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.

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.

Knowledge and practices regarding antibiotic use among small-scale poultry farmers in Enugu State, Nigeria.

This study examined the knowledge and practices regarding antibiotic use among small-scale poultry farmers in Enugu State, Nigeria. A multistage sampling technique was employed to select 88 poultry farmers. The interview schedule was used for data collection. Respondents' indices of knowledge of antibiotic use (KABU), antibiotic resistance (KABR) and antibiotic use practices (PABU) were determined. Binary logistic regression was performed to ascertain the effect of socio-demographics of respondents, knowledge of antibiotic use and knowledge of antibiotic resistance on the likelihood that farmers use antibiotics inappropriately. All poultry farmers studied used antibiotics for growth promotion, disease prevention, and treatment. The mean index of KABU was 0.54 with 48 % of the respondents having good KABU while the mean index of KABR was 0.65 and 70.5 % of the farmers had good KABR. The farmers' mean index of PABU was 0.47 and 83 % of them used antibiotics inappropriately. Farmers with good KABU (OR = 4.2; 95% CI = 1.030-17.222) and KABR (OR = 4.5; 95% CI = 1.258-15.791) were more likely to misuse antibiotics than those with poor knowledge. Antibiotics are routinely, and on many occasions inappropriately, used in small-scale poultry production in Enugu State, Nigeria. Antibiotics are valuable agents whose efficacy can only be preserved if they are handled with care. Training small-scale farmers will allow them to improve their knowledge and practices regarding antibiotic use.

Peste des petits ruminants in Nigeria: identification and variations of lineage II and lineage IV in sheep and goats

Peste des petits ruminants virus (PPRV) is the aetiologic agent of Peste des petits ruminants (PPR), an important viral disease of sheep and goats. PPR is endemic in Nigeria and leads to social and economic losses. The objective of this study was to determine the prevalence of PPR infection and genetically characterize PPRV strains obtained from sheep and goats in three States of Southeast Nigeria. A total of 285 nasal swab samples collected in 2017­2018 were processed for PPRV genome detection by RT­PCR. Sixty­five (22.81%) of the samples were positive for PPRV. Sequence and phylogenetic analyses revealed that the PPRV belonged to lineages II (11/38, 28.9%) and IV (27/38, 71.1%). The N gene fragment sequence showed a 99.77%­100% and 99.98%­100% identity among the strains of lineages II and IV, respectively. Fourteen amino acid substitutions, previously unreported in PPRV strains from Nigeria, were recorded. This study confirms the circulation of PPRV lineages II and IV in Southeast Nigeria, the dominance of strains belonging to lineage IV in recent years, and their close genetic relationship with those previously reported in other parts of Nigeria and neighboring countries.

Potential sources and characteristic occurrence of mobile colistin resistance (mcr) gene-harbouring bacteria recovered from the poultry sector: a literature synthesis specific to high-income countries.

Understanding the sources, prevalence, phenotypic and genotypic characteristics of mcr gene-harbouring bacteria (MGHB) in the poultry sector is crucial to supplement existing information. Through this, the plasmid-mediated colistin resistance (PMCR) could be tackled to improve food safety and reduce public health risks. Therefore, we conducted a literature synthesis of potential sources and characteristic occurrence of MGHB recovered from the poultry sector specific to the high-income countries (HICs). Colistin (COL) is a last-resort antibiotic used for treating deadly infections. For more than 60 years, COL has been used in the poultry sector globally, including the HICs. The emergence and rapid spread of mobile COL resistance (mcr) genes threaten the clinical use of COL. Currently, ten mcr genes (mcr-1 to mcr-10) have been described. By horizontal and vertical transfer, the mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, and mcr-9 genes have disseminated in the poultry sector in HICs, thus posing a grave danger to animal and human health, as harboured by Escherichia coli, Klebsiella pneumoniae, Salmonella species, and Aeromonas isolates. Conjugative and non-conjugative plasmids are the major backbones for mcr in poultry isolates from HICs. The mcr-1, mcr-3 and mcr-9 have been integrated into the chromosome, making them persist among the clones. Transposons, insertion sequences (IS), especially ISApl1 located downstream and upstream of mcr, and integrons also drive the COL resistance in isolates recovered from the poultry sector in HICs. Genes coding multi-and extensive-drug resistance and virulence factors are often co-carried with mcr on chromosome and plasmids in poultry isolates. Transmission of mcr to/among poultry strains in HICs is clonally unrestricted. Additionally, the contact with poultry birds, manure, meat/egg, farmer's wears/farm equipment, consumption of contaminated poultry meat/egg and associated products, and trade of poultry-related products continue to serve as transmission routes of MGHB in HICs. Indeed, the policymakers, especially those involved in antimicrobial resistance and agricultural and poultry sector stakeholders-clinical microbiologists, farmers, veterinarians, occupational health clinicians and related specialists, consumers, and the general public will find this current literature synthesis very useful.

