Masters Degrees (Medical Microbiology)

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Drug resistant eskapee pathogens in clinical isolates and hospital effluent.
(2024) Masalane, Naledi Shakoane.; Mbanga, Joshua.; Bester, Linda.; Essack, Sabiha Yusuf.
Background: The ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp, and Escherichia coli) is a group of Gram-negative and -positive pathogens that exhibit antimicrobial resistance (AMR) to commonly used antibiotics. Aim: This study compared clinical ESKAPEE isolates from patients and hospital effluent in terms of antibiotic resistance patterns, antibiotic resistance genes, mobile genetic elements (MGEs) and phylogenomic relationships. Methodology: Samples were collected and pooled from the final effluent point of a regional hospital in the uMgungundlovu district, Kwa-Zulu Natal, South Africa. Clinical isolates were also collected from the same hospital. Selective culture media was used for isolation and identification. Antimicrobial susceptibility testing (AST) was performed using the VITEK® 2 system. DNA was extracted using the GenElute extraction kits prior to whole genome sequencing. The resistome, mobilome and phylogenetic lineages of sequenced isolates were assessed using bioinformatics analysis. ResFinder, PlasmidFinder, INTEGRALL and PGAP & ISFinder were used to annotate and identify resistance genes, plasmids, integrons and insertion sequences and transposons, respectively. MLST was used to identify sequence types, BV-BRC was used to construct the phylogenetic trees, and iTOL was used to view, edit and annotate the generated phylogenetic trees. Results: A total of 112 presumptive ESKAPEE constituted the sample of which 42 were clinical isolates and 70 were isolates from hospital effluent. Of these, 36 isolates consisting of 16 K. pneumoniae, 9 E. faecium, 7 Enterobacter hormaechei, 3 E. coli and 1 P. aeruginosa were positively identified as ESKAPEE pathogens by WGS. The effluent E. faecium isolates were totally resistant to six of the antibiotics tested (tetracycline, doxycycline, erythromycin, azithromycin, fosfomycin and levofloxacin). They also harboured antibiotic resistance genes (ARGs) that confer resistance to aminoglycosides (aac(6')-Ii, aac(6')-aph(2''), aph(2'')-Ia), macrolides (msr(C)), tetracycline (tet(M), tet(L)), and trimethoprim (dfrG), none of which were carried on any MGEs. In the K. pneumoniae isolates, ARGs conferring resistance to ß-lactam antibiotics were the most common among the clinical isolates while effluent K. pneumoniae carried markedly fewer ARGs. Aminoglycoside resistance genes (aph(6)-Id, aph(3'')-Ib, aac(6')-Ib-cr aadA16), quinolone (qnrB1, qnrB6, qnrS1, OqxA and oqxB), ß-lactam (blaSHV group, blaCTX-M-15, and blaTEM group) and trimethoprim-sulfamethoxazole (sul1, sul2, and dfrA27, drfA14) were common among the clinical K. pneumoniae isolates. ARGs were associated with diverse MGEs, particularly in the clinical isolates and a common plasmid replicon IncFIB(K) was identified in both clinical and effluent isolates. MLST showed no relation between the K. pneumoniae clinical and effluent isolates. The clinical P. aeruginosa isolate harboured ß-lactam [blaOXA-50, blaPAO], aminoglycoside (aph(3')-Iib), and fluoroquinolone (crpP) ARGs that were not associated with MGEs. The isolate had a sequence type ST275, which showed no relation with other isolates it was compared with. The majority of clinical E. hormaechei isolates showed total resistance to aminoglycosides, ß-lactams and tetracycline antibiotics. The E. hormaechei isolates, except one, harboured [blaOXA-1, blaCTX-M-15, blaACT-5 and blaTEM-1B], dfrA14, tet(A), [aph(6)-Id, aph(3'')-Ib, aac(3)-Iia, aac(6')-Ib-cr] and sul2 ARGs conferring resistance to ß-lactams, trimethoprim, tetracycline, aminoglycoside, and sulfamethoxazole, respectively. Clinical and effluent isolates of E. coli displayed similar resistance patterns. ARGs conferring resistance to ß-lactam (blaCTXM-15 and blaOXA-1), aminoglycosides (aph(6)-Id, aph(3'')-Ib, aac(6')-Ib-cr, aadA5 and aac(3)-IId), trimethoprim-sulfamethoxazole (dfrA14, dfrA17, sul1 and sul2), and tetracycline (tet(A) and tet(B)) were observed among the clinical isolates; while the effluent isolate harboured ß-lactam (blaCTXM-15, blaOXA-1, blaTEM-1B and blaOXA-10), aminoglycosides (aac(3)-Iid, aac(3)-IIa, aac(6')-Ib-cr, aph(6)-Id, aph(3'')-Ib), trimethoprim-sulfamethoxazole (dfrA14, dfrA23, sul1 and sul2), and tetracycline (tet(A)). The clinical E. coli isolates had sequence types ST69 and ST131 and the effluent isolate belonged to ST10. The clinical and effluent isolates from this study that did not cluster together were not closely related and belonged to different sequence types. Conclusion: These findings demonstrate the prevalence of ESKAPEE pathogens in hospital effluent. While the effluent did not mirror AMR in the clinical setting, presence of antibiotic-resistant bacteria (ARB) in the effluent cannot be overlooked. This study highlights the need for continuous monitoring of the effluent to track the spread of resistant bacteria from the hospital to the environment.
The phenotypic and clinical characterisation of tigecycline coresistant carbapenem-resistant Enterobacterales observed at Inkosi Albert Luthuli Central Hospital, Durban, South Africa.
(2021) Sheik Aboo, Khathija Bibi.; Swe Swe-Han, Khine.
Background Rising rates of carbapenem-resistant Enterobacterales (CRE) and limited therapeutic options have resulted in clinical dependence on tigecycline with subsequent emergence of tigecycline coresistant CRE (TGC Co-R CRE). Characterisation of TGC Co-R CRE is imperative to limiting its propagation. Objective We sought to determine the frequency, clinical implication, and microbiologic characteristics of TGC Co-R CRE at Inkosi Albert Luthuli Central Hospital from 2017 to 2019. Methodology We undertook a retrospective descriptive study. Data sources comprised the laboratory and hospital information systems. The frequency of TGC Co-R CRE was calculated. Specimen type, species, antimicrobial resistance, infection onset, antimicrobial exposure, age, sex, comorbidities, ward, and clinical outcome were characterised. Results The frequency of TGC Co-R CRE was 2/53 (3.8%) in 2017, 0/90 (0.0%) in 2018 and 4/123 (3.3%) in 2019. The decrease was not significant (p = 0.148). Six isolates were recorded. Acquisition was uniformly healthcare associated. Most cases (5/6; 83,3%) were female and two-thirds (4/6; 66.7%) were paediatric. Most cases were ICU patients (5/6; 83,3%). Most cases (5/6; 83.3%) were carbapenem-exposed. None were tigecycline-exposed. Comorbidities included HIV (2/6; 33.3%), SLE (1/6; 16.7%), burns (1/6; 16.7%) and surgery (2/6; 33.3%). Half the patients (3/6; 50.0%) demised. Specimens comprised peritoneal dialysis fluid (1/6; 16.7%), blood culture (1/6; 16.7%), endotracheal aspirate (2/6; 33.3%), catheter urine (1/6; 16.7%) and wound swab (1/6; 16.7%). Species comprised Klebsiella pneumoniae (3/6; 50.0%), Enterobacter cloacae (2/6; 33.3%) and Serratia species (1/6; 16.7%). All isolates were multidrug-resistant (MDR). Conclusion The advent of TGC Co-R CRE is a phenomenon warranting further research into prevalence, resistance mechanisms and acquisitional risk factors. The results of this study are of hypothesisgenerating value for subsequent research.
