Carriage of linezolid-resistant enterococci (LRE) among humans and animals in Nigeria: coexistence of the cfr, optrA, and poxtA genes in Enterococcus faecium of animal origin

Objectives: In contrast to increasing reports of the emergence of linezolid-resistant enterococci (LRE) emanating from many countries in Europe, Asia, and North America, data on its status and dissemination from the African continent remain scarce, with the information available limited to countries in North Africa. This study investigated the carriage of LRE and the genetic mechanism of resistance among Enterococcus faecium and Enterococcus faecalis strains recovered from humans and animals in Makurdi, Nigeria. Methods: We conducted a cross-sectional study between June 2020 and July 2021 during which 630 nonduplicate human and animal faecal samples were collected and processed for the recovery of LRE. The genetic mechanisms for resistance were investigated using polymerase chain reaction (PCR) and Sanger sequencing. Results: Linezolid-resistant enterococci were recovered from 5.87% (37/630; 95% CI: 4.17–8.00) of the samples, with the prevalence in animals and humans being 6.22% [(28/450); 95% CI: 4.17–8.87] and 5.00% [(9/180); 95% CI: 2.31–9.28], respectively. All isolates remained susceptible to vancomycin. No known point mutation mediating linezolid resistance was detected in the 23S rRNA and ribosomal protein genes; however, acquisition of one or more potentially transferable genes (cfr, optrA, and poxtA) was observed in 26 of the 37 LRE isolates. Co-existence of all three transferable genes in a single isolate was found in four E. faecium strains of animal origin. Conclusion: This study provides baseline evidence for the emergence and active circulation of LRE driven majorly by the acquisition of the optrA gene in Nigeria. To the best of our knowledge, our study is the first to report a co-carriage of all three transferable linezolid resistance determinants in E. faecium. Active LRE surveillance is urgently required to understand the extent of LRE spread across sub-Saharan Africa and to develop tailored mitigation strategies

Identification of Adult Fasciola spp. Using Matrix-Assisted Laser/Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

Fascioliasis is a neglected trematode infection caused by Fasciola gigantica and Fasciola hepatica. Routine diagnosis of fascioliasis relies on macroscopic identification of adult worms in liver tissue of slaughtered animals, and microscopic detection of eggs in fecal samples of animals and humans. However, the diagnostic accuracy of morphological techniques and stool microscopy is low. Molecular diagnostics (e.g., polymerase chain reaction (PCR)) are more reliable, but these techniques are not routinely available in clinical microbiology laboratories. Matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a widely-used technique for identification of bacteria and fungi; yet, standardized protocols and databases for parasite detection need to be developed. The purpose of this study was to develop and validate an in-house database for Fasciola species-specific identification. To achieve this goal, the posterior parts of seven adult F. gigantica and one adult F. hepatica were processed and subjected to MALDI-TOF MS to create main spectra profiles (MSPs). Repeatability and reproducibility tests were performed to develop the database. A principal component analysis revealed significant differences between the spectra of F. gigantica and F. hepatica. Subsequently, 78 Fasciola samples were analyzed by MALDI-TOF MS using the previously developed database, out of which 98.7% (n = 74) and 100% (n = 3) were correctly identified as F. gigantica and F. hepatica, respectively. Log score values ranged between 1.73 and 2.23, thus indicating a reliable identification. We conclude that MALDI-TOF MS can provide species-specific identification of medically relevant liver flukes

Identification of Adult Fasciola spp. Using Matrix-Assisted Laser/Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

Fascioliasis is a neglected trematode infection caused by Fasciola gigantica and Fasciola hepatica. Routine diagnosis of fascioliasis relies on macroscopic identification of adult worms in liver tissue of slaughtered animals, and microscopic detection of eggs in fecal samples of animals and humans. However, the diagnostic accuracy of morphological techniques and stool microscopy is low. Molecular diagnostics (e.g., polymerase chain reaction (PCR)) are more reliable, but these techniques are not routinely available in clinical microbiology laboratories. Matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a widely-used technique for identification of bacteria and fungi; yet, standardized protocols and databases for parasite detection need to be developed. The purpose of this study was to develop and validate an in-house database for Fasciola species-specific identification. To achieve this goal, the posterior parts of seven adult F. gigantica and one adult F. hepatica were processed and subjected to MALDI-TOF MS to create main spectra profiles (MSPs). Repeatability and reproducibility tests were performed to develop the database. A principal component analysis revealed significant differences between the spectra of F. gigantica and F. hepatica. Subsequently, 78 Fasciola samples were analyzed by MALDI-TOF MS using the previously developed database, out of which 98.7% (n = 74) and 100% (n = 3) were correctly identified as F. gigantica and F. hepatica, respectively. Log score values ranged between 1.73 and 2.23, thus indicating a reliable identification. We conclude that MALDI-TOF MS can provide species-specific identification of medically relevant liver flukes