Is Africa ready for mobile colistin resistance threat?

Antimicrobial resistance is a growing public health problem and a threat to effective treatment and prevention of an array of infections caused by bacteria. Africa is already faced with many socio-economic and health crises. Many countries in Africa can seldom boast of a standardized health care facility comparable to those in developed countries. Yet, the non-therapeutic use of COL has been banned in developed countries. However, in Africa, except for South Africa, COL is an over-the-counter (OTC) medication sold and dispensed by non-professionals/without a veterinarian's supervision. The ban of non-therapeutic COL in developed countries has proven to reduce the development of mobile colistin resistance (MCR) in humans and animals. The unregulated use of COL has been proven to select pathogenic and commensal bacteria resistance. A transmissible plasmid-mediated colistin determinant, mobile COL resistance (mcr) gene, which is rapidly transferred/acquired horizontally or laterally intra/inter-species/genera, has been reported. A highly promiscuous mobile genetic element like plasmids containing transposons, insertion sequences, and integrons aid the carriage/rapid transfer and acquisition of these mcr genes. Hence, we highlight the danger posed by escalating colistin (COL) resistance in the continent and the impetus to halt the indiscriminate and non-therapeutic use of COL to protect public health.

Reduction of the Adverse Impacts of Fungal Mycotoxin on Proximate Composition of Feed and Growth Performance in Broilers by Combined Adsorbents.

Synergistic interaction of adsorbents in reducing the adverse impacts of mycotoxin on performance and proximate composition of broiler feeds was investigated. Fungal growth was induced by sprinkling water on the feed. S. cerevisiae + bentonite, kaolin + bentonite or S. cerevisiea + kaolin adsorbent combinations (1.5 g/kg feed) were added and the feeds were stored in black polythene bags. An untreated group was kept as a positive control while fresh uncontaminated feed was used as a negative control. Mycotoxins were extracted from the feeds and quantified using reverse phase HPLC. Proximate composition, nutrient digestibility of the feeds, feed intake and weight gain of the broilers were measured. Deoxynivalenol (DON) concentration in the contaminated/untreated feed was 347 µg/kg while aflatoxin B1 (AFB1) was 34 µg/kg. Addition of bentonite and kaolin in the contaminated feed reduced AFB1 and DON to significantly lower levels. Feed intake and weight gain were low in the broilers fed the contaminated feed. The carbohydrate level was significantly (p < 0.05) reduced from 62.31 to 40.10%, crude protein digestibility dropped from 80.67 to 49.03% in the fresh feed and contaminated feed respectively. Addition of the adsorbents (S. cerevisiae and bentonite) significantly (p < 0.05) improved these parameters.

Prevalence and Traits of Mobile Colistin Resistance Gene Harbouring Isolates from Different Ecosystems in Africa.

The mobile colistin resistance (mcr) gene threatens the efficacy of colistin (COL), a last-line antibiotic used in treating deadly infections. For more than six decades, COL is used in livestock around the globe, including Africa. The use of critically important antimicrobial agents, like COL, is largely unregulated in Africa, and many other factors militate against effective antimicrobial stewardship in the continent. Currently, ten mcr genes (mcr-1 to mcr-10) have been described. In Africa, mcr-1, mcr-2, mcr-3, mcr-5, mcr-8, and mcr-9 have been detected in isolates from humans, animals, foods of animal origin, and the environment. These genes are harboured by Escherichia coli, Klebsiella, Salmonella, Citrobacter, Enterobacter, Pseudomonas, Aeromonas, Alcaligenes, and Acinetobacter baumannii isolates. Different conjugative and nonconjugative plasmids form the backbone for mcr in these isolates; however, mcr-1 and mcr-3 have also been integrated into the chromosome of some African strains. Insertion sequences (ISs) (especially ISApl1), either located upstream or downstream of mcr, class 1 integrons, and transposons, are drivers of mcr in Africa. Genes coding multi/extensive drug resistance and virulence are colocated with mcr on plasmids in African strains. Transmission of mcr to/among African strains is nonclonal. Contact with mcr-habouring reservoirs, the consumption of contaminated foods of animal/plant origin or fluid, animal-/plant-based food trade and travel serve as exportation, importation, and transmission routes of mcr gene-containing bacteria in Africa. Herein, the current status of plasmid-mediated COL resistance in humans, food-producing animals, foods of animal origin, and environment in Africa is discussed.

