR CCG TAC GCA TAC TGG CTT TGC In 55 of 275 non-repetitive stool samples 35 E. coli and MOX MOXMF GCT GCT CAA GGA GCA CAG GAT 50 °C [28] 21 Klebsiella spp. grew on MacConkey agar containing 1 mg/L ceftriaxone, with one sample providing two MOXMR CAC ATT GAC ATA GGT GTG GTG C isolates. The overall prevalence of ceftriaxone-resistant E. coli and Klebsiella spp. colonization was thus 20% Genomic DNA isolation and ERIC-PCR analysis (55/275). There was no statistically significant difference Genomic DNA was isolated and purified using the in the rate of colonization between females (n = 28, 24%) GeneJet Genomic DNA Purification Kit (Thermo and males (n = 28, 17%). All samples displayed growth Scientific, USA). Antibiotic-susceptible E. coli ATCC on the ceftriaxone-free growth control agar. 25922 and beta-lactam resistant SHV-18 K. pneumo- niae ATCC 700603 were used as quality controls. The Phenotypic analyses of antimicrobial susceptibility total PCR reaction volume was 10 μL, which con- All the 56 E. coli (n = 35) and Klebsiella spp. (n = 21) tained 100 ng of template DNA, 50 pmol of each isolates were phenotypically confirmed as ESBL and/or primer, 2.8 μL nuclease-free water and 5 μLof pAmpC producers and showed high rates of resistance DreamTaq PCR Mastermix (2X) (Thermo Scientific). to tetracycline and co-trimoxazole (70%), ceftazidime The primers ERIC 1 and ERIC 2 [21]wereused. PCR (63%), cefoxitin (41%), and ciprofloxacin (34%). Two conditions were as follows: 94 °C for 3 min, 30 cycles isolates displayed carbapenem zone diameters below the of 30 s of denaturation at 94 °C, 1 min of annealing screening breakpoint for carbapenemase production, but at 50 °C, 8 min of extension at 65 °C, and a final were categorized as susceptible by the clinical break- elongation of 16 min at 65 °C, in an Applied Biosys- point. They were subsequently negative in the Carba NP tems 2720 thermal cycler. The ERIC-PCR products test and there were thus no carbapenemase producers were loaded onto 1% (w/v) agarose gels and subjected (Table 2). Eighty-eight percent of the isolates were to electrophoresis at 80 V using 1 × TAE buffer. Amp- defined as multi-resistant. Antimicrobial susceptibility lification products were visualized by UV transillu- results are presented in the Additional file 1: Table S1. mination (Syngene, UK) after staining in 0.1 mg/mL ethidium bromide for 15 min. Genotypic variation ESBL gene identification by PCR and sequencing was analyzed using the Gel Compare II version 6.0 The PCR results for CTX-M and pAmpC genes are software package (Applied Maths, Belgium) by summarized in Table 3. Among 56 isolates, 41% were Jacquard index and Unweighted Pair Group Method positive for at least two genes, 32% were positive for at with Arithmetic Mean (UPGMA) cluster analysis to least three, and 23% were positive for at least four genes. produce a dendrogram. Forty-three percent (24/56) of the isolates contained only CTX-M, 11% (6/56) only pAmpC, and 36% (20/56) both CTX-M and pAmpC sequences. Six isolates (11%) Statistical analyses were negative for CTX-M and pAmpC genes but con- Groups were compared by the Fischer Exact Test tained TEM and/or SHV sequences (data not shown). using the Epi Info StatCalc software version 7.2.2.6 However, as these amplicons were not sequenced it can- (CDC, Atlanta, USA) with statistical significance not be concluded that they encode broad-spectrum defined as p <0.05. beta-lactamases and the basis for ceftriaxone resistance Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 4 of 8 Table 2 Antimicrobial resistance (%) of ESBL-producing E. coli products are the minor amplification products of low and Klebsiella spp intensity [22]. All isolates were typeable using this Antibiotic E. coli (n = 35) Klebsiella spp. (n = 21) fingerprinting technique and band profiles were repro- ducibly obtained under similar experimental conditions Ceftriaxone 100 100 on repeat amplification. Ceftazidime 71 43 The ERIC-PCR profiles allowed differentiation of the Gentamicin 14 43 35 E. coli isolates into 24 ERIC-PCR types which were Cefoxitin 46 33 grouped into 15 clusters (A-O), each of which were Tetracycline 66 76 futher sub-divided into multiple sub-clusters (Fig. 1). Co-trimoxazole 63 76 CTX-M and pAmpC genes were amplified from isolates in different clusters. Isolates with similar profiles demon- Ciprofloxacin 37 29 strated