Rapid detection and molecular epidemiology of ß-lactamase producing Enterobacteriaceae isolated from food animals and in-contact humans in Nigeria.

The emergence and spread of ß-lactamase-producing Enterobacteriaceae poses a significant threat to public health, necessitating the rapid detection and investigation of the molecular epidemiology of these pathogens. We modified a multiplex real-time (RT)-PCR to concurrently detect ß-lactamase genes (blaCTX-M, blaTEM, and blaSHV) and Enterobacteriaceae 16S ribosomal RNA. qPCR probes and primers were validated using control isolates, and the sensitivity and specificity assessed. The optimised multiplex qPCR was used to screen 220 non-clinical Enterobacteriaceae from food animals and in-contact humans in Southeast Nigeria selected on cefotaxime-supplemented agar plates. Binary logistic regression was used to explore factors associated with the presence of the blaTEM and blaSHV genes in these isolates, and a subset of isolates from matched sampling sites and host species were whole genome sequenced, and their antimicrobial resistance (AMR) and plasmid profiles determined. The sensitivity and specificity of the qPCR assay was 100%. All isolates (220/220) were positive for Enterobacteriaceae ribosomal 16S rRNA and blaCTX-M, while 66.4% (146/220) and 9% (20/220) were positive for blaTEM and blaSHV, respectively. The prevalence of blaTEM and blaSHV varied across different sampling sites (farm, animal market and abattoirs). Isolates from Abia state were more likely to harbour blaTEM (OR = 2.3, p = 0.04) and blaSHV (OR = 5.12,p = 0.01) than isolates from Ebonyi state; blaTEM was more likely to be detected in isolates from food animals than humans (OR = 2.34, p = 0.03), whereas the reverse was seen for blaSHV (OR = 7.23, p = 0.02). Furthermore, Klebsiella and Enterobacter isolates harboured more AMR genes than Escherichia coli, even though they were isolated from the same sample. We also identified pan resistant Klebsiella harbouring resistance to ten classes of antimicrobials and disinfectant. Therefore, we recommend ESKAPE pathogens are included in AMR surveillance in future and suggest qPCRs be utilised for rapid screening of Enterobacteriaceae from human and animal sources.

Detection of multidrug-resistant Campylobacter species from food-producing animals and humans in Nigeria: Public health implications and one health control measures.

Antimicrobial-resistant thermophilic Campylobacter species (TCS) pose tremendous public health problems because they are zoonotic, difficult to treat and usually harboured by food-producing animals (FPAs). This study ascertained the phenotypic antimicrobial resistance (AMR) in 56 phenotypically identified TCS from slaughtered cattle, poultry, and humans in Enugu State, Nigeria. The presence of selected AMR and virulence genes harboured by the animal and human isolates were also detected and compared in 36 PCR-confirmed Campylobacter species. All the 56 TCS were multidrug-resistant as none were susceptible to ampicillin, penicillin-G, amoxicillin-clavulanic acid, cephalothin and metronidazole. The isolates were 92.9 %, 62.5 %, 92.9 %, 42.9 %, 26.8 %, 25 %, 28.6 %, 53.7 %, 30.1 %, 32.1 % and 55.4 % resistant to ceftriaxone, nalidixic acid, cefotaxime, enrofloxacin, ciprofloxacin, streptomycin, gentamycin, erythromycin, azithromycin, chloramphenicol and tetracycline, respectively. The top four most effective classes of antimicrobials were aminoglycosides > macrolides > amphenicol > fluoroquinolones. The AMR genes detected and the percentage of the isolates that harboured them were: aadE-1 (33.3 %), aphA-3-1 (36.1 %), tetO (44.4%), Blaoxa-61 (61.1 %) and the multidrug efflux pump, cmeB (86.1%). Virulence genes detected and the corresponding percentage of TCS that harboured them were: cdtB (61.1 %), flaA (47.2 %), ciaB (38.9 %), and pldA (38.9 %). The cmeB was significantly detected in animal isolates (p = 0.018, OR = 5.1, CI = 0.7-6.6) while BlaOXA-61 predominated in human isolates (p = 0.019, OR = 6.2). Likewise, ciaB virulence gene was mostly detected (p = 0.019, OR = 6.4, CI = 1.3-25) in animal isolates. The findings underscore the roles of FPAs in the zoonotic dissemination of Campylobacter-associated AMR and virulence genes in the study area. This warrants the adoption of One Health control strategies to limit spread of the multidrug-resistant zoonotic Campylobacter species.

