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.

Antimicrobials in animal production: usage and practices among livestock farmers in Oyo and Kaduna States of Nigeria.

Antimicrobials have proven to be important for sustainable livestock production by their use as growth promoters and in the control of animal infections. However, injudicious use of antimicrobials could accelerate the emergence and spread of resistant bacterial strains with attendant socioeconomic and public health issues. This work assessed antimicrobial usage in animal production with emphasis on usage and practices by livestock producers in Oyo and Kaduna States of Nigeria. Data on antimicrobial usage were collected through interviews, questionnaire and focus group discussions. Four hundred and fifty-four farmers in 11 communities within 11 Local Government Areas of Oyo and Kaduna States of Nigeria were sampled in a multi-stage sampling procedure. The study showed that antimicrobial agents were widely distributed, readily accessible and commonly used in animal production. Fluoroquinolones and other critically important antimicrobials for human medicine were widely used in animals as prophylactics. Potentially harmful antimicrobials including furazolidones and chloramphenicol already banned for use in humans and animals were freely marketed and used in livestock production. Most of the respondents believed that veterinarians should be responsible for the administration of antimicrobials to animals, but in practice, they buy and administer antimicrobials without consulting veterinary professionals. It was observed that the ready availability of antimicrobial agents promoted the use of antimicrobials in livestock production and may encourage non-adherence to hygienic principles and management laxity in farm operations. The non-involvement of veterinary professionals and laboratory investigations in disease diagnosis prior to antimicrobial use could lead to improper usage that contribute to the development of antimicrobial resistance in bacterial strains. Responsible antimicrobial stewardship and strict regulations are vital to prolonging the benefits derivable from the use of antimicrobials.

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.

Multidrug resistant enterohaemorrhagic Escherichia coli serogroups in the faeces of hunted Wildlife, Abeokuta, Nigeria.

Wildlife play significant roles in the dissemination and zoonotic transmission of pathogens. The enterohaemorrhagic Escherichia coli (EHEC) are associated with complicated cases of food-borne illnesses. This study investigated the presence of EHEC serogroups (O26, O45, O103, O145, O91, O111, O128, O121 and O157) in wildlife species: cane rats (Thryonomys swinderianus), royal antelope (Neotragus pygmaeus), African giant rats (Cricetomys gambianus) and waterbuck (Kobus ellipsiprymnus). EHEC and non-EHEC isolates from these wildlife sources were tested for susceptibility to antimicrobial agents. Overall, 127 (83.0 %) of 153 samples yielded E. coli. Nine (5.9%) samples were positive for EHEC belonging to three serogroups as follows; O26 (n=2), O111 (n=2) and O103 (n=5). The EHEC isolates were from cane rats (n=6) and royal antelope (n=3) and possessed virulence-associated genes stx1 (77.8%), stx2 (100.0%), eaeA (100.0%) and hlyA (100.0%). Overall, 127 E. coli isolates showed resistance to ampicillin (99.2%), ceftiofur (90.6%),  tetracycline (90.0%), cephalexin (87.4%), cefotaxime (50.4%), streptomycin 42.5%, ceftazidime (41.7%), nalidixic acid (37.0%),  ciprofloxacin (43.6%), amoxicillin/clavulanic acid (32.3%), gentamicin (27.6%), sulphamethoxazole/trimethoprim (25.2%), norfloxacin (17.3%) and chloramphenicol (11.0%). The roles of wildlife in the dissemination and transmission of antimicrobial resistant and zoonotic bacteria should not be neglected for effective preventive and control strategies.

Connective tissue disease--associated interstitial lung disease: an underreported cause of interstitial lung disease in Sub-Saharan Africa.

Interstitial lung disease (ILD) occurs in 15% of connective tissue disease (CTD) patients causing considerable morbidity and mortality. Data is scarce regarding its clinical characteristics and outcomes in Africa. We aim to study the frequency, clinico-radiological characteristics, and treatment outcomes of African CTD-ILD patients. A retrospective cross-sectional study of ILD among 318 CTD patients diagnosed using relevant ACR criteria at the rheumatology unit of Lagos State University Teaching Hospital (LASUTH), Lagos from 2012 to 2019. Socio-demographics, clinical features, radiological findings, and treatment outcomes were documented. Data was analyzed using SPSS version 21 with p < 0.05. The LASUTH ethics committee approved the study. Interstitial lung disease occurred in 31 (9.7%) of 318 CTD cases. Their mean age was 38.8 ± 13.3 years, range 19-68 years with 28 (90.3%) females. Proportions of CTD-ILD were Sjogren's syndrome (50%), UCTD (50%), systemic sclerosis (46.7%), MCTD (33.3%), PM/DM (25%), SLE (6.5%), and RA (2.6%). Commonest presentations were cough (93.5%) and bibasal inspiratory crackles (83.9%) with a restrictive pattern in 83.9%. Antinuclear antibody occurred in 100% and anti-ENA in 67.7%. Traction bronchiectasis (89.7%) and ground glass opacities (96.6%) were frequent HRCT findings. Treatments included pulse-dose prednisolone, cyclophosphamide, mycophenolate mofetil, pirfenidone, and rituximab. Outcomes were ambulatory oxygen therapy (12.9%) and mortality (16.1%) with 9.7% lost to follow-up. CTD-ILD is a female predominant disease occurring in 9.7% of CTD patients mostly those with Sjogren's syndrome and systemic sclerosis. Due to significant morbidity and mortality, we advocate routine ILD screening for all CTD patients including those with undifferentiated disease. Key Points: • Interstitial lung disease occurs in 9.7% of patients with underlying connective tissue disease. • Females are predominantly affected especially those with Sjogren's syndrome and systemic sclerosis. • Mortality occurs in roughly 1 in every 6 patients with CTD-ILD.

