Genome Analysis of Multidrug-Resistant Escherichia coli Isolated from Poultry in Nigeria.

Escherichia coli is one of the most common commensal bacteria of the gastrointestinal tract of humans and warm-blooded animals. Contaminated poultry can lead to disease outbreaks in consumers causing massive economic losses in the poultry industry. Additionally, commensal E. coli can harbor antibiotic resistance genes that can be transferred to other bacteria, including pathogens, in a colonized human host. In a previous study on antimicrobial resistance of E. coli from food animals from Nigeria, multidrug-resistant E. coli were detected. Three of those isolates were selected for further study using whole-genome sequencing due to the extensive drug resistance exhibited. All of the isolates carried the extended-spectrum ß-lactamase (ESBL) genes, blaCTX-M15 and blaTEM-1, whereas one isolate harbored an additional ESBL, blaOXA-1. All of the tetracycline-resistant isolates carried tet(A). The genes aac3-IIa and aacA4, conferring resistance to aminoglycosides, were identified in an E. coli isolate resistant to gentamicin and tobramycin. In two E. coli isolates, dfrA14, qnrS1, and sulII, were detected conferring resistance to trimethoprim, fluoroquinolones, and sulfonamides, respectively. The third isolate carried dfrA17, no fluoroquinolone resistance gene, an additional sulI gene, and a chloramphenicol resistance gene, catB3. Mutations in candidate genes conferring resistance to fosfomycin and fluoroquinolones were also detected. Several efflux systems were detected in all the E. coli isolates and virulence-associated genes related to serum resistance, motility, and adhesion. E. coli and non-E. coli origin prophages were also identified in the isolates. The results underline the higher resolution power of whole-genome sequencing for investigation of antimicrobial resistance, virulence, and phage in E. coli.

Characterization of Vibrio cholerae Strains Isolated from the Nigerian Cholera Outbreak in 2010.

We examined clinical samples from Nigerian patients with acute watery diarrhea for Vibrio cholerae during the 2010 cholera outbreak. A total of 109 suspected isolates were characterized, but only 57 V. cholerae strains could be confirmed using multiplex real-time PCR as well as rpoB sequencing and typed as V. cholerae O:1 Ogawa biotype El Tor. This finding highlighted the need for accurate diagnosis of cholera in epidemic countries to implement life-saving interventions.

Genotyping by randomly amplified polymorphic DNA of bacteriocin producing Lactobacillus acidophilus strains from Nigeria.

Yogurt and starter culture producers are still searching strains of Lactobacillus acidophilus to produce healthier yogurt with a longer shelf life and better texture, taste, and quality. This study determined the genotyping of bacteriocin producing Lactobacillus acidophilus strains recovered from Nigerian yogurts. Yogurt samples were collected from four different states of South West regions of Nigeria. Isolates were obtained from MRS Medium and biochemically characterized. This was further confirmed by API50CH. The bacteriocin positivity and activity was determined. Genomic characterization of our Lactobacillus acidophilus strains was done with randomly amplified polymorphic DNA-PCR. All yogurt samples containing Lactobacillus acidophilus strains meet the probiotic requirement of ≥10(6) cfu/mL. The gel picture revealed 6 RAPD clonal types of Lactobacillus acidophilus strains with RAPD type C observed to be more common. Significant differences existed in the mean growth inhibition zone (t = -7.32, P < 0.05 for E. coli ATCC; t = -6.19, P < 0.05 for E. coli clinical isolates; t = -6.16, P < 0.05 for Enterobacter sp; t = -11.92, P < 0.05 for Salmonella typhi, t = -1.10, P > 0.05 Staphylococcus aureus). No correlation between the bacteriocin production, activity, and their RAPD clonal division (X(2) = 7.49, P = 0.1610, df = 5). In conclusion, L. acidophilus isolated in Nigeria samples met the probiotic requirements of ≥10(6) cfu/mL and produce bacteriocins with good spectrum of activity.

Plasmid Replicons and ß-Lactamase-Encoding Genes of Multidrug-Resistant Escherichia coli Isolated from Humans and Food Animals in Lagos, Southwest Nigeria.

