David Olusoga Ogbolu

Molecular mechanisms of antibiotic resistance.

Antibiotic-resistant bacteria that are difficult or impossible to treat are becoming increasingly common and are causing a global health crisis. Antibiotic resistance is encoded by several genes, many of which can transfer between bacteria. New resistance mechanisms are constantly being described, and new genes and vectors of transmission are identified on a regular basis. This article reviews recent advances in our understanding of the mechanisms by which bacteria are either intrinsically resistant or acquire resistance to antibiotics, including the prevention of access to drug targets, changes in the structure and protection of antibiotic targets and the direct modification or inactivation of antibiotics.

High-level and novel mechanisms of carbapenem resistance in Gram-negative bacteria from tertiary hospitals in Nigeria.

To determine the occurrence and molecular basis of carbapenem resistance in Gram-negative bacteria from tertiary hospitals in Nigeria, 182 non-duplicate Gram-negative bacterial isolates were investigated for antimicrobial susceptibility, presence of carbapenemases (tested phenotypically and genotypically), random amplified polymorphic DNA (RAPD) typing, plasmid sizing and replicon typing. Minimum inhibitory concentrations of carbapenems showed a high degree of resistance, with 67 isolates (36.8%) being resistant to all carbapenems, of which 40 (59.7%) produced enzymes able to hydrolyse imipenem. PCR and sequencing identified only 10 isolates (5.5%) carrying known carbapenemase genes, including bla(NDM), bla(VIM) and bla(GES). The majority of phenotypically carbapenem-resistant and carbapenemase-producing isolates did not carry a known carbapenemase gene. Transconjugant or transformant plasmid sizes were estimated to be 115 kb for bla(NDM)- and 93 kb for bla(VIM)-carrying plasmids. These plasmids were untypeable for replicon/incompatibility and transferred various other genes including plasmid-mediated quinolone resistance (PMQR) genes and bla(CTX-M-15). Typing showed that the isolates in this study were not clonally related. There is a high level of carbapenem resistance in Nigeria. As well as the globally relevant carbapenemases (bla(NDM), bla(VIM) and bla(GES)), there are other unknown gene(s) or variant(s) in circulation able to hydrolyse carbapenems and confer high-level resistance.

Dissemination of IncF plasmids carrying beta-lactamase genes in Gram-negative bacteria from Nigerian hospitals

INTRODUCTION Production of beta-lactamases is the predominant cause of resistance to beta-lactam antibiotics in Gram-negative bacteria. We investigated the diversity of plasmid-borne beta-lactamase genes and replicon type of the plasmids carrying the respective genes in Gram-negative bacteria recovered from clinical infection in Nigerian hospitals. METHODOLOGY A total of 134 Gram-negative bacteria of 13 species were analyzed for antimicrobial susceptibility, phenotypic and genotypic detection of various beta-lactamases, and plasmid analysis, including replicon typing. RESULTS Of the 134 isolates, 111 (82.8%) contained beta-lactamases, while 28 (20.9%) carried extended-spectrum beta-lactamases. PCR and sequencing identified TEM-1 in 109 isolates (81.3%), SHV-1 in 33 isolates (24.6%), OXA-1 in 15 isolates (11.2%) and CTX-M enzymes (24 CTX-M-15 and 1 CTX-M-3) in 25 isolates (18.7%). Multiplex PCR showed that 6 isolates carried plasmidic AmpCs (ACT-1, DHA-1 and CMY-2); these enzymes were detected only in isolates possessing CTX-M beta-lactamases. Of 13 (76.9%) representative plasmids investigated in detail, 9 (69.2%) were self-transferable when selected by a beta-lactam and the plasmids once transferred coded for beta-lactam resistance. Replicon typing indicated IncF as the common vector encoding for beta-lactamases. CONCLUSIONS The study showed a diversity of beta-lactamase genes disseminated by conjugative IncF plasmids in Gram-negative bacteria; TEM-1, SHV-1, OXA-1, CTX-M-15, CTX-M-3 and plasmidic AmpC enzymes are in common circulation in Nigeria.