varying beta-lactamase gene content. Imipenem 0 0 ERIC-PCR results for Klebsiella spp. in these isolates was consequently not determined. A Twenty-one Klebsiella spp. isolates were subjected to total of 67 CTX-M/pAmpC genes were found in the 35 ERIC-PCR analysis in comparison to the SHV-18 con- E. coli isolates and 41 in the 21 Klebsiella spp. isolates. taining K. pneumoniae ATCC 700603. Distinct profiles CTX-M was the most common gene detected in both E. were obtained for all isolates tested using ERIC-PCR coli (n = 25) and Klebsiella spp. (n = 16) followed by fingerprinting (Fig. 2). Banding patterns comprised DHA (n =15 in E. coli and n =9 in Klebsiella spp.). between 2 and 16 individual bands. Fragments of differ- DNA sequencing of 19 randomly selected CTX-M ent molecular weights were observed in the ERIC-PCR amplicons from E. coli (n = 13) and Klebsiella spp. (n =6) fingerprints, ranging from 0.5–20 kb (Fig. 2). revealed a predominance of bla (n =14) and occa- The ERIC-PCR profiles allowed differentiation of the 21 CTX-M-15 sional detection of bla (n =3), bla (n =1) isolates into 17 ERIC-PCR types which were grouped into CTX-M-55 CTX-M-3 and bla (n = 1). A full list of CTX-M and pAmpC 12 clusters (A-L), each of which were further sub-divided CTX-M-186 gene content in individual strains is presented in the into multiple sub-clusters (Fig. 2). CTX-M and pAmpC Additional file 1: Table S1. genes were identified in isolates from different clusters, predominantly clusters B-G. Isolates with similar profiles ERIC-PCR results for E. coli demonstrated varying beta-lactamase gene content. Distinct ERIC-PCR profiles were obtained for the 35 E. coli isolates (Fig. 1). The absence or presence of a band Discussion was noted in determining variation among isolates, and Antibiotic-resistant bacteria are an escalating cause of banding patterns comprised between 2 and 14 individual infections in Mozambique and worldwide, but informa- bands. Fragments of different molecular weights were tion regarding the molecular epidemiology of ESBL- and observed in the ERIC-PCR fingerprints, ranging from pAmpC-producing Enterobacteriaceae in the country 0.5–20 kb (Fig. 1). Amplification of different intensities remains scarce. To our knowledge, this is the first study was observed and visual analysis of the ERIC profiles reporting on gastrointestinal colonization with ESBL- included primary, secondary and tertiary amplification and pAmpC-producing Enterobacteriaceae among (Fig. 1). Primary amplification products refer to those university students in Mozambique. products of high intensity, which appear extremely We systematically collected samples from healthy stu- bright on the gels. Secondary amplification products are dents to determine the prevalence of colonization by E. those products that are not as bright as the primary coli and Klebsiella spp. carrying ESBL and/or pAmpC, amplification products but more intense that the tertiary as well as their antibiotic susceptibility. In total, 20% of amplification products, while the tertiary amplification students (55/275) were colonized with ESBL and/or pAmpC positive E. coli and Klebsiella spp. Similar re- Table 3 Distribution of ESBL enzymes according to species sults were found in a study conducted in pre-school ESBL enzymes E. coli (n = 35) Klebsiella spp. (n = 21) Total children attending childcare facilities in the Lao People’s CTX-M 28 16 44 Democratic Republic where the ESBL prevalence was DHA 15 9 24 23% [23] while the prevalence amongst patients in inten- sive care in Korea was 28.2% with a higher frequency in MOX 10 7 17 E. coli (78%) compared with Klebsiella spp. (18%) [24]. A FOX 7 5 12 study recently conducted in Madagascar [18] in commu- CMY 7 4 11 nity settings demonstrated lower rates of colonization by TOTAL 104 57 161 ESBL-positive Gram-negative bacilli compared to this Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 5 of 8 Fig. 1 Dendrogram representing the genetic relatedness and cluster analysis of 35 E. coli strains isolated from stool samples of university students, based on ERIC-PCR fingerprinting patterns using Jacquard index and UPGMA algorithm. The scale at the top represents percentage similarity to E. coli ATCC 25922 study, despite the fact that more species were included facilities [27]. We found 36% co-existence of ESBL and in addition to E. coli and Klebsiella