Prevalence and Retrospective Insight on Tuberculosis in Human Patients in Cross River State, Nigeria; One Health Approach to its Control.

Tuberculosis (TB) is a major zoonotic disease of public health importance worldwide. Its burden is more in developing countries due to inadequate surveillance, co-infection with HIV/AIDS and poor social amenities; hence consumption of unpasteurized milk, contaminated meat and inhalation of infected droplets from animals or patients with active TB are the major risk practices. A survey was conducted on TB prevalence from 2013 to 2014 while patients records from TB and Leprosy units of hospitals in the three Agricultural zones (Ogoja, Ikom and Calabar) of Cross River State, Nigeria were assessed from 2000 to 2010. Out of 1,170 sampled patients, 8 (0.7%) were positive, of which 6 and 2 isolates were identified as M. tuberculosis and M. bovis, respectively. Out of 52,558 studied patients files, 235 (0.4%) were positive with varied annual prevalence; the highest (1.0%) and lowest (0.2%) in 2009 and 2011, respectively. The prevalence was higher in dry (0.9% and 0.5%) than the rainy season (0.5% and 0.4%), in females (0.9% and 0.5%) than the males (0.4% and 0.4%) in the survey and retrospective studies, respectively. The age distribution of TB among the patients were; (0% and 0.3%), (0.4% and 0.4%), (0.7% and 0.4%) and (1.5% and 0.7%) for those (≤ 18), (19-40), (41-60) and (> 60) yrs old in the survey and retrospective study, respectively. TB is prevalent in human patients in Cross River State hence, the need for sustainable campaign, continuous surveillance and private/ public health partnership in accurate and early diagnosis, treatment and one health approach to its control.

Molecular characterization of extended spectrum cephalosporin resistant Escherichia coli isolated from livestock and in-contact humans in Southeast Nigeria.

The rise in antimicrobial resistance (AMR) in bacteria is reducing therapeutic options for livestock and human health, with a paucity of information globally. To fill this gap, a One-Health approach was taken by sampling livestock on farms (n = 52), abattoir (n = 8), and animal markets (n = 10), and in-contact humans in Southeast Nigeria. Extended spectrum cephalosporin (ESC)-resistant (ESC-R) Escherichia coli was selectively cultured from 975 healthy livestock faecal swabs, and hand swabs from in-contact humans. Antimicrobial susceptibility testing (AST) was performed on all ESC-R E. coli. For isolates showing a multi-drug resistance (MDR) phenotype (n = 196), quantitative real-time PCR (qPCR) was performed for confirmation of extended-spectrum ß-lactamase (ESBL) and carbapenemase genes. Whole-genome sequencing (WGS) was performed on a subset (n = 157) for detailed molecular characterisation. The results showed ESC-R E. coli was present in 41.2% of samples, with AST results indicating 48.8% of isolates were phenotypically MDR. qPCR confirmed presence of ESBL genes, with bla CTX-M present in all but others in a subset [bla TEM (62.8%) and bla SHV (0.5%)] of isolates; none harboured transferable carbapenemase genes. Multi-locus sequence typing identified 34 Sequence Types (ST) distributed among different sampling levels; ST196 carrying bla CTX-M-55 was predominant in chickens. Large numbers of single nucleotide polymorphisms (SNPs) in the core genome of isolates, even within the same clade by phylogenetic analysis, indicated high genetic diversity. AMR genotyping indicated the predominant bla CTX-M variant was bla CTX-M-15 (87.9%), although bla CTX-M-55, bla CTX-M-64, and bla CTX-M-65 were present; it was notable that bla CTX-M-1, common in livestock, was absent. Other predominant AMR genes included: sul2, qnrS1, strB, bla TEM-1b, tetA-v2, and dfrA14, with prevalence varying according to host livestock species. A bla CTX-M-15 harbouring plasmid from livestock isolates in Ebonyi showed high sequence identity to one from river/sewage water in India, indicating this ESBL plasmid to be globally disseminated, being present beyond the river environment. In conclusion, ESC-R E. coli was widespread in livestock and in-contact humans from Southeast Nigeria. WGS data indicated the isolates were genetically highly diverse, probably representing true diversity of wild type E. coli; they were likely to be MDR with several harbouring bla CTX-M-15. Surprisingly, human isolates had highest numbers of AMR genes and pigs the least.