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.

Veterinary clinic surfaces as reservoirs of multi-drug- and biocide-resistant Gram-negative bacteria.

This cross-sectional study was carried out to determine the common Gram-negative bacteria (GNB) contaminating veterinary clinic environments, and to evaluate the susceptibility of the isolates to commonly used antibiotics and biocides. A total of 62 swab samples were collected from different frequently touched surfaces in the 4 veterinary clinics visited. The samples were processed for isolation and identification of GNB using standard microbiological procedures. The susceptibility of the isolates to disinfectants and antibiotics was determined using agar dilution and disc diffusion techniques, respectively. A total of 114 GNB were isolated from the 4 clinics with isolation rates of 21.9, 22.8, 23.7 and 31.6% in clinics A, B, C and D, respectively. The surfaces of treatment tables were more contaminated (16.7 %) than receptionist/clinician desks (15.8%), weighing balances (10.5 %), door handles (7.9 %), drip stands (7.9 %), handwashing basins (7.0 %) and client chairs (7.0%). The surface-contaminating isolates were distributed into 20 genera, with members of Enterobacteriaceae predominating (n=97). Fifty-nine per cent of the isolates were resistant to the disinfectant Septol, while 5.3 and 0.9% were resistant to Purit and Dettol disinfectants, respectively. Multiple drug resistance was observed among 99% of the isolates with approximately 100% resistance to beta-lactams. Phenotypic expression of extended-spectrum (3.5 %) and AmpC beta-lactamase (38.6 %) production was detected. These findings highlight the role of clinic environments in serving as reservoirs for potential pathogens and sources for the spread of multi-drug resistant GNB.

Comparison of colorimetric loop-mediated isothermal amplification kit and reverse transcription-polymerase chain reaction in the diagnosis of peste des petits ruminants in sheep and goats in Southeast Nigeria.

BACKGROUND AND AIM: Peste des petits ruminants (PPR) is an acute, extremely contagious transboundary viral disease of small ruminants with severe economic consequences, caused by PPR virus. Cost-effective and rapid diagnosis of the disease is essential for prompt management and control. This study aimed to compare the application of a commercial colorimetric loop-mediated isothermal amplification (cLAMP) kit and reverse transcriptase-polymerase chain reaction (RT-PCR) in the diagnosis of PPR in sheep and goats in Southeast Nigeria. MATERIALS AND METHODS: Nasal swab samples were collected from West African Dwarf sheep and goats showing clinical signs suggestive of PPR (n=80) and those without any clinical signs (n=140) of the disease. The diagnosis was achieved through detection of PPR viral genome in the samples using a cLAMP kit and RT-PCR. cLAMP assay was done directly on nasal swab samples without ribosomal nucleic acid extraction. A set of six primers targeting the matrix gene protein was used for the cLAMP assay. RESULTS: PPR viral genome was detected by both cLAMP and RT-PCR in 51 (63.8%) of the 80 samples from sheep and goats with signs suggestive of PPR while 14 (10%) of those without signs tested positive for PPR by both assay methods. There was a 100% agreement in the cLAMP and RT-PCR results. However, cLAMP was a faster, easier, and less expensive method compared to RT-PCR. CONCLUSION: The cLAMP assay demonstrates the potential for a point of care diagnosis in the field and a valuable diagnostic tool in areas with poor electricity supply as well as in a less equipped diagnostic laboratory. Since the reagents are affordable, cLAMP can be a diagnostic tool of choice in the detection and surveillance of PPR virus in countries with limited resources.

Update on Peste des petits ruminants status in South East Nigeria: serological and farmers' awareness investigation, and potential risk factors.