Phenotypic and molecular characteristics of methicillin-resistant Staphylococcus aureus isolates from Ekiti State, Nigeria

Introduction The characteristics and antimicrobial resistance profiles of Staphylococcus aureus differs according to geographical regions and in relation to antibiotic usage. The aim of this study was to determine the biochemical characteristics of the prevalent S. aureus from Ekiti State, Nigeria, and to evaluate three commonly used disk diffusion methods (cefoxitin, oxacillin, and methicillin) for the detection of methicillin resistance in comparison with mecA gene detection by polymerase chain reaction. Materials and methods A total of 208 isolates of S. aureus recovered from clinical specimens were included in this study. Standard microbiological procedures were employed in isolating the strains. Susceptibility of each isolate to methicillin (5 μg), oxacillin (1 μg), and cefoxitin (30 μg) was carried out using the modified Kirby–Bauer/Clinical and Laboratory Standard Institute disk diffusion technique. They were also tested against panels of antibiotics including vancomycin. The conventional polymerase chain reaction method was used to detect the presence of the mecA gene. Results Phenotypic resistance to methicillin, oxacillin, and cefoxitin were 32.7%, 40.3%, and 46.5%, respectively. The mecA gene was detected in 40 isolates, giving a methicillin-resistant S. aureus (MRSA) prevalence of 19.2%. The S. aureus isolates were resistant to penicillin (82.7%) and tetracycline (65.4%), but largely susceptible to erythromycin (78.8% sensitive), pefloxacin (82.7%), and gentamicin (88.5%). When compared to the mecA gene as the gold standard for MRSA detection, methicillin, oxacillin, and cefoxitin gave sensitivity rates of 70%, 80%, and 100%, and specificity rates of 76.2%, 69.1%, and 78.5% respectively. Conclusion When compared with previous studies employing mecA polymerase chain reaction for MRSA detection, the prevalence of 19.2% reported in Ekiti State, Nigeria in this study is an indication of gradual rise in the prevalence of MRSA in Nigeria. A cefoxitin (30 μg) disk diffusion test is recommended above methicillin and oxacillin for the phenotypic detection of MRSA in clinical laboratories.

Molecular detection of pneumocystis jirovecii in patients with respiratory tract infections.

Background: Pneumocystis jirovecii, formerly known as P. carinii, is an opportunistic fungus causing Pneumocystis carinii pneumonia especially in immunocompromised patients. Aim: The aim of this study was to detect P. jirovecii in sputum samples from patients suspected of having respiratory tract infections. Materials and Methods: In this study, 230 acid fast bacilli negative sputum samples from 230 patients presenting with respiratory tract infections submitted to three teaching hospitals’ medical microbiology laboratories in Osun and Oyo States, Nigeria for routine investigation were examined for P. jirovecii by microscopical and polymerase chain reaction methods. Results: P. jirovecii cysts were observed in 15 (6.5%) samples and polymerase chain reaction was positive for 29 (12.6%) samples out of 230 samples examined. It was observed that the detection of P. jirovecii was associated with age (P < 0.05) while there were no associations between diagnosis, sex, and prevalence of P. jirovecii (P > 0.05). Polymerase chain reaction was showed to be a better method for the detection of P. jirovecii based on the 51.7% sensitivity and 100% specificity of the microscopy. Conclusion: The study concluded that P. jirovecii is prevalent in patients with respiratory tract infections in hospitals from the southwestern part of Nigeria and should be included in diagnosis of these infections in this part of the world.