Antimicrobial resistance in Escherichia coli isolates from frugivorous (Eidolon helvum) and insectivorous (Nycteris hispida) bats in Southeast Nigeria, with detection of CTX-M-15 producing isolates.

Thirty-five Escherichia coli isolates obtained from the liver, spleen and intestines of 180 frugivorous and insectivorous bats were investigated for antimicrobial resistance phenotypes/genotypes, prevalence of Extended-Spectrum beta-lactamase (ESBL) production, virulence gene detection and molecular typing. Eight (22.9 %) of the isolates were multidrug resistant (MDR). Two isolates were cefotaxime-resistant, ESBL-producers and harbored the blaCTX-M-15 gene; they belonged to ST10184-D and ST2178-B1 lineages. tet(A) gene was detected in all tetracycline-resistant isolates while int1 (n = 8) and blaTEM (n = 7) genes were also found. Thirty-three of the E. coli isolates were assigned to seven phylogenetic groups, with B1 (45.7 %) being predominant. Three isolates were enteropathogenic E. coli (EPEC) pathovars, containing the eae gene (with the variants gamma and iota), and lacking stx1/stx2 genes. Bats in Nigeria are possible reservoirs of potentially pathogenic MDR E. coli isolates which may be important in the ecology of antimicrobial resistance at the human-livestock-wildlife-environment interfaces. The study reinforces the importance of including wildlife in national antimicrobial resistance monitoring programmes.

Seroprevalence and Risk Factors of Brucellosis in Dogs in Enugu and Anambra States, Nigeria / Seroprevalencia y factores de riesgo de la brucelosis en perros de los Estados Enugu y Anambra, Nigeria

Abstract Seroprevalence of the antibodies of Brucella canis and Brucella abortus in dogs was assessed using a cross-sectional survey in Anambra and Enugu States, Nigeria. A total of 123 Companion dogs made up of 65 clinic dogs, 34 slaughter dogs and 24 household dogs were screened. For B. abortus antibody assay, the collected serum was used for Rose Bengal plate test (RBPT), Serum agglutination test (SAT) and Solid Phase Immunoassay technique with Immunocomb® Canine Brucellosis Antibody Test Kit was used. Out of the 123 dogs screened, none was positive for Brucella abortus antibodies while 34 (27.7%) of the dogs screened were positive for B. canis antibodies. There was a significant association (P<0.05) between infection and sex, the infection was significantly higher (P<0.05) in female than male dogs. Prevalence was significantly higher (P<0.05) in Exotic breeds than in mixed and local dog breeds. There was no association (P>0.05) between infection and antibody titre levels in the different categories of dogs. However, there was significant association (P<0.05) between the presence of Brucella canis antibodies and free roaming of dogs. This study provides the first serological evidence of B. canis infection in dogs in Enugu and Anambra States. This shows that B. canis is endemic in both states, underscoring the need for further studies. Female dogs, exotic breeds and freely roaming dogs are at a higher risk of Brucella infection in the study area; therefore, preventive and control measures are strongly recommended.

Resumen Se evaluó la seroprevalencia de los anticuerpos de Brucella canis y Brucella abortus en perros usando un sondeo transversal en los Estados Anambra and Enugu, Nigeria. Se examinó un total de 123 perros de compañía, de los cuales 65 eran perros de clínica, 34 perros de matadero y 24 perros caseros. Para el ensayo de anticuerpos de B. abortus, el suero muestreado se usó para la prueba de Rosa de Bengala (RBPT), prueba de aglutinación del suero (SAT) y se usó la técnica de inmunoensayo en fase sólida con el kit de prueba de anticuerpos para brucelosis canina Immunocomb®. De los 123 perros analizados, ninguno dio positivo para los anticuerpos de Brucella abortus mientras que 34 (27.7%) de los perros analizados dieron positivo para los anticuerpos de B. canis. Hubo una asociación significativa (P<0.05) entre infección y género; la infección fue significativamente más alta (P<0.05) en las hembras que en los machos. La prevalencia fue significativamente más alta (P<0.05) en las razas exóticas que en las razas cruzadas y las razas locales. No hubo ninguna relación (P>0.05) entre la infección y los niveles de titulación de anticuerpos en las diferentes categorías de perros. Sin embargo, hubo una relación significativa (P<0.05) entre la presencia de anticuerpos Brucella canis y los perros que andan libremente por doquier. Este estudio provee la primera evidencia serológica de infección con B. canis en perros de los Estados Enugu y Anambra. Esto muestra que la B. canis es endémica en ambos estados, enfatizando la necesidad de hacer más estudios. Las hembras, las razas exóticas y los animales que deambulan libremente se encuentran en el riesgo más alto de infección con Brucella en el área de estudio; por consiguiente, se recomienda enormemente tomar medidas preventivas y de control.

