Molecular characterisation of extended-spectrum ß-lactamase producing Escherichia coli in wild birds and cattle, Ibadan, Nigeria.

BACKGROUND: Antimicrobial resistance (AMR) is an increasing global health concern reducing options for therapy of infections and also for perioperative prophylaxis. Many Enterobacteriaceae cannot be treated anymore with third generation cephalosporins (3GC) due to the production of certain 3GC hydrolysing enzymes (extended spectrum beta-lactamases, ESBLs). The role of animals as carriers and vectors of multi-resistant bacteria in different geographical regions is poorly understood. Therefore, we investigated the occurrence and molecular characteristics of ESBL-producing Escherichia coli (E. coli) in wild birds and slaughtered cattle in Ibadan, Nigeria. Cattle faecal samples (n = 250) and wild bird pooled faecal samples (cattle egrets, Bubulcus ibis, n = 28; white-faced whistling duck, Dendrocygna viduata, n = 24) were collected and cultured on cefotaxime-eosin methylene blue agar. Antimicrobial susceptibility was determined by agar diffusion assays and all 3GC resistant isolates were genotypically characterised for AMR genes, virulence associated genes (VAGs) and serotypes using DNA microarray-based assays. RESULTS: All 3GC resistant isolates were E. coli: cattle (n = 53), egrets (n = 87) and whistling duck (n = 4); cultured from 32/250 (12.8%), 26/28 (92.9%), 2/24(8.3%), cattle, egrets and whistling duck faecal samples, respectively. blaCTX-M gene family was prevalent; blaCTX-M15 (83.3%) predominated over blaCTX-M9 (11.8%). All were susceptible to carbapenems. The majority of isolates were resistant to at least one of the other tested antimicrobials; multidrug resistance was highest in the isolates recovered from egrets. The isolates harboured diverse repositories of other AMR genes (including strB and sul2), integrons (predominantly class 1) and VAGs. The isolates recovered from egrets harboured more AMR genes; eight were unique to these isolates including tetG, gepA, and floR. The prevalent VAGs included hemL and iss; while 14 (including sepA) were unique to certain animal isolates. E. coli serotypes O9:H9, O9:H30 and O9:H4 predominated. An identical phenotypic microarray profile was detected in three isolates from egrets and cattle, indicative of a clonal relationship amongst these isolates. CONCLUSION: Wild birds and cattle harbour diverse ESBL-producing E. coli populations with potential of inter-species dissemination and virulence. Recommended guidelines to balance public health and habitat conservation should be implemented with continuous surveillance.

Draft Genome Sequences of 23 Salmonella enterica Strains Isolated from Cattle in Ibadan, Nigeria, Representing 21 Salmonella Serovars.

To provide a better understanding of the diversity of Salmonella enterica, we report the assembled genome sequences of 23 Salmonella enterica strains isolated from fecal samples of cattle in Nigeria comprising 21 different Salmonella serovars.

Fluoroquinolone-Resistant Enteric Bacteria in Sub-Saharan Africa: Clones, Implications and Research Needs.

Fluoroquinolones came into widespread use in African countries in the early 2000s, after patents for the first generation of these drugs expired. By that time, quinolone antibacterial agents had been used intensively worldwide and resistant lineages of many bacterial species had evolved. We sought to understand which Gram negative enteric pandemic lineages have been reported from Africa, as well as the nature and transmission of any indigenous resistant clones. A systematic review of articles indexed in the Medline and AJOL literature databases was conducted. We report on the findings of 43 eligible studies documenting local or pandemic fluoroquinolone-resistant enteric clones in sub-Sahara African countries. Most reports are of invasive non-typhoidal Salmonella and Escherichia coli lineages and there have been three reports of cholera outbreaks caused by fluoroquinolone-resistant Vibrio cholerae O1. Fluoroquinolone-resistant clones have also been reported from commensals and animal isolates but there are few data for non-Enterobacteriaceae and almost none for difficult-to-culture Campylobacter spp. Fluoroquinolone-resistant lineages identified in African countries were universally resistant to multiple other classes of antibacterial agents. Although as many as 972 non-duplicate articles refer to fluoroquinolone resistance in enteric bacteria from Africa, most do not report on subtypes and therefore information on the epidemiology of fluoroquinolone-resistant clones is available from only a handful of countries in the subcontinent. When resistance is reported, resistance mechanisms and lineage information is rarely investigated. Insufficient attention has been given to molecular and sequence-based methods necessary for identifying and tracking resistant clones in Africa and more research is needed in this area.

A novel plasmid carrying blaCTX-M-15 identified in commensal Escherichia coli from healthy pregnant women in Ibadan, Nigeria.

The aim of this study was to investigate the molecular characteristics of commensal Escherichia coli producing extended-spectrum ß-lactamases and showing fluoroquinolone resistance circulating in a healthy population in Ibadan, Nigeria. In total, 101 faecal samples from healthy pregnant women on the day of admission to hospital were collected and plated on eosin-methylene blue agar supplemented with cefotaxime. Genotyping demonstrated the presence of the blaCTX-M-15 gene in all of the cefotaxime-resistant isolates (n=32), and there was circulation of prevalent clones. The aac(6')-Ib-cr, qnrS1, qepA1 and qnrB1 genes were identified in several strains. A novel plasmid supporting the spread of the blaCTX-M-15, blaTEM-1 and qnrS1 genes was identified in these isolates by complete DNA sequencing.