As resistance to the ß-lactam class of antibiotics has become a worldwide problem, multidrug-resistant (MDR) human (n = 243) and food animal (n = 211) isolates from Lagos, Nigeria were further tested to characterize ß-lactamase-encoding genes and plasmid replicons. Four ß-lactamase-encoding genes (blaCMY, blaCTX-M, blaOXA, and blaTEM) were detected using PCR-based replicon typing, 13 and 17 different replicons were identified using a subset of MDR E. coli from humans (n = 48) and animals (n = 96), respectively. Replicon types FIB and X2 were detected in equal numbers (2/48; 4.2% each) from human isolates, while type Y (16/96; 16.7%) was the most common type from animals. Only two replicon types, FIB and Y, were detected in both groups; all other types were confined to one group or the other, but not both. Using conjugation, replicon type Y, present in three donors, transferred in all three instances, whereas FIA transferred in 75% (3/4) of the matings. This study showed that ß-lactamase genes were prevalent in MDR E. coli from both humans and animals in Nigeria and also contained diverse plasmid replicons. As the replicon-associated genes were mobile, they are likely to continue disseminating among E. coli and facilitating transfer of associated ß-lactamase genes in this region.

Effect of cocoa powder on hypertension and antioxidant status in uninephrectomized hypertensive rats.

BACKGROUND AND AIM: High salt diet and uninephrectomy are associated with high blood pressure with attendant cardiovascular disease conditions such as hypertension, renal damage, myocardial infarction, and stroke. The aim of this study was to investigate the beneficial effects of consumption of cocoa and cocoa-containing products in the management of high blood pressure in uninephrectomized hypertensive rats. MATERIALS AND METHODS: The effect of cocoa powder on blood pressure, markers of inflammation, oxidative stress, and histopathology were investigated in uninephrectomized animals fed with cocoa feed alone or in combination with a high salt diet. Male rats were randomly divided into five groups: Group A was the control group and fed with normal feed alone, Group B was fed with cocoa feed alone, Group C was fed with high salt diet (8% salt), GroupD was fed with cocoa-feed compounded with 8% salt for 4weeks after uninephrectomy, and GroupE was uninephrectomized rats on a normal diet. The left kidneys of animals in GroupsC, D, and E were removed by surgery. After 4weeks of treatment, the systolic, diastolic, and mean arterial blood pressure was measured. The serum markers of renal damage and oxidative stress were determined. Histological examination was also performed on renal and cardiac tissues. RESULTS: Results showed significant increases in biomarkers of oxidative stress, inflammation, and renal damage with a concomitant decrease in antioxidant status in hypertensive uninephrectomized rats. Cocoa feed, however, significantly improved blood pressure and nitric oxide bioavailability, antioxidant status and reduced markers of inflammation and oxidative stress. CONCLUSION: These findings show that cocoa powder could be used to maintain blood pressure levels in hypertensive rats through its antioxidant capacity.

Draft Genome Sequence Analysis of Multidrug-Resistant Escherichia coli Strains Isolated in 2013 from Humans and Chickens in Nigeria.

Here, we present the draft genome sequences of nine multidrug-resistant Escherichia coli strains isolated from humans (n = 6) and chicken carcasses (n = 3) from Lagos, Nigeria, in 2013. Multiple extended-spectrum ß-lactamase (ESBL) genes were identified in these isolates.

Honey: A Therapeutic Agent for Disorders of the Skin.

Problems with conventional treatments for a range of dermatological disorders have led scientists to search for new compounds of therapeutic value. Efforts have included the evaluation of natural products such as honey. Manuka honey, for example, has been scientifically recognised for its anti-microbial and wound healing properties and is now used clinically as a topical treatment for wound infections. In this review, scientific evidence for the effectiveness of honey in the treatment of wounds and other skin conditions is evaluated. A plethora of in vitro studies have revealed that honeys from all over the world have potent antimicrobial activity against skin relevant microbes. Moreover, a number of in vitro studies suggest that honey is able to modulate the skin immune system. Clinical research has shown honey to be efficacious in promoting the healing of partial thickness burn wounds while its effectiveness in the treatment of non-burn acute wounds and chronic wounds is conflicted. Published research investigating the efficacy of honey in the treatment of other types of skin disorders is limited. Nevertheless, positive effects have been reported, for example, kanuka honey from New Zealand was shown to have therapeutic value in the treatment of rosacea. Anti-carcinogenic effects of honey have also been observed in vitro and in a murine model of melanoma. It can be concluded that honey is a biologically active and clinically interesting substance but more research is necessary for a comprehensive understanding of its medicinal value in dermatology.

Plasmodium falciparum Merozoite Surface Protein-1 Polymorphisms among Asymptomatic Sickle Cell Anemia Patients in Nigeria.