Molecular epidemiology, genetic diversity and antimicrobial resistance of Staphylococcus aureus isolated from chicken and pig carcasses, and carcass handlers.

The epidemiology of Staphylococcus aureus in food animals, associated products, and their zoonotic potential in Nigeria are poorly understood. This study aimed to provide data on the prevalence, genetic characteristics and antimicrobial resistance of S. aureus isolated from chicken and pig carcasses, and persons in contact with the carcasses at slaughterhouses in Nigeria. Surface swabs were collected randomly from 600 chicken and 600 pig carcasses. Nasal swabs were collected from 45 workers in chicken slaughterhouses and 45 pig slaughterhouse workers. S. aureus isolates were analyzed by spa typing. They were also examined for presence of the Panton-Valentine Leucocidin (PVL) and mecA genes, as well as for antimicrobial resistance phenotype. Overall, 53 S. aureus isolates were recovered (28 from chicken carcasses, 17 from pig carcasses, 5 from chicken carcass handlers and 3 from pig carcass handlers). Among the isolates, 19 (35.8%) were PVL-positive and 12 (22.6%) carried the mecA gene. The 53 isolates belonged to 19 spa types. The Based Upon Repeat Pattern (BURP) algorithm separated the isolates into 2 spa-clonal complexes (spa-CC) and 9 singletons including 2 novel spa types (t18345 and t18346). The clonal complexes (CC) detected were CC1, CC5, CC8, CC15, CC88 and CC152. CC15-related isolates represented by spa type t084 (32.1%) and CC5 represented by spa type t311 (35.3%) predominated among isolates from chicken carcasses/ handlers, and pig carcasses/ handlers, respectively. Multidrug resistance exhibited by all the CC except CC8, was observed among isolates from chicken carcasses (64.3%), pig carcasses (41.2%), handlers of chicken meat (40.0%) and handlers of pork (33.3%). All the CC showed varying degrees of resistance to tetracycline while CC15 and CC5 exhibited the highest resistance to sulphamethoxazole/trimethoprim and erythromycin, respectively. The predominant antimicrobial resistance pattern observed was penicillin-tetracycline-sulphamethoxazole/trimethoprim (PEN-TET-SXT). In conclusion, food animals processed in Enugu State in Southeast Nigeria are potential vehicles for transmission of PVL-positive multiple-drug resistant S. aureus and methicillin-resistant S. aureus from farm to slaughterhouse and potentially to the human population. Public health intervention programs at pre- and post-slaughter stages should be considered in Nigerian slaughterhouses.

Prevalence, toxigenic potential and antimicrobial susceptibility profile of Staphylococcus isolated from ready-to-eat meats.