Persistence of fluoroquinolone-resistant Salmonella enterica serovar Kentucky from poultry and poultry sources in Nigeria.

INTRODUCTION: This study investigated the antimicrobial resistance and clonality of Salmonella enterica serotype Kentucky in poultry and poultry sources in Nigeria, and compared the isolates with the clone of S. Kentucky STI98-X1 CIPR using (PFGE) and (MIC). METHODOLOGY: Fecal samples from chickens and poultry sources (litter, water, rodent and lizard fecal samples) were collected from  fourteen (14) poultry farms in 2007, 2010 and 2011 and were analyzed for S. Kentucky. RESULTS AND CONCLUSIONS: Six percent of the samples were positive for S. Kentucky - all resistant to nalidixic acid and ciprofloxacin. The isolates are grouped within the PFGE cluster X1 of S. Kentucky STI98 CIPR, indicating the association to the emerging and widely spread CIPR S. Kentucky clone with poultry and poultry sources.

Diversity and antimicrobial susceptibility of Salmonella enterica serovars isolated from pig farms in Ibadan, Nigeria.

Animals including food animals play a significant role in the epidemiology of Salmonella enterica. The control requires identification of sources and institution of targeted interventions. This study investigates the diversity of S. enterica serovars, antimicrobial susceptibility, and occurrence of plasmid-mediated quinolone resistance (PMQR) genes in pigs in Ibadan, Nigeria. Pooled fresh pen floor fecal samples of pigs collected from 31 pig farms were cultured; the Salmonella isolates were serotyped and their antimicrobial susceptibility was determined. PMQR genes were screened by polymerase chain reaction. The 229 Salmonella isolates were made of 50 serovars predominated by rare serovars Salmonella Give (n = 36; 15.7 %), Salmonella Brancaster (n = 17; 7.4 %), Salmonella Colindale (n = 15; 6.6 %), Salmonella Elisaberthville (n = 13; 5.7 %), Salmonella Hillingdon (n = 13; 5.7 %), and Salmonella Kingston (n = 13; 5.7 %). The most widely distributed serovars among the farms were Salmonella Give (six farms) and Salmonella Elisaberthville (six farms). Resistance to chloramphenicol, sulfonamides, nalidixic acid, streptomycin, and tetracycline ranged from 11.6 % (n = 26) to 22.8 % (n = 51). Resistance ciprofloxacin and gentamicin was low (n = 2; 0.9 %). Multiply resistant isolates included Salmonella Kentucky, the most resistant serovar. qnrB19 was found in two isolates of Salmonella Corvallis and one isolate of Salmonella Larochelle, respectively, while qnrS1 was found in two isolates of Salmonella Derby. Other PMQR genes were not detected. Pigs constitute an important source of diverse Salmonella serovars in Ibadan. The isolates were more resistant to old antimicrobials with some multiple resistant. Control measures and regulation of antimicrobials are warranted.

Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo.

INTRODUCTION: Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria. METHODOLOGY: Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars. RESULTS: All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin. CONCLUSION: We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin.

The global establishment of a highly-fluoroquinolone resistant Salmonella enterica serotype Kentucky ST198 strain.

While the spread of Salmonella enterica serotype Kentucky resistant to ciprofloxacin across Africa and the Middle-East has been described recently, the presence of this strain in humans, food, various animal species (livestock, pets, and wildlife) and in environment is suspected in other countries of different continents. Here, we report results of an in-depth molecular epidemiological study on a global human and non-human collection of S. Kentucky (n = 70). We performed XbaI-pulsed field gel electrophoresis and multilocus sequence typing, assessed mutations in the quinolone resistance-determining regions, detected ß-lactam resistance mechanisms, and screened the presence of the Salmonella genomic island 1 (SGI1). In this study, we highlight the rapid and extensive worldwide dissemination of the ciprofloxacin-resistant S. Kentucky ST198-X1-SGI1 strain since the mid-2000s in an increasingly large number of contaminated sources, including the environment. This strain has accumulated an increasing number of chromosomal and plasmid resistance determinants and has been identified in the Indian subcontinent, Southeast Asia and Europe since 2010. The second substitution at position 87 in GyrA (replacing the amino acid Asp) appeared helpful for epidemiological studies to track the origin of contamination. This global study provides evidence leading to the conclusion that high-level resistance to ciprofloxacin in S. Kentucky is a simple microbiological trait that facilitates the identification of the epidemic clone of interest, ST198-X1-SGI1. Taking this into account is essential in order to detect and monitor it easily and to take rapid measures in livestock to ensure control of this infection.

International spread of an epidemic population of Salmonella enterica serotype Kentucky ST198 resistant to ciprofloxacin.

