Antimicrobial resistance and virulence genes of invasive Salmonella enterica from children with bacteremia in north-central Nigeria.

Objectives: Bacteremia due to invasive Salmonella enterica has been reported earlier in children in Nigeria. This study aimed to detect the virulence and antibiotic resistance genes of invasive Salmonella enterica from children with bacteremia in north-central Nigeria. Method: From June 2015 to June 2018, 4163 blood cultures yielded 83 Salmonella isolates. This is a secondary cross-sectional analysis of the Salmonella isolates. The Salmonella enterica were isolated and identified using standard bacteriology protocol. Biochemical identifications of the Salmonella enterica were made by Phoenix MD 50 identification system. Further identification and confirmation were done with polyvalent antisera O and invA gene. Antimicrobial susceptibility testing was done following clinical and laboratory standard institute guidelines. Resistant genes and virulence genes were determined using a real-time polymerase chain reaction. Result: Salmonella typhi 51 (61.4%) was the most prevalent serovar, followed by Salmonella species 13 (15.7%), choleraesuis 8 (9.6%), enteritidis 6 (7.2%), and typhimurium 5 (6.1%). Fifty-one (61.4%) of 83 Salmonella enterica were typhoidal, while 32 (38.6%) were not. Sixty-five (78.3%) of the 83 Salmonella enterica isolates were resistant to ampicillin and trimethoprim-sulfamethoxazole, followed by chloramphenicol 39 (46.7%), tetracycline 41 (41.4%), piperacillin 33 (33.9%), amoxicillin-clavulanate, and streptomycin 21 (25.3%), while cephalothin was 19 (22.9%). Thirty-nine (46.9%) of the 83 Salmonella enterica isolates were multi-drug resistant, and none were extensive drug resistant or pan-drug resistant. A blaTEM 42 (50.6%), floR 32 (38.6%), qnrA 24 (28.9%), tetB 20 (20.1%), tetA 10 (10.0%), and tetG 5 (6.0%) were the antibiotic resistance genes detected. There were perfect agreement between phenotypic and genotypic detection of antimicrobial resistance in tetracycline, ciprofloxacin, and chloramphenicol, while beta-lactam showed κ = 0.60 agreement. All of the Salmonella enterica isolates had the virulence genes invA, sopB, mgtC, and sip4D, while 33 (39.8%), 45 (51.8%), and 2 (2.4%) had ssaQ, spvC, and ljsGI-1, respectively. Conclusion: Our findings showed multi-drug resistant Salmonella enterica in children with bacteremia in northern Nigeria. In addition, significant virulence and antimicrobial resistance genes were found in invasive Salmonella enterica in northern Nigeria. Thus, our study emphasizes the need to monitor antimicrobial resistance in Salmonella enterica from invasive sources in Nigeria and supports antibiotic prudence.

Antimicrobial resistance and virulence genes in Salmonella enterica serovars isolated from droppings of layer chicken in two farms in Nigeria.

AIM: This study aimed to investigate the isolation rate, antibiotic resistance and virulence genes of Salmonella enterica serovar from two commercial farms in Nigeria. METHODS AND RESULTS: Salmonella isolation was performed according to the United States Food and Drug Agency (USFDA) method. Serotyping, antimicrobial susceptibility testing, detection of resistance and virulence genes were done using the Kauffman-White Scheme, disc diffusion, minimum inhibitory concentration and real-time polymerase chain reaction techniques. Salmonella serovars were isolated from only farm A at 22/50 (44.0%) while none were isolated from farm B. Salmonella Typhi, 9 (40.9%); Salmonella Typhimurium, 2 (9.1%), Salmonella Enteritidis, 2 (9.1%), Salmonella Pullorum, 1 (4.5%), Salmonella Kentucky, 4 (18.2%) were identified while 4 (18.2%) were untypable. Sixteen isolates (72.7%) showed multiple drug resistance and 17 different resistance profile types with AMP-CHL-TRM-SXT as the most prevalent pattern. Resistance genes (blaTEM, 12/22 (54.5%) and virulence genes (InvA, sopB, mgtC and spi4D, 22/22 (100.0%), ssaQ, 16/22 (72.7%) and spvC, 13/22 (59.1%) were found, while blaSHV, blaCTX-M, floR, tetA, tetB, tetG and LJSGI-1 genes were absent. CONCLUSION: Pathogenic Salmonella were isolated from the chicken droppings in this study. Most of these strains were resistant to antibiotics and possessed characteristics of virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: Chicken droppings from this study area contained pathogenic strains of Salmonella and a rare occurrence of Salmonella Typhi. The study revealed that the environment and the food chain could be at risk of contamination of highly virulent and antimicrobial-resistant strains of Salmonella. These could affect the profitability of the poultry industry and food consumption. There is a need for caution in indiscriminate disposal of poultry waste and the use of uncomposted chicken droppings in soil amendment.

