Ravesh Singh

Functional ‘omics’ studies previously identified the M. tuberculosis surface located adhesins, heparin-binding hemagglutinin adhesin (HBHA) and curli pili (MTP) as significant potential targets for the design of tuberculosis (TB) point-of-care diagnostics, effective drugs, and vaccines. Little is known on the effect of these adhesins on the pathogen’s transcriptome. The current study, via transcriptomics, elucidated whether the deletion of the single genes, hbhA and mtp, and double genes, hbhA-mtp, via specialised transduction, affected global bacterial gene expression. RNA sequencing of M. tuberculosis wild-type V9124 (WT), single and double deletion HBHA and MTP mutant strains were confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) on selected genes, and a functional bacterial ATP bioluminescence assay. The 43 significantly differentially expressed genes amongst the deletion mutants were functionally categorized into central carbon metabolism (CCM), cell wall biosynthesis and cell wall transport and processes. The increased expression of genes associated with ATP synthase and cell wall processes were confirmed by RT-qPCR. In the absence of the adhesins, a decreased ATP concentration was observed suggesting either increased utilization or alterations to the proton motive force (PMF) that resulted in a potential inhibition of ATP synthesis. Therefore, deletions of the mtp and hbhA genes were associated with significant perturbations in CCM regulation/function, and transport of proteins to the cell wall, indicating the significant contribution of these adhesins in fundamental processes contributing to TB pathogenesis. Thus, this study indicates that MTP and HBHA influence gene expression in M. tuberculosis and represent important targets for TB diagnostic/therapeutic interventions and should be investigated as vaccine and adjunctive therapies.

Background: Antimicrobial resistance (AMR) is a global threat in the public healthcare sector. The emergence of carbapenem-resistant Enterobacterales (CRE) has become a serious public health threat in South Africa. The spread of CRE has led to the use of colistin for treating severe infections. Colistin is a cationic, lipopeptide antibacterial agent that is effective against most Gram-negative bacteria through its disruption of the bacterial cell membrane. This study aims to determine the colistin resistance (MIC) and mobile colistin resistance (mcr-1) gene in clinical isolates from healthcare facilities in Mthatha and its surrounding areas. Methods: Fifty-three CRE isolates were collected from health facilities between January 2019 and June 2021 and stored in skim milk 10% and 5% inositol broth. The carbapenemase confirmatory test involved a RESIST-4 O.K.N.V assay (Coris BioConcept, Gembloux, Belgium), which was conducted following manufacturer protocol. Broth microdilution was performed according to the ISO standard method (20776-1) using A ComAspTM colistin 0.25–16 μg/mL MIC Broth. Conventional polymerase reaction (PCR) was performed for the detection of mcr-1. Results: N = 53 (100%) isolates were used. A total of 53% were defined as Klebsiella pneumoniae, Escherichia coli constituted 8%, Enterobacter cloacae 8%, Serratia marcescens 8%, Serratia fonticola 2%, Enterobacter aerogenes 2%, Klebsiella oxytoca 2%, Citrobacter koseri 2%, and Citrobacter freundii 2%. The specimens were from the following wards: Pediatric and Neonatal 38%, Medical 30%, Gynecology, Labour, and Maternity 11%, OPD and A&E 11%, ENT 4%, and Others—Male TB ward, Trauma, and adult ICU 6%. In total, 13% of the isolates were resistant and 86% were sensitive to colistin. The common CRE genes detected were OXA-48 at 47%, NDM at 13%, VIM at 1%, and a combination of OXA-48 and NDM at 5%. Of the isolates, 66% were positive for the production of carbapenamase. In this study, we found that all N = 53 (100%) isolates did not have the mobile colistin resistance gene (mcr-1). Conclusions: Antimicrobial resistance is associated with the emergence of carbapenemases genes. Increasing resistance to colistin in clinical settings can lead to difficulties in treating CRE infections, which may lead to clinical failure. In our study, 13% of isolates were phenotypically resistant to colistin.