Cytokine immune response profiles during 5 intestinal helminths and Mycobacterium 6 tuberculosis coinfection: An in vitro and human ex vivo study in KwaZulu-Natal.
(2023) Bhengu, Khethiwe Nomcebo.; Mkhize-Kwitshana, Zilungile Lynette.; Singh, Ravesh.; Naidoo, Pragalathan.
Background: There is a striking geographic overlap between helminths and tuberculosis (TB), particularly in developing countries like Africa. Underprivileged communities are more susceptible to these illnesses due to poverty, poor sanitation, and other environmental factor Helminth and tuberculosis infections exhibit distinct immune responses, which may be antagonistic in coinfected hosts and lead to poor prognosis. Helminth infections induce anti422 inflammatory Th2/Treg responses contrary to the pro-inflammatory Th1 responses triggered by Mycobacterium tuberculosis (Mtb) infection. Reduced TB protection has been associated with a strong Th2 response. Uncertainty exists on how helminth infection affects the host’s resistance to TB. This necessitates further investigation of immune responses in helminths and TB coinfection cases, particularly in KwaZulu-Natal (KZN). Aim: To determine the cytokine response profiles during intestinal helminth and TB coinfection using lymphocytic Jurkat and monocytic THP-1 cell lines for the in vitro study and TB and helminth coinfected South African adults for the human ex vivo study. Methods: Lymphocytic Jurkat and monocytic THP-1 cell lines were stimulated for 24 and 48 hours with Mtb H37Rv and Ascaris lumbricoides (A. lumbricoides) excretory-secretory protein extracts for the in vitro study. A cross-sectional study on consenting adult participants (≥18 years) (n = 414) recruited from primary health care clinics was conducted between March 2020 and August 2021 in Durban, KwaZulu Natal, for the pilot human ex vivo study. Blood and stool samples were collected from the recruited participants. The Kato-Katz and Mini-Parasep faecal parasite concentration techniques were used to detect intestinal parasite infections in stool samples. Blood samples were analysed to determine A. lumbricoides-specific immunoglobulin E (IgE) and immunoglobulin G4 (IgG4) levels to improve microscopy sensitivity. In this study, cytokine analysis was undertaken for 164 participants; 96 were HIV infected and had to be excluded, leaving 68 eligible participants. The eligible individuals were subdivided into uninfected controls (no helminth and TB infection) (n = 18), helminth only infected (n = TB only infected (n = 6), and TB and helminth co-infected (n = 6) groups. Thereafter, for both the in vitro and ex vivo study, the gene expression profiles of the T helper type 1(Th1) and transcription factors [Interferon-γ (IFN-γ), Tumour necrosis factor-α (TNF-α), Interleukin-2 (ILxvii 2), Nuclear factor of activated T cells 2 (NFATC2), Eomesodermin 446 (eomes), T helper 2 (Th2) and transcription factors (Interleukin-4 (IL-4), Interleukin5 (IL-5, Transforming growth factor-β (TGF-β), T helper type 17 (Th17) (Interleukin-17 (IL-17), immune protein and proteases (Granzyme B, Perforin), Regulatory T cells (Tregs) (Interleukin-10 (IL-10) and Fork head box P3 (FoxP3)] and the uninfected controls, TB alone, helminth alone and coinfected groups were determined using RT-qPCR. Results: (i) In vitro study: TB-stimulated Jurkat cells had significantly higher levels of IFN-γ, TNF-α, Granzyme B, and perforin compared to unstimulated controls, LPS, A. lumbricoides, 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). TB alone stimulated cells had lower IL-5 and IL-4 levels compared to A. lumbricoides alone stimulated and TB plus A. lumbricoides costimulated Jurkat and THP-1 cells (p<0.0001. A. lumbricoides alone 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 alone stimulated cells compared to TB plus A. lumbricoides costimulated cells. IL-10 levels were lower in TB stimulated Jurkat and THP-1 cells compared to TB plus A. lumbricoides costimulated cells (p<0.0001. (ii) Ex vivo study: Similar results were noted for both the in vitro and the ex vivo study, although the human study had a smaller sample size. Conclusion: Data suggest that helminths induce a predominant anti-inflammatory Th2 and Treg response which may downregulate critical proinflammatory Th1 responses crucial for TB protection.
The impact of point-of-care testing and treatment of sexually transmitted infections and bacterial V aginosis on the genital epithelial barrier integrity.
(2022) Ndlela, Nonsikelelo.; Liebenberg, Lenine Julie.
In sub-Saharan Africa, women and young girls suffer the highest burden of HIV infections. Inflammation at the genital tract is a factor responsible for the increased susceptibility to HIV risk in women, presumably through related epithelial barrier damage and target cell recruitment. Considering the direct contribution of sexually transmitted infections (STIs) and bacterial vaginosis (BV) to this inflammation, their effective treatment could potentially reduce HIV risk in this vulnerable population. It has recently been shown in South African women that a point-of-care (POC) STI/BV detection model, immediate treatment, and expedited partner therapy (EPT) resolved STIs and reduced concentrations of genital proinflammatory cytokines. This study investigated an additional impact of the model on the genital epithelial barrier. Methods POC STI/BV screening was conducted on HIV-negative women (n=238) enrolled in the CAPRISA 083 trial between May 2016 and June 2017. Chlamydia trachomatis and Neisseria gonorrhoeae infections were detected by Xpert CT/NG test, while the OSOM Rapid test detected Trichomonas vaginalis. Women were further tested for Mycoplasma genitalium and BV using PCR and microscopy, respectively. Multiplex ELISA was used to quantify 48 cytokines and five matrix metalloproteinase (MMP) biomarkers of epithelial barrier integrity from menstrual cup (MC) specimens (MMP-1, MMP-2, MMP-7, MMP-9, MMP-10). Mann- Whitney U tests were used to assess the relationship between MMPs and STI/BV at baseline, with ANOVA and multivariable linear mixed models used to determine the impact of treatment on MMP concentrations. Results At baseline, women diagnosed with STI/BV (170/238) had higher concentrations of all MMPs compared to women with neither STI/BV (68/238; p>0.05). Several proinflammatory and chemotactic cytokine concentrations correlated significantly with that of MMPs at baseline. By 12 weeks post-treatment, 31/35 (88.57%) women resolved their baseline STIs, while only 14/57 (24.56%) resolved their baseline BV status. Most participants received concurrent treatment for STIs and BV (n=60), with few receiving treatments for STI (n=3) or BV alone15 (n=25). Significant reductions in MMP-1 concentrations were observed after 6 weeks in women treated for STI and BV (2.763 pg/ml; p= 0.0066) or STIs regardless of BV status (2.760 pg/ml; p= 0.0048). No changes in MMP concentrations were observed in women treated for BV. Conclusion POC STI/BV treatment was associated with a reduction in MMP-1 concentrations. This implies that although POC STI/BV treatment may treat STIs and reduce inflammation, the integrity of the genital epithelial barrier may not be fully restored, and women remain susceptible to genital infections, including HIV, as a result. Additional strategies may be needed to repair the genital epithelium after treatment.
The Impact of vaginal microbiota on human Papillomavirus infection.
(2022) Ntuli, Lungelo.; Ngcapu, Sinaye.; Mtshali, Andile Ntombikhona.; Mzobe, Gugulethu Favourate.