National Salmonella surveillance systems from France, England and Wales, Denmark, and the United States identified the recent emergence of multidrug-resistant isolates of Salmonella enterica serotype Kentucky displaying high-level resistance to ciprofloxacin. A total of 489 human cases were identified during the period from 2002 (3 cases) to 2008 (174 cases). These isolates belonged to a single clone defined by the multilocus sequence type ST198, the XbaI-pulsed-field gel electrophoresis cluster X1, and the presence of the Salmonella genomic island 1 variant SGI1-K. This clone was probably selected in 3 steps in Egypt during the 1990s and the early 2000s and has now spread to several countries in Africa and, more recently, in the Middle East. Poultry has been identified as a potential major vehicle for infection by this clone. Continued surveillance and appropriate control measures should be implemented by national and international authorities to limit the spread of this strain.

Plasmid-mediated quinolone resistance and ß-lactamases in Escherichia coli from healthy animals from Nigeria.

OBJECTIVES: The animal reservoir of plasmid-mediated quinolone resistance (PMQR) and ß-lactamases is still controversial and little information is available on the prevalence of these resistance determinants in developing countries. The aim of this study was to identify and characterize PMQR and ß-lactamases in a collection of commensal ampicillin-resistant Escherichia coli isolated from healthy chickens and pigs at slaughter, collected in November-December 2006, in Ibadan, Nigeria. METHODS: One hundred and sixty-two ampicillin-resistant E. coli were obtained from healthy chickens and pigs at slaughter in Ibadan, Nigeria. Strains were tested for antimicrobial susceptibility by disc diffusion assay. MICs of ciprofloxacin were determined by Etest. Resistance genes were screened by PCR and DNA sequencing. Clonal relatedness of the isolates was determined by enterobacterial repetitive intergenic consensus-PCR. Plasmids were transferred by conjugation and transformation and characterized by PCR-based replicon typing and plasmid multilocus sequence typing. RESULTS: PMQR genes were detected in 18 E. coli strains; 11 of them showed reduced susceptibility to ciprofloxacin. Twelve strains carried qnrS1, three strains carried qnrB19, one strain carried qnrB10 and three strains carried qepA; one strain carried both qepA and qnrB10. All strains carried the bla(TEM) gene; one strain was positive for the CTX-M-15 extended-spectrum ß-lactamase. CONCLUSIONS: Our findings suggest that food animals could represent an important reservoir of PMQR in this region of Africa. Previous studies reported high prevalence of qnr genes in clinical isolates from humans in Nigeria, suggesting that the spread of these resistance determinants in this country could be particularly relevant.

Antimicrobial susceptibility and serovars of Salmonella from chickens and humans in Ibadan, Nigeria.

BACKGROUND: This study determines the prevalence and antibiotic resistance of Salmonella serovars from humans and chickens in Ibadan, Nigeria, in 2004-2007. METHODOLOGY: A total of 991 blood samples were collected from patients in 2004 to 2005 and 641 fecal samples were collected from poultry farms in 2007. All Salmonella isolates were serotyped and tested for antimicrobial susceptibility. RESULTS: Thirty-nine (4%) Salmonella isolates were obtained from human blood and 70 (11%) from chicken fecal samples. The human isolates revealed nine different serovars; 82% were non-typhoidal Salmonella and 18% were (S. Typhi). The majority of serovars from humans were S. Enteritidis (33%), S. Dublin (18%), and S. Typhimurium (18%). Resistance to chloramphenicol, sulfamethoxazole, trimethoprim, and ampicillin ranged from 36% to 59% for the human isolates. Eight different serovars were obtained from chickens; S. Virchow (71%) predominated. A high frequency (87%) of reduced susceptibility to ciprofloxacin was observed among the chicken isolates. A high frequency of resistance to tetracycline (93%), nalidixic acid (81%), and sulfamethoxazole (87%) was observed. Rare serovars such as S. Apapa, S. Mouschaui, S. Jukestown, S. Oritamerin, and S. Onireke were isolated from both humans and chickens. Identical serovars were not found among human and chicken isolates. CONCLUSIONS: This study indicates that chickens are not a reservoir of Salmonella causing bacteraemia among humans in Ibadan, Nigeria. Studies locating the reservoirs responsible for invasive salmonellosis in humans are needed. Controls and targeted interventions against S. Virchow and the frequent occurrence of antimicrobial resistance in chickens should be initiated to prevent the spread of this serovar.

Characterization of ESBL (SHV-12) producing clinical isolate of Enterobacter aerogenes from a tertiary care hospital in Nigeria.

BACKGROUND: We studied the beta-lactamases of an E. aerogenes isolate recovered from the blood of a two-year-old patient. The isolate demonstrated a disk-diffusion phenotype typical for an AmpC-ESBL co-producer. METHODS: Microbiology studies were performed according to standard protocols. The resistance gene was identified by transconjugation and cloning experiments. RESULTS: By transconjugation only a narrow spectrum beta-lactamase (TEM-1) encoded on a small plasmid was transmitted. The ESBL was cloned and expressed in an E. coli host. Sequence analysis of the recombinant plasmid revealed blaSHV-12 associated to the insertion sequence, IS26. CONCLUSION: This is the first study demonstrated the occurrence of SHV-12 in Nigeria.