Background: The emergence of carbapenem-resistant Enterobacterales is prevalent and poses a significant threat to health systems worldwide. This study aimed to conduct a molecular analysis of tigecycline resistance in 100 CRE isolates from Mthatha Hospital and surrounding hospitals. Methods: A retrospective study among patients who attended Nelson Mandela Academic Hospital (NMAH) and Mthatha Regional Hospital (MRH), Eastern Cape, South Africa. Enterobacterales isolates were identified using the Vitek2^® system (bioMérieux); an E-test was performed on 100 CRE isolates according to the manufacturer’s instructions. PCR assays for rapid detection of tet(X) and its variants, including tet(X1) and tet(X2), and high-level tigecycline resistance genes tet(X3), tet(X4), and tet(X5) were developed. Results: The results show a notably high prevalence of CRE infections in neonatal, male surgical, and maternal and pediatric wards, predominantly driven by Klebsiella species (53.4%), followed by Enterobacter species (20.5%) and then Escherichia coli (6.7%), and 7.2% of CRE isolates were resistant to tigecycline (E-test). In this study, tet(X) genes were not identified as the primary mechanism of tigecycline resistance. The risk factors associated with tigecycline resistance in CRE include age, pre-exposure to antibiotics, prolonged hospitalization, and undergoing invasive procedures, indicated by strong r = 0.9501. Conclusions: CRE gradually evolves, posing a significant threat to patients of all ages; early detection of carbapenemase production in clinical infections, carriage states, or both is essential to prevent hospital-based outbreaks.

Background: The emergency of carbapenem-resistant Enterobacterales is prevalent and poses a significant threat to health systems worldwide. This study aimed to conduct a molecular analysis of tigecycline resistance in 100 CRE in hospital isolates from Mthatha and surrounding hospitals. Methods: A retrospective study among patients who attended Nelson Mandela Academic Hospital (NMAH) and Mthatha Regional Hospital (MRH), Eastern Cape, South Africa. Enterobacterales isolates were identified using the Vitek2® system (bioMérieux), E-test was performed in 100 CRE hospital isolates according to manufacturer’s instructions, A multiplex SYBR green-based PCR assay for rapid detection of tet(X) and its variants, including tet(X1), tet(X2), and high-level tigecycline resistance genes tet(X3), tet(X4), and tet(X5) were developed. Results: The results show a notable high prevalence of CRE infections in neonatal, male surgical, and maternal and pediatric wards, predominantly driven by Klebsiella spp. (53.4%), followed by Enterobacter spp. (20.5 %), then Escherichia coli (6.7%), 7.2% of CRE were resistant to Tigecycline (E-TEST), Tet(X) genes were Not responsible for tigecycline resistance in this setting. The risk factors associated with tigecycline resistance in CRE include age, pre-exposure to antibiotics, prolonged hospitalization, and undergoing invasive procedures indicated by strong r2 =0.9501. Conclusion: CRE gradually evolves, posing a significant threat to patients of all ages; early detection of carbapenemase production in clinical infections, carriage states, or both is essential to prevent hospital-based outbreaks.