Background: Cervical human papillomavirus (HPV) infection is the most common sexually transmitted infection (STI) in sub-Saharan African women of reproductive age. While most women clear HPV infection, persistent infection with high-risk HPV is the most common nonsystem biological risk factor for cervical cancer development. Increased levels of proinflammatory cytokines and overgrowth of diverse microbial communities have been implicated in undermining the clearance of the infection and promoting oncogenesis. Here we aimed to evaluate the role of vaginal microbiota composition in the persistence and clearance of HPV infections in women. Methods: This study included the assessment of 56 women who participated in the CAPRISA 083 cohort. The CAPRISA 083 study evaluated point of care STI testing immediate treatment and expedited partner therapy. Sexually transmitted infections (STIs) and BV were screened using the GeneXpert system or OSOM Trichomonas rapid test and Nugent score, respectively. Vaginal swabs and SoftCup genital secretions were collected at enrolment, 6 weeks, and 13 weeks posttreatment. The Roche Linear Array was used for HPV genotyping, and the vaginal microbiome was characterized using 16S rRNA sequencing. Results: The study demonstrated a 36/56 (64 %), 28/56 (50 %), and 36/56 (64 %) prevalent of HPV at baseline, 6 weeks and 13 weeks, respectively. The prevalence of high-risk HPV infection at baseline was 58%, 61% at 6 weeks, and 45% at 13 weeks. HPV 16, 45, 58, and 59 were the most dominant high-risk genotypes in all visits, while HPV 6 was the least common. Overall, 46% (26/56) of participants cleared any HPV genotype, while 45% (25/56) acquired and 38% (21/56) had persisted any HPV genotype at follow-up visits. Alpha diversity of the vaginal microbiome of women with HPV (p value= 0.57) and high-risk HPV (p value= 0.6) infection did not differ significantly to that in vaginal microbiome from uninfected women. LEfSe analysis identified Lactobacillus spp. (particularly L. iners) as potential biomarkers for HPV clearance between visits, whereas HPV persistence was associated with enrichment of Sneathia amnii and other BVassociated bacteria. Conclusion: While our data do not indicate the causal link between the diverse genital microbiome and HPV clearance or persistent, L. iners and Sneathia abundance were associated with HPV clearance and persistent, respectively. These data suggest the need for longitudinal investigation to confirm a biological mechanism for this relationship, which will likely benefit cervical cancer management.
The characterization of a putative DNA repair protein in Mycobacterium Tuberculosis, encoded rv2414c.
(2023) Maleke, Dieketseng Palesa.; Senzani, Sibusiso.
Mycobacterium tuberculosis (MTB) is a causative agent of the communicable disease tuberculosis (TB), which is regarded as one of the top ten causes of death worldwide. Globally, TB accounts for over 10 million infections and over 1.8 million deaths annually. These statistics are subject to a constant increase due to the emergence of drug resistant strains. Although, the recent use of next generation sequencing technology has generated complete genome sequences and functional genomic data for various organisms (MTB included), to this point, the biological functions of several proteins encoded for in the MTB genome are not known or characterized hence they are called hypothetical proteins. Characterization of these hypothetical proteins is essential, as they could be involved in key regulatory processes of MTB, which is essential for the pathogen to retain a successful life cycle and disease progression. For successful invasion of the host and disease progression, it is important for MTB to retain genomic stability. Therefore, the degree of survival of MTB in the host environment is largely dependent on the bacterium’s ability to retain genomic stability. DNA repair mechanisms protect bacterial DNA from damage that can be induced by numerous stress factors. The hypothetical protein rv2414c encodes a gene amongst the MTB immunogenic protein identified in a study by Chiliza et al., (2019) which is closely associated with genes involved in MTB DNA repair, suggesting a possible role in DNA repair pathways. Therefore, the present study is aimed at characterizing a conserved hypothetical protein encoded rv2414c in Mycobacterium tuberculosis to elucidate the proteins biological function. For in-silico characterization, three bioinformatics tools were used, namely; Mycobroswer, I-TASSER and STRING online tools. Thereafter, a CRISPR-cas9 gene silencing mechanism was developed to elucidate the biological role of rv2414c. CRISPR system entails the co-expression of the silenced form of RNA-guided DNA endonuclease from the type II CRISPR system (dCas9) and a small guide RNA specific to a target sequence, leading to the DNA recognition complex resultant in transcription interference of corresponding DNA sequence. This CRIPSR mechanism was achieved by generating knockdown mutants. Phenotypic characterization of the mutants was accomplished by monitoring the mutants’ growth kinetics and biological assays were done to assign a possible biological function to rv2414c. Bioinformatics analysis suggests rv2414c is involved in DNA repair based on the structural networks it forms with 3 genes (dprA, recA and cinA) involved in MTB DNA repair and 3 proteins (rv3242c, rv3737 and rv2897c) that are involved in mainly aiding in the mechanism of DNA repair. However, the growth kinetics showed that rv2414c has no impact on the MTB growth rate, as all strains grew in a similar growth pattern with no statistical significance (p > 0.05) observed at the different time points. Additionally, UV biological assay showed that rv2414c is not a major role player in DNA repair, as UV exposure did not have an effect on bacterial survival rate even in the knockdown strain. A slight decrease in cell survival rate was noticed after addition of Mitomycin C (MMC) between Δrv2414c (100 ng/ml) + MMC and Δrv2414c + MMC, however, the difference was not significant. This implies that rv2414c is not involved in MTB DNA repair.
Activation of silent biosynthetic gene clusters and profiling of secondary metabolites secreted by Endophytic Fungi for use as potential anti-HIV agents.
(2022) Makhwitine, John Phuti.; Ndlovu, Sizwe Innocent.; Mkhwanazi, Nompumelelo Prudence.
The continuous burden of Human Immunodeficiency Virus-1 in Sub-Saharan Africa, coupled with the inability of antiretroviral agents to eradicate HIV-1 from viral reservoirs, the potential risks of drug resistance development, and the development of adverse effects, emphasizes the need to develop a new class of HIV-1 inhibitors. Here, we cultivated five endophytic fungi isolated from Albizia adianthifolia with the addition of small epigenetic modifiers, sodium butyrate and valproic acid, to induce the expression of biosynthetic gene clusters encoding active secondary metabolites with probable anti-HIV activities. We identified a non-toxic crude extract of the endophytic fungus Penicillium chrysogenum treated with sodium butyrate to possess significantly greater anti-HIV activity than the untreated extracts. Single-round fractionated extracts of treated P.chrysogenum showed potent anti-HIV activity with an IC₅₀ of 5.90 μg/mL and a 5-fold increase compared to the untreated fraction. The active fractionated extracts were subjected to gas chromatography-mass spectrometry (GCMS), and more bioactive compounds were detected in treated P.chrysogenum fractions than in untreated fractions. These results indicate that treatment of endophytic fungi with small epigenetic modifiers enhances the secretion of secondary metabolites with anti-HIV-1 properties, acknowledging the feasibility of epigenetic modification as an innovative approach for the discovery of cryptic fungal metabolites as therapeutic compounds
The role of specific adhesins in the regulation of other adhesin genes associated with Mycobacterium tuberculosis pathogenicity.
(2022) Mthembu, Johannes Nkanyiso Thandabantu.; Pillay, Manormoney.; Senzani, Sibusiso.