AIM: An epidemiological surveillance for Staphylococci contamination of ready-to-eat (RTE) meats from Enugu State, Nigeria, was carried out to determine the prevalence, species distribution, toxigenic potential and antimicrobial susceptibility profile of the organisms and hence the microbiological and toxicological safety of the meats. MATERIALS AND METHODS: Isolation and phenotypic Staphylococcus detection were done according to standard microbiological methods. Phenotypic resistance to 17 commonly used antimicrobial agents was determined by disc diffusion method. Molecular characterization of the isolates to species level and detection of selected toxigenic and antimicrobial-resistance genes were done by PCR methods. RESULTS: Twenty-four (9.4%) of the 255 meat samples investigated were contaminated with Staphylococcus species. Twenty-four Staphylococcus isolates belonging to six species of coagulase-negative Staphylococcus (CoNS) were identified. Four (16.7%) isolates harbored genes coding for exfoliative toxin-A. Ten (41.7%) isolates were multidrug resistant, while mecA, tetK, mphC, ermT and ermC were the antimicrobial-resistance genes detected in the isolates. Meat samples sourced from motor parks (16.7%) and open markets (8.5%) were the most contaminated. CONCLUSION: 9.4% of RTE meats sampled were contaminated with toxigenic and multidrug resistance CoNS. Beef was the most contaminated RTE meat type and harbored all the toxigenic and most of the antibiotic-resistant genes detected. Meat samples from motor parks had the highest staphylococcal contamination (16.7%), while those from mechanic village had the least (2.4%). Majority (79.2%) of the isolates were not susceptible to fusidic acid but none exhibited antimicrobial-resistance to chloramphenicol, ciprofloxacin, linezolid or teicoplanin. Food safety authorities in the study area should work proactively to massively improve the hygienic practices of meat vendors; in order to limit staphylococcal contamination of RTE meats and the associated public health problems.

Assessment of antimicrobial drug administration and antimicrobial residues in food animals in Enugu State, Nigeria.

Imprudent administration of antimicrobial drugs in food-producing animals can facilitate the development and spread of antimicrobial-resistant organisms and also enhance the occurrence of antimicrobial residue in animal products. This study was undertaken to assess antimicrobial drug administration to food animals in livestock farms in Enugu State and determine livestock farmers' awareness on the consequences of imprudent antimicrobial administration to food animals and finally the prevalence of antimicrobial drug residues in edible tissues of cattle and pigs in the state. Structured questionnaire was used to extract information on antimicrobial drug administration and consequences of irresponsible use of antimicrobials in food animals from 109 livestock farms/farmers randomly selected using multi-stage sampling technique. Premi® test technology (R-Biopharm, Germany) was used to screen for antimicrobial residues in edible tissues from 300 carcasses consisting of 165 cattle and 135 pigs slaughtered for human consumption in two major slaughterhouses in Enugu State. Tetracyclines (90.8%), penicillins and beta-lactams (89.9%), and aminoglycoside (57.8%) were the classes of antimicrobials most frequently administered to food animals in the farms surveyed. Withdrawal period was not observed in 65% of the farms. About 30% of cattle and 23% of pig carcasses screened contained detectable amounts of antimicrobial residues. There is widespread indiscriminate administration of antimicrobial drugs in food animals in Enugu State. This underscores the need for public enlightenment on prudent use of antimicrobial drugs in food-producing animals in order to preserve the therapeutic efficacy for sustainable livestock production and to safeguard human health.

Methicillin-resistant coagulase-negative staphylococci from healthy dogs in Nsukka, Nigeria.

The occurrence, resistance phenotype and molecular mechanisms of resistance of methicillin-resistant staphylococci from groin swabs of 109 clinically healthy dogs in Nsukka, Nigeria were investigated. The groin swab samples were cultured on mannitol salt agar supplemented with 10 µg of cloxacillin. Sixteen methicillin-resistant coagulase negative staphylococci (MRCoNS), all harbouring the mecA gene were isolated from 14 (12.8%) of the 109 dogs studied. The MRCoNS isolated were: S. sciuri subspecies rodentium, S. lentus, S. haemolyticus, and S. simulans with S. sciuri subspecies rodentium (62.5%) being the predominant species. Thirteen (81.3%) of the MRCoNS were resistant to tetracycline while 12 (75%) and 10 (62.5%) were resistant to kanamycin and trimthoprim-sulphamethoxazole respectively. None of the isolates was resistant to fusidic acid, linezolid and vancomycin. Thirteen (81.3%) of the MRCoNS were multi-drug resistance (MDR). Other antimicrobial genes detected were: blaZ, tet(K), tet(M), tet(L), erm(B), lnu(A), aacA-aphD, aphA3, str, dfr(G), cat pC221 , and cat pC223 . Methicillin-resistant staphylococci are common colonizers of healthy dogs in Nigeria with a major species detected being S. sciuri subsp. rodentium.

Comparative assessment of postmortem inspection and immunochromatographic techniques for the detection of bovine tuberculosis in slaughter cattle in Nigeria.