Asymptomatic malaria (ASM) has been implicated in the development of hemolytic crisis in infected sickle cell anemia (SCA) patients worldwide. This study surveyed steady state SCA Nigerian patients for ASM to investigate the influence of malaria prevention behaviors and age on parasitaemia and multiplicity of infection (MOI). A total of 78 steady SCA patients aged 5 - 27 years on routine care at three health facilities in Lagos were investigated for ASM by light microscopy and PCR with a multiplicity of infection determined by genotyping block 2 of merozoite surface protein 1 (msp1) gene of Plasmodium falciparum (P. falciparum). Use of malaria prevention measures was captured using a semi-structured questionnaire. The prevalence rates of ASM (due to Pf only) by microscopy and PCR were found to be 27.3% and 47.4% respectively (P < 0.05) with a Mean + SEM parasite density of 2238.4 + 464.3 parasites/uL. Five distinct msp1 genotypes [K1 (2), MAD20 (2), RO33 (1)] were detected and significant (P<0.05) disparity in allele frequencies (K1, 91.8%, MAD20, 32.4%; RO33, 18.9%) was found. The overall MOI was 1.43 and 37.8% of infections were polyclonal (P<0.05). ASM was associated with non-use of preventive measures and occurred in 62.1% of SCA patients aged < 10y with lower MOI of 1.3 compared to 38.1% in older patients with a higher MOI of 1.5 (P<0.05). We conclude that PCR improved the diagnosis of ASM among Nigerian SCA patients with infections being of low complexity and associated with non-use of preventive interventions and R033 msp1 allele selection.

Distribution of plasmid-mediated quinolone resistance in Gram-negative bacteria from a tertiary hospital in Nigeria.

BACKGROUND: Until recently, mechanisms of resistance to quinolones in Gram-negative bacteria were believed to be only chromosome encoded. However, emergence of plasmid-mediated quinolone resistance (PMQR) has been reported worldwide. AIM: This study investigated distribution of PMQR in Gram-negative bacteria from a tertiary hospital in eastern part of Nigeria. MATERIALS AND METHODS: Seventy-one nonduplicate Gram-negative bacterial isolates of eight species were analyzed for antimicrobial susceptibility, genotypic detection of various PMQRs, typed by random amplified polymorphic DNA (RAPD) and analysis of plasmids present, including replicon typing. RESULTS: The minimum inhibitory concentrations showed MIC90values as high as 256 µg/ml for fluoroquinolones. Carriage of PMQR was found to be 35.2%. Twenty (28.2%) isolates carried various qnr genes, of which seven (9.9%) qnrA1; four (5.6%) qnrB1; eight (11.3%) qnrS1 while one (1.4%) encoded qnrD1. Eighteen (25.4%) isolates were positive for aac(6')-Ib-cr while carriage of multiple genes exists in some strains. Similarly, 13 isolates (18.7%) were found to carry PMQR efflux pump gene, qepA. Conjugation experiments revealed that the plasmids once transferred coded for fluoroquinolone resistance. The transconjugant strains carried a common plasmid estimated to be 65 kb. These plasmids were untypable for replicon/incompatibility. Typing revealed high diversity among all species tested with no identical RAPD pattern seen. CONCLUSION: This study further confirms high level resistance to many antimicrobials in different species of Gram-negative bacteria including fluoroquinolones and spread of PMQR genes in Southern Nigeria.

Prevalence and multidrug resistance of Escherichia coli from community-acquired infections in Lagos, Nigeria.

INTRODUCTION: The emergence of multidrug resistance (MDR; resistance to ≥ 2 more antimicrobials) in Escherichia coli is of concern due to complications encountered in treatment. METHODOLOGY: In this study, prevalence, antimicrobial resistance, and genetic characteristics of MDR community isolates of E. coli from Lagos, Nigeria were determined. Urine and stool samples were obtained from outpatients attending Lagos State hospitals and from animal handlers in abattoirs, poultries, and open markets, from December 2012 to July 2013. RESULTS: Approximately 50% of urine (200/394) and 88% of stool samples (120/136) were positive for E. coli. Based upon ß-lactamase production, a subset of those isolates was selected for further study. Of the 22 antimicrobials tested, E. coli exhibited resistance to all antimicrobials except amikacin and piperacillin/tazobactam. The highest levels of resistance were to tetracycline (182/247; 73.7%), trimethoprim/sulfamethoxazole (152/247; 61.5%), and ampicillin (147/247; 59.1%). Resistance to the cephalosporins ranged from 1.6%-15% including the third- and fourth-generation cephalosporins, cefpodoxime (20/247; 8.1%) and cefepime (4/247; 1.6%), respectively. MDR was observed in 69.6% (172/247) of the isolates. Forty-eight E. coli resistant to at least five antimicrobials were selected for further analysis using pulsed-field gel electrophoresis; seven distinct clusters were observed among the diverse patterns. Of the 48 MDR E. coli, 30 different sequence types (ST) were detected using multilocus sequence typing, including four ST131. CONCLUSIONS: This study demonstrated circulating MDR E. coli in the Nigerian community. Monitoring of antimicrobial resistance in developing countries is necessary to optimize empiric treatment and the prudent use of antimicrobials.