spp. The present pAmpC beta-lactamase genes which is high compared to study showed a higher prevalence of ESBL and pAmpC reports from clinical isolates in Turkey (13.9%) [28]. colonization compared to a study conducted in rural The CTX-M positive isolates carried different genotypes; communities in France where the frequency of ESBL bla (n =14), bla (n =3), bla CTX-M-15 CTX-M-55 CTX-M-186 colonization was 5.3% [25]. This large difference may be (n =1) and bla (n = 1). These results are similar to CTX-M-3 attributed to poorly controlled antibiotic consumption findings in a study conducted in Tanzania [29]that found and sub-optimal hygiene conditions in developing coun- bla genes in 95% of the carriers, but differ from a CTX-M-15 tries [26]. In Mozambique, antibiotic therapy is mostly study in Kenya [30] which demonstrated only 29% of the empirical because of scarce diagnostic microbiology isolates carrying bla and 4% carrying bla . CTX-M-15 CTX-M-3 Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 6 of 8 Fig. 2 Dendrogram representing the genetic relatedness and cluster analysis of 21 Klebsiella spp. strains isolated from stool samples of university students, based on ERIC-PCR fingerprinting patterns using Jacquard index and UPGMA algorithm. The scale at the top represents percentage similarity to Klebsiella pneumoniae ATCC 700603 No isolates harbored bla or bla in the The prevalence in different studies may vary depending CTX-M-55 CTX-M-186 Kenyan study. bla is endemic in many Asian on socio-economic status of individuals involved [32], CTX-M-55 countries, and the detection of this variant in Mozambique and this makes it difficult to estimate the prevalence in may reflect travel and trade with this part of the world [31]. the general population in Mozambique. One may In this study of healthy students, none of the partici- suppose that transmission between students is facilitated pants had a history of hospitalization 6 months prior to because of the conditions they live under with many the study, and 87.5% had not consumed any antibiotics at individuals sharing the same bathroom and kitchen. least 3 months prior to the study. This suggests that the However, the diversity of strains demonstrated using high antibiotic resistance rates are not related to antibiotic ERIC-PCR indicates that there is at present no wide- consumption or hospitalization, and may indicate that the spread clonal outbreak, although there could be horizon- E. coli and Klebsiella ESBL- and pAmpC-producers tal transfer of plasmids or other mobile genetic isolated in this study were acquired in the community. In elements. One may speculate that students are exposed the university residences there is a mixture of students to ESBL and/or pAmpC isolates from some external from different courses including medicine and health source like dissemination in the food supply. Very little sciences, but the study was not powered to analyse differ- is known about the occurrence of ESBL and pAmpC ences between student groups (data not shown). ESBL- strains in the food supply in Mozambique, but it is and/or pAmpC-colonized students working in health reported from other countries that ESBL and pAmpC E. institutions may constitute a reservoir for further spread coli and Klebsiella spp. may disseminate in food animals of multi-resistant microorganisms among patients. and environmental sources [33–37]. To control the rapid dissemination of resistant Entero- bacteriaceae among students and, consequently, the gen- Conclusions eral population, it is necessary to educate students about This study demonstrated a 20% prevalence of colonization the importance of personal hygiene. It is also necessary by multi-drug resistant, non-clonally-related ESBL- and/ to perform further studies to determine the prevalence or pAmpC positive E. coli and Klebsiella spp. isolates of colonization in different groups of the population. among male and female university students in Chirindze et al. BMC Infectious Diseases (2018) 18:244 Page 7 of 8 Mozambique. Colonization of ESBL/pAmpC in the popu- Received: 8 January 2018 Accepted: 18 May 2018 lation of study was not related to prior antimicrobial consumption or hospitalization. Similar studies should be References done to further explore the epidemiology of multi-drug 1. Al-Jasser AM. Extended-spectrum beta-lactamases (ESBL’s): global problem. resistant Enterobacteriaceae in the population as a whole. Kuwait Med J. 2006;38:171–85. 2. Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis. Additional file 2008;8(3):159–66. 3. Rawat D, Nair D. Extended-spectrum beta-lactamases in gram-negative Additional file 1: Table S1. Resistance genes identified and sensitivity bacteria. J Glob Infect Dis. 2010;2(3):263–74. results of E. coli and Klebsiella spp. (DOCX 38 kb) 4. Villa L, Pezzella C, Tosini F, Visca P, Petrucca A, Carattoli A. Multiple-antibiotic resistance mediated by structurally related IncL/M plasmids carrying an extended spectrum beta-lactamase gene and class 1 integron. Antimicrob Abbreviations Agents Chemother. 2000;44(10):2911–4. ATCC: American Type Culture Collection; DNA: Deoxyribonucleic acid; ERIC- 5. Coudron PE, Hanson ND, Climo MW. Occurrence of extended-spectrum and PCR: Enterobacterial repetitive intergenic consensus PCR; ESBL: Extended- AmpC beta-lactamases in bloodstream isolates of Klebsiella pneumoniae: spectrum beta-lactamases; pAmpC: Plasmid-mediated AmpC beta-lactamase; isolates harbor plasmid-mediated FOX-5 and ACT-1 AmpC beta-lactamases. PCR: Polymerase chain reaction; TAE: Buffer containing Tris base, acetic acid J Clin Microbiol. 2003;41(2):772–7. and EDTA (Ethylenediaminetetraacetic acid); UPGMA: Unweighted pair group 6. Dolejska M, Villa L, Hasman H, Hansen L, Carattoli A. Characterization of IncN method with arithmetic mean plasmids carrying blaCTX-M-1 and qnr genes in Escherichia coli and Salmonella from animals, the environment and humans. J Antimicrob Acknowledgements Chemother. 2013;68(2):333–9. The authors would like to acknowledge all participants in the study, as well 7. Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical as technical staff involved in the collection and analysis of samples. update. Clin Microbiol Rev. 2005;18(4):657–86. 8. Kothari C, Gaind R, Singh LC, Sinha A, Kumari V, Arya S, Chellani H, Saxena S, Funding Deb M. Community acquisition of beta-lactamase producing The study was funded by the Norwegian Agency for Development Enterobacteriaceae in neonatal gut. BMC Microbiol. 2013;13:136. Cooperation under the Norwegian Programme for Capacity Development in 9. de Kraker ME, Davey PG, Grundmann H, BURDEN study group. Mortality and Higher Education and Research for Development (NORHED) Grant QZA 0484 hospital stay associated with resistant Staphylococcus aureus and RSA 13/0010 entitled “Antibiotic Stewardship and Conservancy in Africa”. The Escherichia coli bacteremia: estimating the burden of antibiotic resistance in funding body had no role in the design of the study, collection, analysis, or Europe. PLoS Med. 2011;8(10):e1001104. interpretation of data, or in writing the manuscript. 10. Blomberg B, Jureen R, Manji KP, Tamim BS, Mwakagile DS, Urassa WK, Fataki M, Msangi V, Tellevik MG, Maselle SY, Langeland N. High rate of fatal cases Availability of data and materials of pediatric septicemia caused by gram-negative bacteria with extended- All data generated or analysed during this study are included in this spectrum beta-lactamases in Dar es Salaam, Tanzania. J Clin Microbiol. published article and its supplementary information files. 2005;43(2):745–9. 11. Storberg V. ESBL-producing Enterobacteriaceae in Africa - a non-systematic Authors’ contributions literature review of research published 2008-2012. Infect Ecol Epidemiol. The study was designed LMC, TFZ, AS, SYE and GSS. Collection of data and 2014;4:20342. laboratory analyses were performed by LMC, JOS, UG and HYC. All authors 12. Tansarli GS, Poulikakos P, Kapaskelis A, Falagas ME. Proportion of extended- contributed to the interpretation of the data and writing of the manuscript. spectrum β-lactamase (ESBL)-producing isolates among Enterobacteriaceae All authors read and approved the final manuscript. in Africa: evaluation of the evidence–systematic review. J Antimicrob Chemother. 2014;69(5):1177–84. 13. Sangare SA, Rondinaud E, Maataoui N, Maiga AI, Guindo I, Maiga A, Ethics approval and consent to participate Camara N, Dicko OA, DaoS,Diallo S,Bougoudogo F,Andremont A, Ethical approval was received from the Biomedical Research Ethics Committee Maiga II, Armand-Lefevre L. 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BMC Infectious DiseasesSpringer Journals

Published: May 30, 2018

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