Background: Drug‐resistant pathogens are a global health concern. Of particular concern are methicillin‐resistant Staphylococcus aureus (MRSA) and carbapenem‐resistant Pseudomonas aeruginosa (CRPA), as they leave clinicians with limited treatment options. Aim: This study was aimed at determining the prevalence of MRSA and CRPA at an academic referral hospital in KwaZulu‐Natal from January 2019 to December 2023. Method: The hospital laboratory information system provided the antimicrobial susceptibility profiles of S. aureus and P. aeruginosa . Statistical Package for the Social Sciences (SPSS, Version 21) was used to analyze data on the prevalence of MRSA and CRPA in the hospital over the past 5 years. Results: This study identified 18% (249/1,409) prevalence of MRSA in 2019 to 18% (237/1,284) by 2023. Also, the prevalence of CRPA decreased from 15% (98/673) in 2019 to 14% (85/618) by 2023. MRSA isolates in this study exhibited over 50% resistance to gentamicin, erythromycin/azithromycin, clindamycin, and trimethoprim–sulfamethoxazole. All the tested antibiotics (amikacin, cefepime, ceftazidime, ciprofloxacin, gentamicin, and piperacillin–tazobactam) against CRPA have been met by resistance of greater than 30%, except for 3% resistance to colistin. There was no statistically significant difference between females and males ( p = 0.135) and between intensive care unit (ICU) and non‐ICU wards ( p = 0.156). Conclusion: The growing threat of MRSA and CRPA in hospitals highlights the need for continuous surveillance to track their spread and strengthen infection prevention efforts.

Editorial: STI awareness day: diagnosis and control of STIs in LMICs

Introduction. Intestinal helminths and microbiota share the same anatomical niche during infection and are likely to interact either directly or indirectly. Whether intestinal helminths employ bactericidal strategies that influence their microbial environment is not completely understood. Hypothesis. In the present study, the hypothesis that the adult hookworm Nippostrongylus brasiliensis produces molecules that impair bacterial growth in vitro, is tested. Aim. To investigate the in vitro bactericidal activity of Nippostrongylus brasiliensis against commensal and pathogenic bacteria. Methodology. The bactericidal effect of somatic extract and excretory-secretory products of adult Nippostrongylus brasiliensis on Gram-positive ( Staphylococcus aureus ) and Gram-negative ( Escherichia coli , Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae ) bacteria was assessed using growth assays. Minimum inhibitory concentration and minimum bactericidal concentration assays were performed using excretory-secretory products released from the pathogen. Results. Broad-spectrum in vitro bactericidal activity in excretory-secretory products, but not somatic extract of adult Nippostrongylus brasiliensis was detected. The bactericidal activity of excretory-secretory products was concentration-dependent, maintained after heat treatment, and preserved after repeated freezing and thawing. Conclusion. The results of this study demonstrate that helminths such as Nippostrongylus brasiliensis release molecules via their excretory-secretory pathway that have broad-spectrum bactericidal activity. The mechanisms responsible for this bactericidal activity remain to be determined and further studies aimed at isolating and identifying active bactericidal molecules are needed.

Background: Intestinal helminth parasites are potent stimulators of T helper type 2 (Th2) and regulatory Th3 anti-inflammatory immune responses, while human immunodeficiency virus (HIV) infections are activators of predominantly T helper type 1(Th1) pro-inflammatory responses. Studies investigating the immune profiles of individuals coinfected with helminths and HIV are scarce. Although it is well known that helminths cause a type 2 immune response during the chronic stage of infection that is characterised by Th2 cell differentiation, eosinophil recruitment, and alternative macrophage activation, the immune mechanisms that regulate tissue damage at the time of parasite invasion are poorly understood. Aim: The aim of the study was to determine the cytokine gene expression profiles during HIV and helminth coinfection in underprivileged South African adults living in a peri-urban area with poor sanitary conditions and a lack of clean water supply. Method: Study participants (n = 164) were subdivided into uninfected controls, HIV-infected, helminth-infected, and HIV and helminth-coinfected groups. The Kato–Katz and Mini Parasep techniques and Ascaris lumbricoides-specific Immunoglobulin E (IgE) and Immunoglobulin G4 (IgG4) levels were used to detect helminth infections. Participants’ HIV status was determined using two HIV1/2 antibody test kits. RNA was isolated from white blood cells for cytokine (Th1-, Th2-, and Th17-related) and transcription factor gene expression profiling using real-time PCR. Results: Multivariate regression data were adjusted for age, gender, BMI, antiretroviral treatment (ART), and nutritional supplement intake. The HIV and helminth-coinfected group had significantly higher tumour necrosis factor alpha (TNF-α) (adjusted β = 0.53, p = 0.036), interleukin 2 (IL-2) (adjusted β = 6.48, p = 0.008), and interleukin 17 (IL-17) (adjusted β = 1.16, p = 0.001) levels and lower GATA binding protein 3 (GATA3) levels (adjusted β = −0.77, p = 0.018) compared to the uninfected controls. No statistical significance was noted for Th2-related cytokines. Conclusion: The coinfected group had higher proinflammatory Th1- and Th17-related cytokine gene expression profiles compared to the uninfected controls. The findings suggest that pro-inflammatory responses are elevated during coinfection, which supports the hypothesis that helminths have a deleterious effect on HIV immune responses.