Peste des petits ruminants (PPR) is a highly contagious, trans-boundary viral disease of sheep and goats that have hindered successful small ruminant farming. Its current status in South East Nigeria with respect to its prevalence and farmers' awareness was studied. Three states, Anambra, Ebonyi, and Enugu, were randomly selected for the study. Sera samples from 113 goats and 172 sheep (collected from December 2017 to June 2018) were randomly collected and analysed for the presence of PPRV antibodies, while structured interview schedules were conducted to elicit information on farmers' awareness of the disease and PPR vaccination and use of veterinary services. An overall seroprevalence of 42.5% (121/285) was recorded. The seroprevalence in decreasing order was 62.2% (Enugu), 34.8% (Anambra) and 20.3% (Ebonyi). There was a significant association (X2 = 36.08, df = 2, p = 0.0001) between seroprevalence and the state sampled. Lack of awareness of PPR vaccination among small ruminant farmers, their limited use of veterinary services (38% consult veterinarians) and non-availability of the vaccine at veterinary establishments in the sampled states are potential risk factors of PPR prevalence in South East Nigeria. Consequently, an effective control measure like mass vaccination is recommended for the study area. Also, there is a need for an extension program for stakeholders and farmers in the study area and country on the grave importance and economic benefits of PPR vaccination and the use of veterinary services.

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.

Chitosan/alginate microparticles for the oral delivery of fowl typhoid vaccine: Innate and acquired immunity.

Oral fowl typhoid (FT) vaccine is necessary for improved flock vaccinations and economic growth. This study was undertaken to evaluate the immune responses of birds given oral fowl typhoid vaccine coated with chitosan/alginate microparticles and comparing it with the conventional subcutaneous route of administration. Preliminary studies were done to evaluate the particle size, encapsulation efficiency and agglutination. Sixty day-old chicks were divided into three groups of twenty birds each. This comprised a negative control group NEG 451 (non-vaccinated and non-challenged used as control for cytokine quantification), SC 634 (live 9R vaccine by the injection route) and OCV 567 (live 9R vaccine coated with chitosan/alginate microparticles). Vaccination was done at 10 weeks and 14 weeks of age followed by challenge at 16 weeks of age. IgG was measured using ELISA. mRNA fold expression of IFN-γ in spleen was calculated using qRT-PCR. Particle sizes ranged between 0.55 µm and 10 µm. Encapsulation efficiency was above 60%. ELISA showed E-values of 0.10 ±â€¯0.14, 0.07 ±â€¯0.01 and 0.02 ±â€¯0.01 for OCV 567, SC 634 and NEG 451 respectively after primary vaccination. Also E-values were 0.25 ±â€¯0.16, 0.19 ±â€¯0.04 and 0.0008 ±â€¯0.005 for SC 634, OCV 567 and NEG451 respectively after boost vaccination. The expression of IFN-γin spleen using 2-ΔΔ CT calculation was upregulated with values of 1.97 and 0.75 for OCV 567 and SC 634 respectively. After challenge with the 85-kb virulence plasmid SG9, there was 100% protection of the birds in both OCV 567 and SC 634 groups with no mortality. In conclusion, there was no significant difference at p < 0.05 of the means ±â€¯SD in immune responses between the oral fowl typhoid vaccine coated with chitosan/alginate microparticles and the subcutaneous route of administration. However, it is noteworthy to mention that the protective efficacy of the oral route is due to the chitosan/alginate biopolymers which coated the vaccine preventing destruction in the gastrointestinal tract.

Detection and molecular characterisation of extended-spectrum ß-lactamase-producing enteric bacteria from pigs and chickens in Nsukka, Nigeria.