Background/Aim: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), remains one of the most common causes of death throughout the world. The lack of rapid diagnostics, effective vaccines, and drugs contributes to the high number of deaths recorded every year. The presence of multiple surface adhesins is crit ical for M. tuberculosis survival because they initiate and sustain host-pathogen interactions. Amongst other adhesins is the M. tuberculosis curli pili (MTP), which aid in the adhesion/invasion of host cells and the development of biofilms. The heparin-binding haemagglutinin adhesin (HBHA) facilitates the spread of M. tuberculosis away from the site of infection. Since L, D-transpeptidase (Ldt), which is encoded by Rv0309, has been shown to bind to laminin and fibronectin and to be an adhesin, it may serve as a biomarker for the development of novel therapeutic approaches. Studying the impact triggered by the three adhesins, MTP, HBHA, and Rv0309, on the regulation of other adhesins that bind to macrophages; 19-kilodalton (19 kDa), M. tuberculosis Phosphate-binding protein (PstS‐1), Chaperone chaperonin 60.2 (Cpn 60.2), Alanine and proline-rich antigenic glycoprotein (Apa), antigen 85 complexes (Ag85A), chaperone DnaK, and M. tuberculosis type IV pili, will further substantiate their use in biomarker development. Therefore, the purpose of this study was to elucidate the role of MTP, HBHA, and Rv0309 adhesins in regulating adhesins that bind to macrophages during infection. This was achieved using in vitro infection assays with gene knockout and complemented mutant strains of M. tuberculosis, real-time quantitative PCR (RT-qPCR), and a dot blot assay. Methods: M. tuberculosis wild-type, mtp-deletion mutant (Δmtp), hbhA-deletion mutant (ΔhbhA), mtp-hbhA-deletion mutant (Δmtp-hbhA), ΔRv0309 mutant and the respective complemented strains that had been constructed in previous studies, were confirmed using polymerase chain reaction (PCR). The strains were individually cultured in supplemented Middlebrook 7H9 broth till an optical density of 600 (OD)600 of 1 was reached. THP-1 monocytic cells were differentiated into macrophages and infected at a multiplicity of infection (MOI) of five. At the end of 4-h and 24-h post-infection, cells were lysed with TritonX-100. The lysate from the infected cells was collected for RNA extraction and bacterial protein extraction. To quantify the internalized bacteria, serial dilutions of the lysate from the infected cells were plated on 7H11 agar plates for CFUs. To confirm MOI, the bacterial inoculum was serially diluted and plated for CFUs. Intracellular pathogen RNA was extracted using the TriZol method and converted into cDNA using the High Capacity cDNA Reverse Transcription kit. Primers for adhesin genes: Rv0350, Rv0440, Rv0934, Rv1860, Rv3660, Rv3763, and 2 Rv3804 were designed using Primer3plus web. To assess the impact triggered by MTP, HBHA, and Rv0309 adhesin on the expression of Rv0350, Rv0440, Rv0934, Rv1860, Rv3660, Rv3763, and Rv3804, RT-qPCR- was performed using 2X SYBR green supermix in a 7500 RT-qPCR Detection System. The gene expression data was normalized using 16S rRNA and analysed using the absolute quantification method. Proteins were isolated using the TriZol method and resuspended in 0.1 % SDS. Extracted proteins were resolved in SDS-PAGE. Western blot was attempted with Cpn60.2 and PstS-1 primary antibodies used against the Goat anti-mouse HRP secondary antibody. Dot blot was used to determine the optimal antibody dilutions and protein concentration. GraphPad Prism version 8 software was used to determine significance values. Results and Discussion: Infection with mutant mtp resulted in a decrease in the number of bacteria that infected THP-1 cells at both 4-h (p <0.001) and 24-h (p=0.002) time points compared to the wild-type. At 4-h post-infection, four of the seven genes, Rv3763, Rv3804, Rv1860 and Rv0440, were expressed in all three strains. The expression of three of these genes, Rv3763 (p=0.049), Rv3804 (p=0.003) and Rv0440 (p=0.004), was significantly increased in the Δmtp compared to the wild-type strains. Rv3660 was expressed in the Δmtp but not in the wildtype (p=0.012). At 24-h post infection. the expression of five genes was significantly increased in the wild-type compared to the Δmtp strain; Rv3763 (p=0.049), Rv0934 (p=0.006), Rv3804 (p=0.005), Rv0350 (p=0.012) and Rv0440 (p=0.004). The mtp mutant strain induced lower expression of the genes at 24-h (Rv3763, Rv0934, Rv3804, Rv1860 and Rv0440) in contrast to 4-h. Low expression of the genes Rv3763, Rv0934, Rv1860 and Rv3804 in the mutant mtp strain are associated with cell wall activities and are important virulence factors of M. tuberculosis. The decrease in the CFUs and altered gene expression in the mutant suggests that mtp is required for the expression of the genes associated with cell wall processes and that the deletion of the mtp gene may result in a decrease in cell wall activities. The deficiency of mtp gene in the mutant resulted in the reduced capability of the strain in infecting the THP-1 cells implying a decrease in the virulence of the strain. Infection with the hbhA mutant resulted in a decrease in the number of bacteria that infected THP-1 cells at both 4-h (p=0.049) and 24-h (p =0.034) time points compared to the wild-type. At 4-h post-infection, five of the seven genes, Rv3763, Rv0934, Rv3804, Rv1860 and Rv0440, were expressed in all three strains. The hbhA mutant induced the expression of all the genes at both 4-h and 24-h. Rv3660 (p=0.001) and Rv0350 (p<0.001) were expressed in the ΔhbhA but not by the wild-type at 4-h. At 24-h, all seven genes, Rv3763, Rv0934, Rv3804, Rv0350, Rv3660, Rv1860 and Rv0440, were expressed across all strains. The expression of four of these 3 genes was significantly increased in the wild-type compared to the ΔhbhA strain Rv3763 (p=0.023), Rv0934 (p=0.001), Rv3804 (p=0.002), Rv1860 (p=0.017). The deletion of hbhA induced the expression of all the adhesin genes to compensate for the loss of this gene in the mutant. There was a significant reduction in the number of bacteria that infected THP-1 cells in the Δmtp-hbhA compared to the wild-type at the 4-h (p =0.002) and 24-h (p =0.047) time points. The double knockout induced a low expression of Rv3763, Rv0934, Rv0350, Rv3804, and Rv3660 at 4-h. The expression of Rv3763 (p=0.039), Rv0934 (p = 0.002) and Rv3804 (p=0.000) was significantly increased in the wild-type strains compared to the Δmtp-hbhA. There was a higher expression of Rv1860 and Rv0440 in the mutant at 4-h compared to the 24-h time point. At 24-h, all seven genes, Rv3763, Rv0934, Rv3804, Rv0350, Rv3660, Rv1860 and Rv0440, were expressed across all strains. The expression of these genes was significantly increased in the wild-type compared to the Δmtp-hbhA. Rv3763 expression was higher in the mutant at 24- h compared to the 4-h. The observed expression in the mutant suggests the importance of both hbhA and mtp in the virulence of M. tuberculosis. Deletion of mtp-hbhA resulted in a decrease in the capability of M. tuberculosis in initiating infection in THP-1 cells. There was a significant difference in Rv0309 mutant in infecting THP-1 cells in comparison to the wild-type strains at the 4-h and 24-h time points (p = 0.001). This suggests that deletion of the Rv0309 gene in the mutant might have reduced the infecting capability of the strains. There was an upregulation of the Rv3763, Rv0934, Rv1860, Rv3804, and Rv0440 in the mutant at 4- h. Upregulation of these genes suggests that Rv0309 is an important virulence factor of M. tuberculosis. Rv1860 expression at 24-h was doubled compared to the expression of 4-h in the mutant to compensate for the loss of Rv0309 in the mutant. This may suggest that in the absence of Rv0309, the M. tuberculosis expresses Rv1860 which binds to laminin and fibronectin to promote infection. Confirmatory studies by protein detection using anti-Cpn 60.2 and anti-PstS1 antibodies with western blot failed. Numerous attempts for troubleshooting were conducted using the dot blot assay and optimisation of various factors, such as the use of positive control, different antibody dilutions, protein concentration, and optimized buffers. This showed that the purchased antibodies did not work. Conclusion: The findings demonstrated that MTP, HBHA and Rv0309 play a role in the regulation of other adhesin genes, as evidenced by the deletion of the three major genes that may have disrupted several metabolic and cell wall processes, potentially reducing the virulence of the M. tuberculosis strain. The findings of this work add to the growing evidence that the adhesins, MTP, HBHA, and Rv0309 as well as the related adhesins they interact with during macrophage infection, are promising targets for TB diagnostic or therapeutic interventions.