Antimicrobial Resistance and Virulence of Methicillin-Resistant Staphylococcus aureus from Human, Chicken and Environmental Samples within Live Bird Markets in Three Nigerian Cities.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major threat to public health. This study investigated the occurrence of MRSA in humans, chickens, chicken meat and environmental samples within poultry farms and live bird markets in southwestern Nigeria. Methods: MRSA were isolated using selective culture and tested for antimicrobial susceptibility by broth microdilution. Selected isolates were characterized by whole genome sequencing (WGS). From WGS data, spa, dru, multilocus sequence typing (MLST) and SCCmec types, but also virulence and antimicrobial resistance genes, were identified. Results: Fifty-six MRSA isolates were detected in 734 samples. They showed resistance to ß-lactams (100%), tetracycline (60.7%), ciprofloxacin (33.9%), erythromycin (28.6%), gentamicin (32.1%), and trimethoprim/sulfamethoxazole (10.7%). All 30 isolates investigated by WGS carried mecA, dfrG, and tet(38) genes. Other resistance genes detected were blaZ (83.3%), fosB (73.3%), tet(K) (60.0%), aacA-aphD (36.6%), aphA3 (33.3%), msr(A) (30.0%), mph(C) (30.0%), dfrS1 (3.3%), and sat4 (3.3%). Seven spa types (t091, t314, t657, t1476, t2331, t4690 and t12236), four known (dt9aw, dt10ao, dt10cj, and dt11a) and two novel (dt10dr and dt11dw) dru types, as well as five sequence types (ST8, ST121, ST152, ST772 and ST789) were found among the MRSA isolates. All ST121 isolates carried an SCCmec type IV cassette and were not dru-typeable. ST152 and ST121 were found only in specific sample categories within defined locations, while ST8 and ST772 were distributed across most sample categories and locations. Three SCCmec types, IVa, V and Vc, were identified. All MRSA isolates possessed virulence genes including aur, clpP, coa, fnbA, esaA, hly, hla, ica, isdA, srtB, sspA, and vWbp, among others. The toxic shock syndrome toxin gene (tst) was not detected in any isolate, whereas the Pantone-Valentine leukocidin genes lukF-PV/lukS-PV were present in all ST121, all ST772, and all but one ST152 isolates. Conclusion: The results of this study (i) showed that chicken meat is contaminated by MRSA and (ii) suggested that live bird markets may serve as focal points for the dissemination of MRSA within the community.

Standardized outcome measures for cerebral palsy among physiotherapists in southwestern Nigeria: awareness, use, barriers, and facilitators.

The use of outcome measures for assessing progress in the management and treatment of cerebral palsy (CP) is widely recommended. This study was, therefore, carried out to determine awareness and use of standardized outcome measures among physiotherapists managing CP in Nigeria. Barriers to and facilitators for the use of outcome measures were also investigated. This was a descriptive study involving 138 physiotherapists from selected hospitals in southwestern Nigeria. A self-administered questionnaire was used to obtain relevant information on socio-demographics, awareness, use, barriers to, and facilitators for the use of seven standardized outcome measures. The Gross Motor Function Measure was the most recognized (78.9%) and commonly used (58%) outcome measure, while the Paediatric Outcomes Data Collection Instrument (23.2%) and the Paediatric Evaluation of Disability Inventory (10.9%) were the least recognized and least used, respectively. The greatest perceived facilitators were familiarity (87.7%), positive attitude (87.7%), and that outcome measures allow for a balanced clinical assessment (89.1%). The greatest perceived barriers identified were the need for extra accommodation to apply outcome measures (63%) and time consumption on the part of patients (44.2%). Many physiotherapists in this study identified the standardized outcome measures, but fewer used them irrespective of educational status and years of work experience. Generally, there was a positive attitude toward the use of outcome measures. The use of outcome measures should be promoted among physiotherapists in Nigeria, through training programs and translation into the native languages, to effectively assess, manage, and monitor the progress of patients with CP, putting into consideration barriers and facilitators.

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.

Shiga toxin-producing Escherichia coli O157:H7 in milk and milk products in Ogun State, Nigeria.

Shiga toxin producing Escherichia coli (STEC) O157 is a major cause of food-borne illnesses in humans. This study investigated the presence of STEC O157 in milk and milk products in Ogun State, Nigeria. Of a total of 202 samples 10 (5%) were positive for STEC O157 including 1 (2%) of 50 raw milk samples, 3 (6%) of 50 samples of fresh local cheese, 1 (2%) of 50 samples of fried local cheese and 5 (9.6%) of 52 fermented milk samples. There was no significant difference (p>0.05) in the prevalence of STEC O157 among the sample types. Of 10 isolates, shiga toxin 1 gene (stx1) was detected only in 2 samples (20%), shiga toxin 2 (stx2) was extracted only in 6 samples (60%), stx1 /stx2 in 2 samples (20.0%), intimin gene (eaeA) in 5 samples (50%), and enterohaemolysin (E-hlyA) gene was isolated in 7 (70%) samples. Rates of resistance of the STEC O157 isolates were: amoxicillin/clavulanic acid 100%, ampicillin 100%, chloramphenicol 60%, nalidixic acid 20%, norfloxacin 10%, streptomycin 30%, sulphamethoxazole/trimethprim 20%, and tetracycline 90%. The isolates were all susceptible to ciprofloxacin and neomycin. The presence of virulent multidrug resistant E. coli O157 strains in milk and milk products as revealed by this study unveils a risk of human exposure to these potentially fatal pathogens following consumption of contaminated products.