Toll-like receptor (TLR4) Asp299Gly and Thr399Ile polymorphisms in relation to clinical falciparum malaria among Nigerian children: a multisite cross-sectional immunogenetic study in Lagos.

INTRODUCTION: This study determined the association of TLR4 Asp299Gly and Thr399Ile with uncomplicated and severe malaria among Nigerian children of similar ethnic background in Lagos. The association of these SNPs with high parasite density, malnutrition, hyperpyrexia and anaemia was also investigated. METHODS: Genomic DNA of the study participants was screened for the genotypes of TLR4 Asp299Gly and Thr399Ile by PCR-RFLP. Anthropometric measurement was performed on the Pf infected children stratified into asymptomatic malaria (control), uncomplicated and severe malaria (case). Parasites were detected by light microscopy and Hardy Weinberg Equilibrium (HWE) of SNP genotypes was also determined. RESULTS: A total of 279 children comprising 182 children (62.1 % male; mean ± SEM age, 57.3 ± 1.7 months) with clinical falciparum malaria and 97 children (55.7 % male; mean ± SEM age, 55.6 ± 2.5 years) with asymptomatic falciparum malaria were enrolled. All the genotypes of both TLR4 SNPs were found in the study population with their minor alleles: 299Gly and 399Ile, found to be 17.6 % and 14.7 % in severe malaria children. Unlike in asymptomatic population, the genotype distribution of TLR4 Asp299Gly SNP was not in HWE in the clinical malaria group but did not condition susceptibility. However, Asp299Gly and Thr399Ile polymorphisms were found to increase the risk of severe malaria 3-fold and 8-fold respectively (P < 0.05). They also increased the risk of severe anaemia, high parasite density and severe malnutrition 3.8 -5.3-fold, 3.3 - 4.4-fold and 4-fold respectively. CONCLUSIONS: Based on the above findings, we conclude that TLR4 Asp299Gly and Thr399Ile polymorphisms may modulate susceptibility to severe malaria among Nigerian children of Yoruba ethnic background.

Prevalence and Antimicrobial Resistance in Escherichia coli from Food Animals in Lagos, Nigeria.

Foodborne bacteria are often associated with human infections; these infections can become more complicated to treat if the bacteria are also resistant to antimicrobials. In this study, prevalence, antimicrobial resistance, and genetic relatedness of Escherichia coli among food producing animals from Lagos, Nigeria, was investigated. From December 2012 to June 2013, E. coli were isolated from fecal samples of healthy cattle, chicken, and swine. Antimicrobial susceptibility testing against 22 antimicrobials was performed using broth microdilution with the Sensititre™ system. Clonal types were determined by pulsed-field gel electrophoresis (PFGE). From the analysis, 211/238 (88.7%), 170/210 (81%), and 136/152 (89.5%) samples from cattle, chicken, and swine, respectively, were positive for E. coli. A subset of those isolates (n=211) selected based on ß-lactamase production was chosen for further study. Overall, E. coli exhibited the highest resistance to tetracycline (124/211; 58.8%), trimethoprim/sulfamethoxazole (84/211; 39.8%), and ampicillin (72/211; 34.1%). Approximately 40% of the isolates were pan-susceptible, and none of the isolates were resistant to amikacin, cefepime, ceftazidime, ertapenem, meropenem, or tigecycline. Among the resistant isolates, 28 different resistance patterns were observed; 26 of those were characterized as multi-drug resistant (MDR; resistance to ≥2 antimicrobials). One isolate was resistant to 13 different antimicrobials representing five different antimicrobial classes. Using PFGE, MDR E. coli were genetically diverse and overall did not group based on source; identical PFGE patterns were detected among isolates from different sources. These results suggest that isolates cannot be attributed to specific sources, and some may be present across all of the sources. Results from this study indicate that food-producing animals in Nigeria are a reservoir of MDR E. coli that may be transferred to humans via the food chain.

Bacteriologic and plasmid analysis of etiologic agents of conjunctivitis in Lagos, Nigeria.