Characterization of the function of RV1268c, an ATP binding cassette transporter in Mycobacterium tuberculosis.
(2023) Hallom, Pumelela.; Senzani, Sibusiso.
Tuberculosis (TB) is an epidemic disease that is caused by a bacterium called Mycobacterium tuberculosis (Mtb). This disease infects and kills millions of people globally. Anti-Tuberculosis (anti-TB) drugs such as isoniazid, ethambutol, pyrazinamide, and fluoroquinolones have been discovered and produced for TB treatment but regardless, TB persists because of the resistance to these drugs, leading to the development of multidrug-resistant (MDR) Mtb and extensively-drug resistant (XDR) Mtb. One of the key areas to focus on for the development of new effective anti- TB drugs are efflux systems, because they transport molecules outside cells and have a role in the resistance against TB treatment. This study aimed to identify the biological function of the Mtb protein, Rv1268c. RV1268c was one of the identified proteins in a study that was done by Chiliza et al., 2019 where a few protein biomarkers that are recognized by both active TB and latent TB patient antibodies. Some of these biomarkers that were studied are TreY, Bfr, and TrpG, which are biomarkers that are specific to ATP and play an important role in pathogenesis. Other biomarkers included MoaE, PonA1, and NarG, which are specific to latent TB and play a role in dormancy. The Rv1268c protein of Mtb is classified as a hypothetical membrane protein of the cell envelope and its associated proteins are Rv1267c and RV1269c, which are regulatory protein and a conserved putatively exported protein, respectively. The Rv1268c protein is hypothesised to be an ATP-binding cassette (ABC) transporter. The nucleotide and protein sequences of Rv1268c were v downloaded from the database of Mtb H37Rv using Mycobrowser. To create a knock down strain, annealed oligos were ligated to PLJR965 plasmid and transformed into XL10-Gold ultracompetent cells and grown on kanamycin-containing plates. Extracted plasmids were electroporated into Mtb, and after 4 weeks of incubation, the colonies were screened to check if they carried the knock down plasmid . DNA was then extracted to characterize the function of the Rv1268c Mtb protein. The results showed that Rv1268c had no effect on the in vitro growth of Mtb, while the Ethidium bromide (EtBr) assay displayed a difference on the extrusion of EtBr as the knock down and the knock down with anhydrotetracycline (aTc) had lower fluorescence as compared to the wild type which implies that Rv1268c is not an ABC transporter. The statistical analysis showed that the was no significant difference on the drug susceptibility between the wild type, knock down, and the knock down with aTc strains . The growth of neither the wild type or knock down strains was completely inhibited by either of the drugs tested. Keywords:
External ventricular drain infections: a retrospective analysis at a central hospital in KwaZulu-Natal, South Africa.
(2022) De Meyer, Jenine Naomi.; Mahabeer, Yesholata.; Swe Swe-Han, Khine.
Objectives We describe clinical, laboratory and microbiological characteristics of patients with suspected EVD-related infections (EVDRIs), colonisation and contamination. Methods An observational analytical retrospective descriptive cohort study was conducted on all positive cerebrospinal fluid (CSF) cultures from external ventricular drains (EVDs) at a referral hospital from 2019 - 2021. Episodes were categorised as infection, colonisation or contamination based on pre-defined clinical and laboratory characteristics. Demographic data, clinical information and identification and susceptibility results of microbes isolated from CSF were analysed for each of these episodes. Results One hundred and sixty-three patients were included with 337 positive CSF cultures. Positive cultures were grouped into 213 episodes; 76 (36%) infection, 13 (6%) colonisation and 124 (58%) contamination. The median duration of EVD insertion to infection was 17 days. In the infection group 58 episodes (76%) had low CSF glucose, persistently low or decreasing CSF glucose. Sixty-seven patient episodes (88%) had high CSF protein or increasing protein. The CSF white cell count (WCC) was higher in the infection group versus colonisation and contamination groups with a wide range. The causative organisms of EVDRIs were predominantly Enterobacterales (33%), extensively drug-resistant (XDR) A. baumannii (27.6%) and coagulase-negative staphylococci (CoNS) (13%). The causative organisms of EVD-related colonisation and contamination were predominantly CoNS. Conclusion Laboratory parameters (CSF glucose, protein and WCC) were useful to distinguish EVDRIs from colonisation and contamination. However clinical manifestations require correlation with laboratory abnormalities to diagnose EVDRIs. Multidrug resistant (MDR) Enterobacterales and extensively drug-resistant (XDR) A. baumannii were the commonest cause of EVDRIs.
Characterizing the function of the Rv3218 gene in Mycobacterium tuberculosis.
(2023) Canham, Khumbuzile.; Senzani, Sibusiso.
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a primordial affliction that continues to torment humankind since its known history and prehistory. TB is among the major causes of ill-health and death in the world with an estimated 1.8 million cases of death recorded yearly. The situation is worsened by the emergence of the strains of TB that are regarded as resistant. Recently, Mycobacterium bovis bacillus Calmette-Guerin (BCG), has been the only available vaccine for TB. An intense understanding of Mtb’s biology, should reveal new perceptions that can lead to the improved treatment, diagnostics, vaccines and highly needed control measures. Throughout infection, Mtb produces some proteins into the host environment to play critical role in pathogen host interactions. Close to half of the Mtb genome consists of genes with unknown functions. Among those genes is Rv3218 gene which was identified in the study by Chiliza et al., 2019. The Rv3218 gene is hypothesised to have a Diacylglycerol kinase activity. This study aimed at characterising the function of Rv3218 gene in Mtb with the purpose of coming up with ideas of how that can be used in the development of more effective and convenient diagnostic tools, therapeutics, or the total elimination of TB. There is a vast amount of molecular techniques that are currently used to characterise unknown genes. Here we employed a CRISPRi dCas9 system for the silencing of the Rv3218 gene in Mtb. We also used a number of Bioinformatics tools for in silico analysis of the gene and construction of all relevant primers necessary for this molecular cloning. The Rv3218 knockdown repressed by Anhydrotetracycline (ATc) was constructed for assaying the effect of this gene silencing compared to the MtbH37Rv wild type. We then conducted growth curves and MICs (Minimum Inhibitory Concentrations) to check if this gene has an impact on antimicrobial susceptibility and growth of Mtb. We also tested its activity as a diacylglycerol kinase via osmolarity assay as it is said that dgk mutants do not grow well on nutrient media of low osmolarity. On bioinformatics analysis, we found that the gene has cell wall and transcription regulatory functions and possesses a similar structure as diacylglycerol kinase. However, the in vitro analysis was contradictory to these findings. We found that the Rv3218 gene has no impact on the growth of Mtb and it’s susceptibly to the antimicrobial drugs that were used in this study. On the osmolarity assay, there was no observable difference between the growth of the wild type and the knockdown strain in all the concentrations of osmolarity. Judging from these findings, we then concluded that this gene does not function as a diacylglycerol kinase. We then suggested that, more advanced experimental studies still need to be conducted in order to confirm this hypothesis as we were unable to do them due to the short time frame for this study.