Antimicrobial usage and presence of extended-spectrum ß-lactamase-producing Enterobacteriaceae in animal-rearing households of selected rural and peri-urban communities.

This study examined socioeconomic and cultural factors relating to animal husbandry, antimicrobial usage and household hygiene in 320 animal-keeping households of 16 rural and peri-urban communities of Ogun State, Nigeria. The occurrence of extended-spectrum ß-lactamase-producing Enterobacteriaceae in 457 samples from animal and environmental sources within the households was investigated. Chickens (41.6%), goats (35.3%), dogs (33.8%) and sheep (14.4%) were the most common household animals. Animals were reared mainly for income generation (73.9%) and for household consumption (18.3%). They were reared predominantly (60.2%-100%) under the extensive system with unrestricted access to human space, cooking utensils and foods. Households were assessed as having good (59.4%), fair (22.2%) and poor (18.4%) hygiene. The rate of household non-prescriptional antimicrobial usage was 69.4% in humans and 60.6% in animals. Overall, ESBL-producing Enterobacteriaceae were detected in 53 (11.6%) of 457 samples. The ESBL-producing isolates were identified as Escherichia coli (n = 49) and Klebsiella pneumoniae (n = 4). They harboured the ESBL gene variants blaCTX-M-15 (n = 49), blaCTX-M-14 (n = 2), blaCTX-M-27 (n = 1) or blaCTX-M-55 (n = 1). Forty-eight ESBL-producing E. coli were assigned into phylogenetic groups A (n = 17), B1 (n = 14), D (n = 13) and F (n = 4). All ESBL-producing isolates demonstrated multidrug resistance to antimicrobial agents belonging to at least three different classes of antimicrobials. Poor regulation of antimicrobial marketing and inadequate access to veterinary care contributed to non-prescriptional use of antimicrobials in humans and animals. Free-range household animals harboured ESBL-producing bacteria and may facilitate the dispersal of the organisms within the community.

Prevalence and renal pathology of pathogenic Leptospira spp. in wildlife in Abeokuta, Ogun State, Nigeria.

There is paucity of information on the prevalence of leptospirosis in wildlife in Nigeria. This study investigated the prevalence and renal pathology of leptospirosis in wild animals in Southwest Nigeria. One hundred and five kidney samples were examined from 10 different wildlife species (antelope) greater cane rat (GCR), hare, African giant rat (AGR), tree hyrax, civet cat, monitor lizard, python, bushbuck and partridge) using a combination of Ellinghausen McCullough Johnson Harris (EMJH) medium, microscopic agglutination test (MAT), Warthin- Starry silver stain (WSss) and immunohistochemistry. Chi-square test was used with confidence level set at 0.05 to ascertain associations between positive cases and sex and species. Eightytwo (78.1%) samples were culturally positive, while 67.7% (63/93), 57.0% (16/28) and 66.7% (8/12) were WSss, MAT and immunohistochemically positive, respectively. Interstitial nephritis (41.0%) and tubular nephrosis (81.0%) were the most prominent histopathological changes. Pathogenic Leptospira organisms were highest in GCR (32.1%) and antelope (14.3%). Serovars hardjo (11.54%), bratislava (3.9%), canicola (3.9%), icterohaemorrhagiae (15.4%), pomona (7.14%) gripptotyphosa (19.2%) and undetermined isolates were also detected in other animals. The result showed high prevalence of Leptospira infection in the wild and the possibility of domestic animals and humans contracting the disease. This study is the first documentation of evidence of pathogenic Leptospira species in wildlife in Nigeria.