BACKGROUND: Conjunctivitis, an inflammation of the conjunctiva, is one of the most common eye problems affecting all age groups in Nigeria. A better understanding of its epidemiology and the antibiotic susceptibility of etiologic bacterial agents is crucial for the initiation of preventive and therapeutic measures. This study determined the distribution and patterns of bacterial infections in Nigerian patients with conjunctivitis. Antibiotic resistance patterns and the plasmid profiles of these pathogens were also investigated. METHODOLOGY: A total of 83 consecutive and non-duplicate conjunctival specimens were collected from patients attending eye clinics at three different hospitals in Lagos, Nigeria, between February and September 2010. Specimens were cultured on standard bacteriologic media and the recovered isolates speciated using standard techniques. Susceptibility of pathogens to antibiotics and plasmid DNA extraction were carried out by disk diffusion and alkaline lysis methods. Conjugation experiment was done with rifampicin-resistant Escherichia coli DH5α as the recipient cell. Data were analyzed using the chi-square test. RESULTS: All the specimens were culture-positive, yielding a total of 155 bacterial isolates. Gram-positive cocci comprising Staphylococcus aureus (27.7%) and coagulase-negative Staphylococcus sp. (22.6%) accounted for 50.3% (78 of 155) of conjunctivitis cases, followed by Gram-positive bacilli (22.6%), Gram-negative bacilli (21.3%), and Gram-negative cocci (4.5%). Corynebacterium spp. were the most commonly isolated Gram-positive bacilli accounting for 16.1% of conjunctivitis cases. Pseudomonas aeruginosa topped with 9.7% as the most commonly isolated Gram-negative bacilli. Other Gram-negative bacilli in order of their isolations were E. coli (6.5%), Proteus sp. (3.2%), Klebsiella sp. (1.9%), and Enterobacter aerogenes (1.9%). Moraxella spp. were the only Gram-negative cocci isolated, and they accounted for 4.5% of the total conjunctival infections. Further analysis of the complexity of infections showed that 25 specimens elicited mono-infections, while cases of polymicrobial infections caused by two pathogens and three or more pathogens constituted 51.8% and 18.1% of conjunctivitis specimens screened, respectively. The disparity in the percentage contribution of three infection patterns was significant (P < 0.05). Antibiotic susceptibility testing revealed chloramphenicol and ofloxacin as the least and most active antibiotics tested as 99 (63.9%) and 149 (96.1%) of the 155 recovered isolates were sensitive to them. On the whole, the least susceptible pathogen was P. aeruginosa with sensitivities ranging from 20% to 80%, while Moraxella sp. represented the most sensitive pathogen with sensitivities ranging from 71.4% to 100%. Other bacterial isolates also elicited antibiotic sensitivities in the range of 33.3-100%. A total of 101 isolates were screened for plasmids, of which 45 harbored plasmids, yielding a plasmid frequency of 44.6%. Conjugal transfer of resistance to chloramphenicol, ampicillin, and streptomycin was detected in the transconjugants after the mating experiment. The antibiotic resistances were transferred either singly or in combination from six of the seven selected donor strains. The antibiotic resistance pattern transferred by these donor strains was partial and was associated with the transfer of R plasmids of sizes 21.3, 15.2, and 5.0 kb from three of the six transferable strains. The frequencies of transfer of antibiotype or R plasmids to the transconjugants ranged from 1.8 × 10(-7) to 1.4 × 10(-5) transconjugants per donor strain. CONCLUSION: Conjunctivitis as an eye problem in Lagos is polymicrobial with infections associated with transferable R plasmids for chloramphenicol, ampicillin, and streptomycin. Continuous surveillance of conjunctivitis in relation to etiology, drug susceptibility, and plasmid transferability in the study area is therefore recommended.

Specific viruses detected in nigerian children in association with acute respiratory disease.

Occurrence of different viruses in acute respiratory tract infections of Nigerian children was examined. Respiratory swabs were collected from 246 children referred to hospital clinics because of acute respiratory symptoms from February through May 2009. Validated real-time RT-PCR techniques revealed nucleic acids of at least one virus group in 189 specimens (77%). Human rhinoviruses and parainfluenza viruses were present each in one third of the children. Adenoviruses, enteroviruses, human metapneumovirus, human bocavirus, and influenza C virus were also relatively common. Possibly due to their seasonal occurrence, influenza A and B virus, and respiratory syncytial virus were detected rarely. We conclude that all major groups of respiratory tract viruses are causing illness in Nigerian children.