Impact of Chlamydia trachomatis on inhibitory capabilities of broadly neutralizing antibodies in vitro.
(2021) Magini, Stanley Nzuzo.; Ngcapu, Sinaye.; Mzobe, Gugulethu Favourate.; Ndlovu, Bongiwe Goodness.
Abstract available in PDF.
Causes of meningitis in the era of HIV.
(2021) Ramjathan, Praksha.; Swe Swe-Han, Khine.
No abstract available.
Microbial profile and antimicrobial susceptibility patterns of neonatal blood stream infections in Durban, South Africa.
(2020) Pillay, Dharshni.; Mahabeer, Yesholata.
Objectives Antimicrobial resistance (AMR) has emerged as a global threat to healthcare resulting in an increase in morbidity and mortality. Neonatal sepsis is ranked as the third highest cause of neonatal demise globally, in which AMR accounted for 31.0% of deaths. This study analysed the aetiology and antimicrobial resistance patterns of bloodstream infections within the neonatal intensive care unit (NICU) at a tertiary hospital in Durban, South Africa. Methods A retrospective data review was conducted on all positive blood cultures at three time periods: 2014, 2016 and 2018. The organisms and antimicrobial susceptibilities were analysed for significant trends using Poisson and logistic regression. Results A preponderance of gram-positive organisms (68.7%) over gram-negatives (26.8%) and fungi (4.5%) was detected. Common pathogens included coagulase-negative staphylococci (53.5%), Klebsiella pneumoniae (11.6%), enterococci (9.3%), and Acinetobacter baumannii (7.7%). Late-onset sepsis (86.8%) predominated over early-onset sepsis (13.2%). High rates of resistance to first- and second-line antibiotics were noted among gram-positive and gramnegative organisms. Multidrug resistant organisms included extended-spectrum betalactamase (ESBL) K. pneumoniae (7.6%) and multi-drug resistant A. baumannii (7.0%). A statistically significant decrease in ESBL-producing organisms was documented between 2014 and 2018 (p = 0.005). Conclusion High resistance rates were seen for first- and second-line antibiotics used for the treatment of neonatal sepsis. Ongoing microbial surveillance is essential to tailor empiric antimicrobial choices in individual units.
Molecular epidemiology of antibiotic resistant salmonella spp. from farm to fork in an intensive pig production system in KwaZulu-Natal South Africa.
(2021) Tshakane, Nozipho Pamela.; Essack, Sabiha Yusuf.; Abia Akebe, Luther King.; Amoako, Daniel Gyamfi.
Antibiotic resistance (ABR) is a worldwide challenge, and, if not resolved, can be a danger to humans, animals and the ecosystem. The inappropriate use and misuse of antibiotics in food animal production creates selection pressure for the development of bacterial resistance. We investigated the molecular epidemiology, antibiotic resistance and virulence of Salmonella spp. from farm-to-fork in an intensive pig production system in KwaZulu-Natal. A herd of pigs was followed from birth to slaughter over a period of 4 months. Following ethical approval, a total of 408 samples were collected, which consisted of feces, litter, slurry, hand and nasal swabs from occupationally exposed workers, carcass swabs and rinsate, caecal samples and pork for retail purposes. Salmonella was putatively identified using selective media, i.e., Brilliance Salmonella Agar and Salmonella Shigella Agar (SS agar). Identification to species and sub-species level was confirmed by polymerase chain reaction (PCR), where the invA gene was used to confirm Salmonella spp. and the iroB gene for Salmonella enterica. Isolates were subjected to antibiotic susceptibility testing using the Kirby-Bauer disk diffusion method against a panel of 14 antibiotics. Isolates were screened for selected virulence genes, misL, spiC, orfL, sopB, pipD, hilA and stn, conferring intracellular survival (misL), type III secretion system (spiC), adhesion and autotransporter (orfL), type III secreted effector protein (sopB), type III secreted effector associated with SPI-1 system (pipD), host cell invasion (hilA), and enterotoxin production (stn) by PCR. Genetic relatedness of the isolates was determined by ERIC-PCR. A total of 399 putative Salmonella spp. were detected by selective media, of which 49% (n= 197) were confirmed by the presence of the invA gene and 45% (n=179) were identified as Salmonella enterica by the presence of the iroB gene. The largest number of Salmonella were isolated from retail meat samples. Antibiotic susceptibility testing showed 10% (n=19) resistance to cefoxitin, 8% (n=16) to amoxicillin and 0.5% (n=1) to gentamicin and chloramphenicol. The isolates carried the hilA (91%), stn (91%), misL (89%), pipD (88%), spiC (87%), orfL (85%) and sopB (72%) virulence genes. The isolates were clonally diverse with 26 ERIC-types and four major ERIC-type groups. The large number of isolates in retail meat samples, their virulence, and, to a lesser extent their antibiotic resistance profiles poses a challenge to the food safety system and requires a comprehensive understanding of molecular epidemiology of the organism so that it’s incidence spread can be reduced and better controlled from the primary source within the food chain.
Defining the role of high-dose isoniazid in the treatment of multi-drug resistance tuberculosis: isoniazid resistant profiling.
(2021) Ngema, Senamile Lale.; Dookie, Navisha.; Naidoo, Kogieleum.
Background: High-dose isoniazid is recommended in short-course regimens for multidrug-resistant tuberculosis (MDR-TB). However, there is no substantial evidence supporting its use in the presence of INH resistant mutations. Therefore, this study aimed to establish the efficacy of INH in the presence INH resistance associated mutations. Methods: We selected 94 clinical isolates obtained from 65 patients from the IndEX (CAP020) study specimen biorepository. Isolates were selected based on whole genome sequencing results showing evidence of INH resistant conferring mutations. Twenty-one isolates had inhA promoter gene and/ inhA coding region mutations, 35 had katG mutations, and 20 had both inhA promoter and/ inhA coding region plus katG mutations. Additionally, 18 INH susceptible clinical isolates were included in this analysis. Minimum inhibitory concentrations (MICs) were done in different concentration ranges depending on the mutation present. INH susceptible and H37Rv (0.016-0.256) μg/ml, inhA (0.256-4.0) μg/ml, katG (1.0-16.0) μg/ml and inhA plus katG (4.0-16) μg/ml. Results: Among 94 isolates, 36 were excluded: 11 MPT64 antigen negative, 23 non-growers and two were contaminated. Fifty-eight isolates from 55 patients were left for analysis. Eleven isolates had inhA mutations, 23 katG mutations, 12 had double mutations in inhA and katG, and 12 were INH susceptible. MICs obtained varied within isolates ranging from 0.016 to >64.0 μg/ml. InhA, katG, inhA plus katG mutations and INH susceptible isolates had median INH MIC of 8.0 (4.0-64.0), 4.0 (95% CI, 4.0-8.0), 64.0 (95% CI, 64.0-64.0), and 0.48 (95% CI, 0.32-1.0) μg/ml, respectively, confirming the association between INH MICs and genotypic profile. The MDR-TB and pre/XDR-TB had median INH MIC of 8.0 (95% CI, 8.0-32.0) and 48.0 (4.0-64.0) μg/ml, respectively. We found association between cavitary disease and increase in INH MICs for inhA mutants, median of 64.0 (64.0-64.0) μg/ml, and previous TB history and increased INH MICs (8.0[95% CI, 8.0-64]. Conclusion: This study demonstrated highly variable MIC range with significant overlap in MIC range among the mutant groups. Furthermore, inhA mutants demonstrated unexpectedly high MICs raising a concern for the ongoing use of the high-dose INH in our setting. Our findings suggest that the current one-size-fits all approach to MDR-TB short-course regimen requires urgent review.