OBJECTIVES: This study screened chickens and pigs slaughtered for human consumption for the presence and characteristics of extended-spectrum ß-lactamase (ESBL)- and plasmid-encoded AmpC (pAmpC) ß-lactamase-producing enteric bacteria. METHODS: Faecal samples from 410 broiler chickens and 100 pigs were cultured on MacConkey agar supplemented with 2µg/mL cefotaxime. Antimicrobial resistance phenotypes of the recovered isolates were determined by disk diffusion. PCR and sequencing were performed to identify the ESBL and pAmpC gene variants and other associated resistance determinants. Genetic diversity of the isolates was analysed by phylotyping and multilocus sequence typing. RESULTS: ESBL-producing Escherichia coli, Klebsiella pneumoniae, Enterobacter asburiae and Providencia spp. were isolated from 17 (4.1%) and 2 (2.0%) of the samples from chickens and pigs, respectively. One pAmpC-producing E. coli isolate was obtained from a chicken. Resistance to tetracycline, trimethoprim/sulfamethoxazole, chloramphenicol and gentamicin was exhibited by 95%, 80%, 60% and 55% of the ESBL/pAmpC-producing strains, respectively. tet(A) and aac(3)-II were the predominant genes detected in tetracycline- and aminoglycoside-resistant strains, respectively. blaCTX-M, encoding CTX-M-15 (15 isolates) or CTX-M-1 variants (3 isolates), was present in all but one ESBL-producer, either alone or in combination with blaSHV and/or blaTEM. The remaining ESBL-producer, a Providencia spp. recovered from a chicken, harboured blaVEB. The only pAmpC-positive E. coli strain carried blaCMY-2. The 11 ESBL-producing E. coli strains belonged to five lineages (ST226-A, ST3625-B1, ST10-A, ST46-A and ST58-B1). CONCLUSIONS: Healthy chickens and pigs act as reservoirs of ESBL/pAmpC-producing enterobacteria that can potentially be transmitted to humans through direct contact or ingestion of contaminated meat.

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.

Diversity of Ochrobactrum species in food animals, antibiotic resistance phenotypes and polymorphisms in the blaOCH gene.

Twenty-six lactose non-fermenting, oxidase, urease and citrate-positive Gram-negative rods, isolated from broiler chickens, pigs and cattle at slaughter, were subjected to the matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and 16S rDNA sequencing for identification. Susceptibility to 14 antimicrobials was determined by the disc diffusion method. Ochrobactrum isolates resistant to third-generation cephalosporins were PCR-screened for the presence of the Ochrobactrum anthropi ampC gene (blaOCH). A 547-bp internal segment of blaOCH in the Ochrobactrum spp isolates was amplified with a newly designed primer set, and a phylogenetic reconstruction based on the complete amino acid sequence of blaOCH obtained from nine Ochrobactrum strains in our collection and 20 O. anthropi available in the GenBank was undertaken. All the Ochrobactrum isolates were resistant to the expanded-spectrum beta-lactams and streptomycin. None of the isolates was resistant to imipenem while 41.7% to 50.0% of them were resistant to fluoroquinolones. The blaOCH gene was detected in 16 (66.7%) and 20 (83.3%) of the 24 Ochrobactrum isolates (O. intermedium/O. tritici species), using primers designed for O. anthropi and the newly designed primer set, respectively. Six blaOCH variants grouped into two divergent clusters were identified. This is the first report of the complete nucleotide sequence of the blaOCH gene in non-antropi Ochrobactrum species.

Characterization of mannitol-fermenting methicillin-resistant staphylococci isolated from pigs in Nigeria.

This study was conducted to determine the species distribution, antimicrobial resistance pheno- and genotypes and virulence traits of mannitol-positive methicillin-resistant staphylococci (MRS) isolated from pigs in Nsukka agricultural zone, Nigeria. Twenty mannitol-positive methicillin-resistant coagulase-negative staphylococcal (MRCoNS) strains harboring the mecA gene were detected among the 64 Staphylococcus isolates from 291 pigs. A total of 4 species were identified among the MRCoNS isolates, namely, Staphylococcus sciuri (10 strains), Staphylococcus lentus (6 strains), Staphylococcus cohnii (3 strains) and Staphylococcus haemolyticus (one strain). All MRCoNS isolates were multidrug-resistant. In addition to ß-lactams, the strains were resistant to fusidic acid (85%), tetracycline (75%), streptomycin (65%), ciprofloxacin (65%), and trimethoprim/sulphamethoxazole (60%). In addition to the mecA and blaZ genes, other antimicrobial resistance genes detected were tet(K), tet(M), tet(L), erm(B), erm(C), aacA-aphD, aphA3, str, dfrK, dfrG, cat pC221, and cat pC223. Thirteen isolates were found to be ciprofloxacin-resistant, and all harbored a Ser84Leu mutation within the QRDR of the GyrA protein, with 3 isolates showing 2 extra substitutions, Ser98Ile and Arg100Lys (one strain) and Glu88Asp and Asp96Thr (2 strains). A phylogenetic tree of the QRDR nucleotide sequences in the gyrA gene revealed a high nucleotide diversity, with several major clusters not associated with the bacterial species. Our study highlights the possibility of transfer of mecA and other antimicrobial resistance genes from MRCoNS to pathogenic bacteria, which is a serious public health and veterinary concern.