Carriage of linezolid-resistant enterococci (LRE) among humans and animals in Nigeria: coexistence of the cfr, optrA, and poxtA genes in Enterococcus faecium of animal origin

ABSTRACT: Objectives: In contrast to increasing reports of the emergence of linezolid-resistant enterococci (LRE) emanating from many countries in Europe, Asia, and North America, data on its status and dissemination from the African continent remain scarce, with the information available limited to countries in North Africa. This study investigated the carriage of LRE and the genetic mechanism of resistance among Enterococcus faecium and Enterococcus faecalis strains recovered from humans and animals in Makurdi, Nigeria. Methods: We conducted a cross-sectional study between June 2020 and July 2021 during which 630 non-duplicate human and animal faecal samples were collected and processed for the recovery of LRE. The genetic mechanisms for resistance were investigated using polymerase chain reaction (PCR) and Sanger sequencing. Results: Linezolid-resistant enterococci were recovered from 5.87% (37/630; 95% CI: 4.17–8.00) of the samples, with the prevalence in animals and humans being 6.22% [(28/450); 95% CI: 4.17–8.87] and 5.00% [(9/180); 95% CI: 2.31–9.28], respectively. All isolates remained susceptible to vancomycin. No known point mutation mediating linezolid resistance was detected in the 23S rRNA and ribosomal protein genes; however, acquisition of one or more potentially transferable genes (cfr, optrA, and poxtA) was observed in 26 of the 37 LRE isolates. Co-existence of all three transferable genes in a single isolate was found in four E. faecium strains of animal origin. Conclusion: This study provides baseline evidence for the emergence and active circulation of LRE driven majorly by the acquisition of the optrA gene in Nigeria. To the best of our knowledge, our study is the first to report a co-carriage of all three transferable linezolid resistance determinants in E. faecium. Active LRE surveillance is urgently required to understand the extent of LRE spread across sub-Saharan Africa and to develop tailored mitigation strategies

Scavenger Receptor Class B Type I and Hepatitis C Virus Infection of Primary Tupaia Hepatocytes

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes

Inhibition of Hepatitis C Virus-Like Particle Binding to Target Cells by Antiviral Antibodies in Acute and Chronic Hepatitis C

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies

Concurrent resistance to carbapenem and colistin among enterobacteriaceae recovered from human and animal sources in Nigeria is associated with multiple genetic mechanisms

Resistance to last resort drugs such as carbapenem and colistin is a serious global health threat. This study investigated carbapenem and colistin resistance in 583 non-duplicate Enterobacteriaceae isolates utilizing phenotypic methods and whole genome sequencing (WGS). Of the 583 isolates recovered from humans, animals and the environment in Nigeria, 18.9% (110/583) were resistant to at least one carbapenem (meropenem, ertapenem, and imipenem) and 9.1% (53/583) exhibited concurrent carbapenem-colistin resistance. The minimum inhibitory concentrations of carbapenem and colistin were 2-32 mu g/mL and 8 to >64 mu g/mL, respectively. No carbapenem resistant isolates produced carbapenemase nor harbored any known carbapenemase producing genes. WGS supported that concurrent carbapenem-colistin resistance was mediated by novel and previously described alterations in chromosomal efflux regulatory genes, particularly mgrB (M1V) ompC (M1_V24del) ompK37 (I70M, I128M) ramR (M1V), and marR (M1V). In addition, alterations/mutations were detected in the etpA, arnT, ccrB, pmrB in colistin resistant bacteria and ompK36 in carbapenem resistant bacteria. The bacterial isolates were distributed into 37 sequence types and characterized by the presence of internationally recognized high-risk clones. The results indicate that humans and animals in Nigeria may serve as reservoirs and vehicles for the global spread of the isolates. Further studies on antimicrobial resistance in African countries are warranted