Animals with tuberculosis pose some risks to humans, especially in developing countries of the world. In this study, postmortem inspection (PMI) and immunochromatographic assay (ICA) techniques were compared for the detection of bovine tuberculosis (BTB) in slaughter cattle in Enugu State, Nigeria using culture as the gold standard. A cross-sectional study was conducted from January-June, 2011 on animals presented at four purposively selected slaughterhouses in the study area, involving a total of 500 randomly selected animals. Blood samples were collected from the jugular veins of selected animals and serum samples harvested for ICA. Thorough PMI was carried out and tissue samples from the lung, liver, intestine, and lymph nodes were collected, with or without lesions for culture; from the animals examined, culture detected 11 positive cases giving a prevalence rate of 2.2 %, whereas PMI detected 22 positive cases including 7 (out of the 11) positive cases detected by culture, giving a prevalence rate of 4.4 %. Fifteen of the cases detected as positive by PMI were negative by culture. Therefore, the sensitivity and specificity of PMI were 64 and 97 %, respectively. ICA detected 59 positive cases including 10 of the 11 positive cases detected by culture, hence, a prevalence rate of 11.8 %. Forty-nine of the cases detected as positive by ICA were negative by culture. Hence, the sensitivity and specificity of ICA were 91 and 90 %, respectively. In conclusion, the performance of ICA was found sufficiently high to support its use in BTB surveillance and control in cattle in Enugu State, Nigeria.

Methicillin-resistant coagulase-negative staphylococci from healthy dogs in Nsukka, Nigeria

The occurrence, resistance phenotype and molecular mechanisms of resistance of methicillin-resistant staphylococci from groin swabs of 109 clinically healthy dogs in Nsukka, Nigeria were investigated. The groin swab samples were cultured on mannitol salt agar supplemented with 10 µgof cloxacillin. Sixteen methicillin-resistant coagulase negative staphylococci (MRCoNS), all harbouring the mecA gene were isolated from 14 (12.8%) of the 109 dogs studied. The MRCoNS isolated were: S. sciuri subspecies rodentium, S. lentus, S. haemolyticus, and S. simulans with S. sciuri subspecies rodentium (62.5%) being the predominant species. Thirteen (81.3%) of the MRCoNS were resistant to tetracycline while 12 (75%) and 10 (62.5%) were resistant to kanamycin and trimthoprim-sulphamethoxazole respectively. None of the isolates was resistant to fusidic acid, linezolid and vancomycin. Thirteen (81.3%) of the MRCoNS were multi-drug resistance (MDR). Other antimicrobial genes detected were: blaZ, tet(K), tet(M), tet(L), erm(B), lnu(A), aacA-aphD, aphA3, str, dfr(G), cat pC221,and cat pC223. Methicillin-resistant staphylococci are common colonizers of healthy dogs in Nigeria with a major species detected being S. sciuri subsp. rodentium.

Prevalence of antimicrobial residues in raw table eggs from farms and retail outlets in Enugu State, Nigeria.

The use of antimicrobial agents in poultry production results in their accumulation in the body tissues and products such as milk and egg. The subsequent accumulation of these drugs and their metabolites in body cells is known as drug residue. This study was designed to determine the prevalence of antimicrobial residues in eggs from poultry farms and retail outlets in Enugu State, Nigeria. Eggs from 25 selected commercial farms and ten retail outlets were screened for the prevalence of antimicrobial residue. Also, structured questionnaires were administered to 25 commercial farms in the state to determine the management practices and the most widely used antimicrobial drugs in farms and possible association between the management practices and the occurrence of antimicrobial residues in eggs from these farms. All the 25 farms surveyed use oxytetracycline. Eggs from nine of the surveyed farms tested positive for antimicrobial residue and three of the ten surveyed farms also tested positive for antimicrobial residue. No association was observed (p 0.05; Fisher's exact test) between the occurrence of antibiotic residues in eggs and farm size, feed source and housing systems. This study was able to demonstrate the presence of antimicrobial residues in eggs destined for human consumption. Drugs like nitrofurans which has been banned for use in food animals are still very much in use in Enugu State, Nigeria. Antibiotics given as feed additives may give rise to drug residues in food animals.