Background: Helminth infections are widespread in tuberculosis-endemic areas and are associated with an increased risk of active tuberculosis. In contrast to the pro-inflammatory Th1 responses elicited by Mycobacterium tuberculosis (Mtb) infection, helminth infections induce anti-inflammatory Th2/Treg responses. A robust Th2 response has been linked to reduced tuberculosis protection. Several studies show the effect of helminth infection on BCG vaccination and TB, but the mechanisms remain unclear. Aim: To determine the cytokine response profiles during tuberculosis and intestinal helminth coinfection. Methods: For the in vitro study, lymphocytic Jurkat and monocytic THP-1 cell lines were stimulated with Mtb H37Rv and Ascaris lumbricoides (A. lumbricoides) excretory-secretory protein extracts for 24 and 48 h. The pilot human ex vivo study consisted of participants infected with Mtb, helminths, or coinfected with both Mtb and helminths. Thereafter, the gene transcription levels of IFN-γ, TNF-α, granzyme B, perforin, IL-2, IL-17, NFATC2, Eomesodermin, IL-4, IL-5, IL-10, TGF-β and FoxP3 in the unstimulated/uninfected controls, singly stimulated/infected and costimulated/coinfected groups were determined using RT-qPCR. Results: TB-stimulated Jurkat cells had significantly higher levels of IFN-γ, TNF-α, granzyme B, and perforin compared to unstimulated controls, LPS- and A. lumbricoides-stimulated cells, and A. lumbricoides plus TB-costimulated cells (p < 0.0001). IL-2, IL-17, Eomes, and NFATC2 levels were also higher in TB-stimulated Jurkat cells (p < 0.0001). Jurkat and THP-1 cells singly stimulated with TB had lower IL-5 and IL-4 levels compared to those singly stimulated with A. lumbricoides and those costimulated with TB plus A. lumbricoides (p < 0.0001). A. lumbricoides-singly stimulated cells had higher IL-4 levels compared to TB plus A. lumbricoides-costimulated Jurkat and THP-1 cells (p < 0.0001). TGF-β levels were also lower in TB-singly stimulated cells compared to TB plus A. lumbricoides-costimulated cells (p < 0.0001). IL-10 levels were lower in TB-stimulated Jurkat and THP-1 cells compared to TB plus A. lumbricoides-costimulated cells (p < 0.0001). Similar results were noted for the human ex vivo study, albeit with a smaller sample size. Conclusions: Data suggest that helminths induce a predominant Th2/Treg response which may downregulate critical Th1 responses that are crucial for tuberculosis protection.

Sub-Saharan Africa is heavily burdened with human immunodeficiency virus (HIV) and helminth infections, which are potent activators of pro-inflammatory and anti-inflammatory immune responses, respectively. Considering that helminths (such as Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Schistosoma haematobium, and Schistosoma mansoni) can potentially dampen the production and expression of vital anti-viral pro-inflammatory cytokines, leading to enhanced HIV replication and severity, well-designed intervention studies are needed which will offer conclusive data on the nature of these interactions and the impact of deworming in HIV-infected patients. Such research will impact governmental health policies and deworming programmes, allowing for the implementation of integrated systems that will contribute to the overall improvement of African health systems.