Association of virulence genes with mecA gene in Staphylococcus aureus isolates from Tertiary Hospitals in Nigeria

INTRODUCTION Staphylococcus aureus is the etiological agent for a wide range of human infections, and its pathogenicity largely depends on various virulence factors associated with adherence, evasion of the immune system and damage of the host. This study determined the prevalence of methicillin-resistant S. aureus (MRSA) and some selected virulence genes in clinical isolates of S. aureus from South-Western Nigeria. MATERIALS AND METHODS The antibiotic susceptibility of 156 S. aureus isolates to various antibiotics was determined. Moreover, polymerase chain reaction detection of the mecA gene was performed including SCCmec typing, and the isolates were screened for selected genes (alpha hemolysin [hla], intracellular adhesion A [icaA], Panton-Valentine leukocidin [PVL], fibronectin binding protein A [fnbA], bone sialoprotein binding protein [bbp], exfoliative toxin A [eta], exfoliative toxin B [etb], and collagen binding adhesion [cna]) associated with virulence. RESULTS The prevalence of mecA gene was 42.3% (66 out of 156 S. aureus), and SCCmec typing showed that 24 (36.4%) carried the SCCmec II element, 4 (6.1%) with type III, 10 (15.2%) with SCCmec IV, and 28 (42.4%) harbored type V. The proportion of S. aureus with the following genes was ascertained: Hla (55.1%), icaA (42.3%), PVL (34.6%), fnbA (8.3%), bbp (4.5%), and eta (3.8%). All the isolates were etb and cna negative. The prevalence of the PVL gene in methicillin susceptible Staphylococcus aureus (MSSA) was 53.3% compared with 9.1% of MRSA. An association between virulence genes (eta and icaA) and mecA positive S. aureus; and significant difference in the distribution of virulence genes in in-patients and out-patients were found. The MRSA strains in South-Western Nigeria were dominated by SCCmec II and SCCmec V. CONCLUSION The study concluded that there is a high prevalence of MRSA in Nigeria with association of eta and icaA genes with mecA gene in S. aureus isolates.

Opening Pandora box - high level resistance to antibiotics of last resort in Gram negative bacteria from Nigeria

Antimicrobial resistance (AMR) is a global problem but information about the prevalence and mechanisms of resistance in sub-Saharan Africa are lacking. We determined the percentage of drug resistant isolates and resistance mechanisms in 307 Gram negative isolates randomly collected from south western Nigeria. Susceptibility testing revealed 78.1%, 92.2% and 52.6% of all isolates were resistant to fluoroquinolones, third generation cephalosporins and carbapenems respectively. There were more resistant isolates from the stools of uninfected patients than from specimens of patients with symptoms of infections. Only a small proportion of E. coli (10%) and Klebsiella (7%) isolates produced a carbapenemase. Whole genome sequencing of selected isolates identified the presence of globally disseminated clones. This depicts a crisis for the use of first line therapy in Nigerian patients, it is likely that Nigeria is playing a significant role in the spread of AMR due to her high population and mobility across the globe.

High seroprevalence of asymptomatic viral haemoparasites among prospective blood donors in Nigeria

OBJECTIVE To determine the prevalence of viral haemoparasites in prospective Nigerian blood donors. METHODS Ethical clearance was obtained and informed consent questionnaires were distributed to blood donors to obtain their demographical data. A total of 186 blood donors from LAUTECH Teaching Hospital, Osogbo were tested for hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) using rapid test kit and enzyme linked immunosorbent assay. RESULTS The highest prevalence of blood transmitted infections was 182 (97.85%) while the prevalence of HIV, HAV, HBV and HCV were 6.45%, 97.85%, 14.52% and 3.23%, respectively. Highest seroprevalence for hepatitis A, B and C occurred among low risk occupation. There was no significant association between all the hepatitis viruses and demographic factors except occupation with P value of 0.002 7. Hepatitis A, B and C seropositive blood donors on average tend to have PCV within the normal reference range. Out of the 27 hepatitis B positive blood donors, 22 were donating blood for the first time while 5 were repeat donors. None of the hepatitis C seropositive donors have been exposed to blood or any form of its products and were all donating blood for the first time. However, the distribution of donor type for HAV is random. CONCLUSIONS The prevalence of HAV, HBV, HCV and HIV among prospective donors in Nigeria is alarming particularly HAV. These infections can be transmitted to recipients if proper screening is not carried out, hence they should be included as a routine test for blood donors.