Detection and characterization of extended-spectrum ß-lactamase-producing Escherichia coli from chicken production chains in Nigeria.

A total of 405 Escherichia coli from the chicken production chains in Nigeria were investigated for ESBL-production and 4 isolates were identified as ESBL producers. They were characterized by XbaI-PFGE, multilocus sequence typing (MLST), phylotyping, sequencing of porin and regulatory genes and of the regulatory region of chromosomal ampC genes. Transformed ESBL gene-carrying plasmids were characterized by S1-nuclease, replicon typing, conjugation, digestion and PCRs for detection of the genetic environment of ESBL genes. Susceptibility testing, PCRs for the resistance genes, integrons, and the DNA microarray were performed with both, the original isolates and the transformants. All ESBL-producing isolates harboured blaCTX-M-15 genes located on non-conjugative plasmids (120-155kb). Three isolates with closely related/indistinguishable XbaI-patterns belonged to phylogroup A, and MLST sequence type ST10 and the fourth to phylogroup D and ST405. Resistance to aminoglycosides, sulfonamides/trimethoprim, quinolones, and tetracyclines were seen in all isolates. Incompatibility group IncFIB blaCTX-M-15-carrying plasmids were detected in the three related isolates which carried also a class 1 integron (aadA2-orfF-dfrA12) and the resistance genes blaOXA-1, blaTEM-1, aac(3')-IIa, aac(6')-Ib-cr, sul1, sul2, and tet(A). The IncFIA-IncFIB-IncI1 blaCTX-M-15-carrying plasmid harboured additionally the resistance genes aac(3')-IIa and tet(B). The blaCTX-M-15 genes were associated with ISEcp1 and Δorf477. ESBL-producing isolates showed elevated MICs to cefoxitin (16-64mg/L) and ertapenem MICs (0.5-2.0mg/L) mainly due to alterations in the porin genes. The virulence genes astA and prfB were detected. Although a low prevalence of ESBL-producing isolates was found, co-located resistance genes on the ESBL gene-carrying plasmids may facilitate the dissemination of them.

Phylogenetic analysis of Dermatophilus congolensis isolated from naturally infected cattle in Abeokuta and Ilorin, Nigeria.

Dermatophilus congolensis, the aetiological agent of dermatophilosis, is a pleomorphic, Gram-positive actinomycete, which infects animals and humans. Often, there is a wrong diagnosis of the infection in animals because of the close resemblance of the organism with other members of the family Actinomycetaceae. In this study, molecular tools were applied to suspected isolates of D. congolensis obtained from naturally infected cattle in Nigeria for confirmation of dermatophilosis. DNA extraction from 54 suspected pure colonies of D. congolensis was carried out using the QIAamp® DNA Mini extraction kit. PCR targeted at the 16S rRNA gene was employed for the confirmation of D. congolensis using 5'-ACATGCAAGTCGAACGATGA-3' and 5'-ACGCTCGCACCCTACGTATT-3' as forward and reverse primers, respectively. Positive amplicons were then sequenced directly using Big Dye Terminator Cycle Sequencing Kit with the forward primers and AmpliTaq-FS DNA Polymerase. Nucleotide sequences were aligned using bioedit (Ibis Biosciences Carlsbad, CA USA) and the phylogenetic analysis was carried out using mega 5.2 (Center for Evolutionary Medicine and Informatics, The Biodesign Institute, Tempe, Arizona, USA) software programme. The aligned nucleotide sequences of 10 positive D. congolensis isolates had between 94% to 99% homology with the sequences of D. congolensis satellite DNA in GenBank. This result also revealed that the sequenced D. congolensis are of different strains. Phylogenetic analysis revealed that D. congolensis, though closely related to Nocardia brasiliensis (NR 074743.01) and Streptomyces sp. (JN 400114.1), belongs to different genus. In conclusion, molecular tools employed in the study were able to confirm the identity of the test organisms as D. congolensis. It can also be concluded that two strains of D. congolensis obtained from the study can still be accommodated within the previously listed strains available in GenBank while the remaining eight may be different strains of D. congolensis not yet listed in GenBank.