The role of Mycobacterium tuberculosis curli pili (MTP) and heparin-binding hemagglutinin adhesin (HBHA) on global in vitro bacterial transcriptomics.
(2021) Naidoo, Tarien Jael.; Pillay, Manormoney.; Pillay, Balakrishna.
Background/Aim: Tuberculosis (TB), is an infectious, airborne disease caused by Mycobacterium tuberculosis (M. tuberculosis). TB remains one of the most devastating bacterial causes of human mortality, especially in low-income countries. Surface located adhesins are crucial for M. tuberculosis survival, as they initiate and perpetuate host-pathogen interactions. The adhesin, M. tuberculosis curli pili (MTP), plays a role in adhesion and invasion of host cells and biofilm formation, whilst heparin-binding hemagglutinin adhesin (HBHA) promotes M. tuberculosis dissemination from the site of infection. The use of transcriptomics promises to enhance current knowledge on MTP and HBHA as virulence factors, thereby substantiating their role as biomarkers for the development of accurate TB diagnostics and therapeutics. Therefore, this study aimed to elucidate the role of MTP and HBHA in the regulation of M. tuberculosis transcriptome, and to identify novel biomarkers. This was achieved by analysing the transcriptomic perturbations in the strains lacking the MTP adhesin, HBHA adhesin or both MTP-HBHA adhesins and the strains containing the aforementioned adhesins. Methods: Polymerase chain reaction (PCR) confirmed strains of M. tuberculosis wild-type (WT), mtp-deletion mutant (Δmtp), hbhA-deletion mutant (ΔhbhA), mtp-hbhA-deletion mutant (Δmtp-hbhA), and the respective complemented strains, were standardized and cultured until log phase. Thereafter, the bacterial cultures were prepared for RNA extraction. RNA was extracted via an optimized TRIzol method and sequenced using the Illumina 2×150 HiSeq ×10 platform. The sequenced reads were analysed by FastQC toolkit (version 0.11.8), pre-processed using Trimmomatic (version 0.36), mapped to the custom-built M. tuberculosis H37Rv genome index using hierarchical indexing for spliced alignment of transcripts (HISAT version 2.1.0), assembled by Stringtie (version 1.2.1), and further annotated and assembled the transcripts into known and novel categories by Gffcompare, located within Stringtie. The output files were annotated in R (version 1.2.1578) using the Ballgown package to generate the respective fold changes (FC) between the deletion mutants and the WT, and q-values and p-values for the differential expression. The generated results were filtered using a FC cut-off value ≥ 1.3 (to indicate a 1.3-fold up-regulation) and ≤ 0.5 (to indicate a 2-fold down-regulation) to identify significant genes and pathways. Thereafter, relevant databases and literature were reviewed to categorize the genes into pathways. Real time quantitative PCR (RT-qPCR) was performed on 10 selected genes, as a genotypic method to functionally confirm the RNA sequencing data. A bacterial bioluminescence cell viability assay was performed to elucidate the concentration of adenosine triphosphate (ATP) in the deletion mutants and complements, relative to the WT. Results: A total of 43 genes were significantly differentially expressed amongst the deletion mutants. These genes were functionally categorized into: intermediary metabolism and respiration metabolism, cell wall biosynthesis, cell wall transport and processes, lipid metabolism, and virulence; stable RNA’s; conserved hypotheticals; proline-glutamate (PE) or proline-proline-glutamate (PPE); insertion sequences and phages; and information pathways. The bioluminescence assay functionally confirmed the increased utilization of ATP in the absence of MTP and HBHA. Discussion/Conclusion: Adhesin gene deletions caused major perturbations to the central carbon metabolism, cell wall biosynthesis, cell transport process, lipid biosynthesis, and virulence pathways, leading to potentially increased energy requirements; compensatory transport of proteins to the cell wall, altered cell wall biosynthesis and decreased virulence and pathogenicity. Additionally, deletion of these adhesins resulted in the disruption of many processes potentially attenuating growth and replication. Thus, this study further corroborates the adhesins, MTP and HBHA, and associated pathway genes as potential suitable targets for TB diagnostic/therapeutic interventions.
Fosfomycin: an oral treatment option for multi drug resistant uropathogens.
(2021) Naidoo, Alicia.; Swe Swe-Han, Khine.; Mvelase, Nomonde Ritta.
Introduction Urinary tract infections (UTIs) are commonplace in both the community and hospital environment where it is accepted clinical practice to treat empirically. Consequently, this has led to an alarming increase in antimicrobial resistance in frequently prescribed oral treatment options. In light of the global shortage of newly discovered antibiotics, there is a need to relook at older antimicrobial agents, such as fosfomycin, to fill the gap in therapeutic guidelines. Purpose of the Study It was the purpose of this study to ascertain the extent of antimicrobial resistance in commonly isolated urinary pathogens to frequently prescribed antibiotics. This was done with the intention of bringing to light the severity of the situation. We also looked at fosfomycin as a possible alternative to the currently recommended treatment options most especially in multi drug resistant (MDR) infections. Method A retrospective analysis of antimicrobial susceptibility data of positive urine specimens collected during 2018 – 2020 was performed. Additionally, fosfomycin susceptibility testing was performed in 178 stored MDR uropathogenic isolates using the gold standard agar dilution method. We also compared agar dilution (reference method) to disk diffusion and E test as they are less labour intensive. All data and isolates were obtained from RK Khan Laboratory located in KwaZulu Natal, South Africa. The Clinical and Laboratory Standards Institute guidelines was utilised as the guiding document. Results While conducting the laboratory information system (LIS) based review, it was determined that within the study time frame, 3044 common urinary pathogens were isolated, with Escherichia coli being the most frequent cause of UTI (1603: 53%), followed by Klebsiella spp (437: 14%). Both organisms showed high rates of resistance to amoxicillin clavulanic acid (AMC) (29.8% and 42.3%) and ciprofloxacin (37.7% and 30.4%) which are popular treatment options. Our study on fosfomycin susceptibility in MDR uropathogenic isolates revealed that of the 178 isolates, E. coli was the most prevalent isolate (97: 55%), followed by Klebsiella spp (55: 30.9%). E. coli had a susceptibility rate of 93.8% to fosfomycin, while Klebsiella spp had susceptibility rate of 78%. Categorical agreement was achieved between agar dilution and disk diffusion at 91%, although there was a high rate of false susceptibility at 42.9%. Categorical agreement between agar dilution and E tests at only 89% did not meet CLSI guideline. Conclusion While E. coli remains the most commonly isolated uropathogen, resistance rates for both E. coli and Klebsiella spp to frequently prescribed oral treatment options are alarmingly high, leaving clinicians very little in the way of viable treatment options. There is a need to keep abreast of current antimicrobial resistance trends as well as look at alternative treatment options. To that end, the use of fosfomycin as an alternative oral treatment option in a UTI is a viable solution, most especially when the infection is caused by a MDR E. coli. Lastly, though laborious, agar dilution remains the most reliable method in establishing antimicrobial susceptibility in fosfomycin.
Molecular surveillance of Staphylococcus aureus on frequently touched sites in public hospitals in KwaZulu-Natal, South Africa.
(2021) Mkhize, Siyethaba.; Bester, Linda Antoinette.; Zishiri, Oliver Tendayi.
There is an escalation in the prevalence of hospital-acquired infections (HAI) due to the transfer of pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), within the hospital environment. Pathogens contaminate inanimate objects and are transmitted in the hospital environment through direct hand contact, of which healthcare workers and patients act as vectors. This study aimed to conduct surveillance of methicillin-resistant S. aureus (MRSA) on frequently touched hospital environment sites of selected public hospitals from different healthcare levels in KwaZulu-Natal, South Africa. Eleven predetermined frequently touched sites in the general, paediatric and ICU wards were swabbed viz. occupied and unoccupied beds, ward telephones, drip stands, nurses’ tables, door handles of laundry rooms, mops, sinks, ventilators, blood pressure machines and patient files. The swabs were plated on selective chromogenic media for Staphylococcus aureus (S. aureus) isolation and phenotypic identification. Total genomic DNA was extracted using the conventional boiling method. The presence of the S. aureus thermo-nuclease nuc gene was confirmed using the polymerase chain reaction (PCR). Antibiotic susceptibility tests were conducted by performing the Kirby-Bauer disk diffusion assays, according to CLSI guidelines, to determine the isolates' resistance profiles to nine antibiotics. The mecA gene, an MRSA indicator, and genes encoding resistance and virulence were identified through PCR. Genomic DNA was extracted using the Quick-DNATM Miniprep Plus kit, and ERIC-PCR was conducted to determine the clonality of the isolates. Pearson’s correlation, Fisher’s exact test and Chi-Square test were implemented using the SPSS software version 25 (IBM SPSS Statistics) for statistical analyses. The statistical significance determined by a probability value that was less than 0.05 (p < 0.05). An overall prevalence of 12.7 % (99/777) for S. aureus isolates was obtained. Of these, 89.9 % (89/99) were MRSA, and only 10.1 % (10/99) of the total collected isolates were identified as methicillin-susceptible S. aureus (MSSA). The sites with the highest prevalence were the occupied beds (16.2 % (16/99)), unoccupied beds (16.2 % (16/99)), patient files (14.1 % (14/99)), ward telephones (13.1 % (13/99)) and nurses’ tables (14.1 % (14/99)). The sites with the lowest prevalence were the blood pressure machines (6.1 % (6/99)), drip stands (6.1 % (6/99)), ventilator (6.1 % (6/99)), door handle (4 % (4/99)), mop (3.0 % (3/99)) and sink (1.0 % (1/99)). The Pearson’s Chi-square and Fischer’s exact test indicated a significant relationship (p < 0.05) between the mecA gene and the collection site. A significant relationship (p < 0.05) was identified between the hospital and the tetK, ermC, aac (6')-aph (2") and LukS/F-PV genes. ERIC-PCR produced bands for 87.8 % (87/99) of the isolates; 12.1 % (12/99) were non-typeable. Our findings have highlighted the S. aureus contamination on frequently touched hospital sites, virulence and resistance, and the clonal diversity of S. aureus isolates in the hospital environment of four KwaZulu Natal public hospitals in the eThekwini district. Our findings may be used as a baseline for future surveillance initiatives to improve hospital hygiene through IPC strategies centred around S. aureus in KwaZulu-Natal public hospitals.
Distribution, virulence and antimicrobial resistance profile of Bacillus species from the environment of four public hospitals in South Africa.
(2021) Mbhele, Zamile Nompumelelo.; Bester, Linda Antoinette.; Zishiri, Oliver Tendayi.
Hospital-acquired infections (HAIs) are counted as the most crucial global health crisis. The hospital environment may be colonized by opportunistic pathogens, which can lead to HAIs in hospitalized patients. As a result, microbial monitoring of the hospital environment is important in managing these pathogenic organisms. This research aimed to investigate the prevalence, antimicrobial resistance, virulence genes, and genetic diversity of Bacillus spp. on hospital surfaces in public hospitals in KwaZulu-Natal (KZN), South Africa (SA). A total of 777 swabs were collected from four different public hospitals classified as A (Central), B (Tertiary), C (Regional), and D (District), within three different wards (paediatric, general ward, and intensive care unit (ICU) and 11 touchable surfaces belonging to medical devices and well used equipment. Samples were assessed for the existence of Bacillus spp in these four public hospitals. Bacillus was isolated using the microbial plating method on a selective Bacillus medium, and the biochemical characteristics confirmed, including oxidase, catalase, motility, and triple sugar iron (TSI). Molecular confirmation was also performed using polymerase chain reaction (PCR) targeting the MotB gene for Bacillus cereus and 16s rRNA gene for Bacillus subtilis. The minimum inhibitory concentration (MIC) technique evaluated the antimicrobial resistance profile using the Clinical Laboratory Standards Institute (CLSI) guidelines. Molecular characterization was conducted for seven antimicrobial resistance genes and 11 virulence factors using PCR. Genetic relatedness between the isolates across the four hospitals was evaluated by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). A total of 777 samples were collected in the hospital environments, of which 135 were positive for Bacillus spp. Species identification revealed 4 % (6) as Bacillus subtilis and 96 % (129) as B. cereus. The overall prevalence of Bacillus spp. per hospital was 24 % (32/135) for Central (hospital A), 33 % (45/135) for Tertiary (hospital B), 27 % (36/135) for Regional (hospital C) and 16 % (22/135) for District hospital (hospital D). A statistically significant difference in Bacillus prevalence (p = 0.044) was found between tertiary and regional hospitals (p = 0.000).The prevalence among the wards was averaging 33 % (45/135), noting ICU with the highest prevalence of 35 % (47/135). In terms of the wards, no statistically significant differences were found (p = 0.133). The hospital and the wards had a strong correlation (r = 0.525, p = 0.000).The highest prevalence on frequently touched sites was a bed with 15 % (20/135), drip stands and sinks with 12 % (16/135) respectively, ward phones with 11 % (15/135), and nurses’ tables with 10 % (14/135). Complete resistance was observed against ampicillin (100 %; 135/135), and high resistance against ciprofloxacin (99 %; 134/135), amoxicillin (97 %; 131/135), tetracycline (82 %; 112/135), cefotaxime (51 %; 69/135), and erythromycin (43 %; 59/135). Lower resistance was observed against meropenem (20 %; 27/135) and imipenem (25 %; 26/135). A total of 43 different antibiograms were detected, with 97 % (132/135) of the isolates found to be multidrug resistance (MDR). No statistically significant difference was observed between the antibiotics tested (p ≥ 0.05). The observed resistance genes were ermB (56 %; 33/59) (conferring erythromycin resistance), tetracycline resistance-conferring genes were tetM (5 %; 5/112) and tetK (4 %; 4/112). No tetA, tetB, tet39, and the blm gene (beta-lactamase resistance-conferring gene) were detected. Different toxins produced by Bacillus are associated with the pathogenicity of Bacillus species. Prevalence of the virulence enterotoxin genes associated with diarrhea prevailed in 88 % (99/135) of hblD, 77 % (104/135) in hblA and CytK respectively, nheC 67 % (90/135), nheB 65 % (88/135), nheA 64 % (89/135), hblC 55 % (74/135), bceT 44 % (59/135), hlyII 37 % (50/135), and finally EntFM 27 % (37/135) of the isolates housed the gene. No cereulide (ces gene) causing emetic syndrome was detected. Typing using ERIC-PCR noted type clusters composed of isolates from different wards, environmental sites, and equipment and showing high genetic diversity, indicating no common infection sources. This study revealed a moderate prevalence of Bacillus spp. collected from the environment of the four public hospitals. High resistance was observed for some antibiotics that are usually effective against Bacillus; this may serve as a potential risk for effective treatment of Bacillus infections. Most isolates harboured virulence factors that cause diarrheal syndrome rather than those causing the emetic syndrome. These findings highlight the need for microbial surveillance of hospital environments to inform and improve current intervention programs for cleaning methods in public hospitals to reduce environmental contamination and transmission of pathogenic Bacillus spp. infections associated with HAI’s in hospitalised patients.

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