Biolog identification of non-sorbitol fermenting bacteria isolated on E. coli O157 selective CT-SMAC agar

E. coli O157:H7 is recognised as an important human pathogen world-wide and has been associated with diseases such as haemorrhagic colitis (HC), haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopaenic purpura (TTP). Accurate laboratory detection of E. coli O157:H7 is important for diagnostic purposes and to justify epidemiological data on E. coli O157:H7. A well-known phenotypic characteristic of E. coli O157:H7 bacteria is their inability to ferment sorbitol. This characteristic is often used to isolate these organisms from food and water using selective agar medium such as SMAC. However, the high number of false positive results obtained by a number of researchers when selectively screening for E. coli O157: H7 on CT-SMAC has prompted an investigation to determine which other sorbitol-negative bacteria also grow on CT-SMAC. The agar medium used for the investigation consisted of Sorbitol MacConkey agar (SMAC) supplemented with Cefiximetellurite (CT). All sorbitol-negative colonies obtained from CT-SMAC, after selective enrichment and IMS were identified using the Biolog microbial identification system. The majority of sorbitol-negative isolates identified were Burkholderia, Pseudomonas, Vibrio and Aeromonas spp. Only two E. coli O157:H7 isolates were identified with Biolog and confirmed with a polymerase chain reaction (PCR) specific for the shiga toxin 1 (Stx1) genes and with O157 and H7 antisera. The inability of the CT-SMAC agar medium to specifically select for E.coli O157:H7 was confirmed by the results of this study. These observations call for further improvement of affordable methods for the selective isolation of E. coli O157:H7 in the presence of large numbers of interfering bacteria capable of growing on CT-SMAC.This work was supported with a grant awarded by the National Research Foundation of South Africa

Diversity and antimicrobial susceptibility profiling of staphylococci isolated from bovine mastitis cases and close human contacts

The objectives of this study were to examine the diversity of Staphylococcus spp. recovered from bovine intramammary infections and humans working in close contact with the animals and to evaluate the susceptibility of the staphylococcal isolates to different antimicrobials. A total of 3,387 milk samples and 79 human nasal swabs were collected from 13 sampling sites in the KwaZulu-Natal province of South Africa. In total, 146 Staph. aureus isolates and 102 coagulase-negative staphylococci (CNS) were recovered from clinical and subclinical milk samples. Staphylococcus aureus was isolated from 12 (15.2%) of the human nasal swabs and 95 representative CNS were recovered for further characterization. The CNS were identified using multiplex- PCR assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and tuf gene sequencing. Seven Staphylococcus spp. were identified among the CNS of bovine origin, with Staph. chromogenes (78.4%) predominating. The predominant CNS species recovered from the human nasal swabs was Staph. epidermidis (80%) followed by Staph. chromogenes (6.3%). The antimicrobial susceptibility of all staphylococcal isolates was evaluated using disk diffusion and was supplemented by screening for specific antimicrobial resistance genes. Ninety-eight (67.1%) Staph. aureus isolates of bovine origin were pansusceptible; 39 (26.7%) isolates were resistant to a single class, and 7 (4.8%) isolates were resistant to 2 classes of antimicrobials. Two Staph. aureus (1.4%) isolates were multidrug-resistant. Resistance to penicillin was common, with 28.8% of the bovine and 75% of the human Staph. aureus isolates exhibiting resistance. A similar observation was made with the CNS, where 37.3% of the bovine and 89.5% of the human isolates were resistant to penicillin. Multidrug-resistance was common among the human CNS, with 39% of the isolates exhibiting resistance to 3 or more classes of antimicrobials. The antimicrobial susceptibility results suggest that resistance among staphylococci causing bovine intramammary infections in South Africa is uncommon and not a significant cause for concern. In contrast, antimicrobial resistance was frequently observed in staphylococcal isolates of human origin, highlighting a possible reservoir of resistance genes. Continued monitoring of staphylococcal isolates is warranted to monitor changes in the susceptibility of isolates to different classes of antimicrobials.University of Pretoria, RESCOM, the National Research Foundation (NRF) of South Africa, and the KZN Department of Agriculture & Rural Development (South Africa).http://www.journals.elsevier.com/journal-of-dairy-science2016-09-30hb201

Identification and characterization of Staphylococcus devriesei isolates from bovine intramammary infections in KwaZulu-Natal, South Africa

BACKGROUND : Coagulase-negative staphylococci (CoNS) are among the leading bacterial causes of bovine mastitis in many dairy-producing countries. Among the challenges associated with the specific diagnosis of CoNS infections is the biochemical heterogeneity of the species in the genus and the unavailability of accurate, cost-effective and up-to-date diagnostic tests. A previous study investigating the diversity of CoNS associated with cases of bovine mastitis in South Africa, resulted in six CoNS isolates which could not be identified despite the use of a combination of different molecular assays. The identification and characterisation of the isolates was pursued further in this study. RESULTS : The six CoNS isolates in question were identified by sequencing multiple housekeeping genes (dnaJ, hsp60, rpoB, 16S rRNA) and characterized through the use of matrix-assisted laser/desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and the Biolog GEN III Microplate™ bacterial identification system. Sequencing of housekeeping genes identified the isolates as S. devriesei. This Staphylococcus species was only described in 2010 and this is the first report documenting the isolation of S. devriesei from cases of bovine IMIs in South Africa. Analysis of mass spectra generated by the six isolates showed intra-species variation which was also observed when evaluating the metabolic profiles of the isolates using the Biolog GEN III system. Neither the MALDITOF MS nor the Biolog database are currently populated with data relating to S. devriesei, resulting in the isolates not being identified, in the case of MALDI-TOF MS analysis, or mis-identified as was observed with the Biolog GEN III system. CONCLUSIONS : The phenotyping data collected during this investigation provides useful information concerning Staphylococcus devriesei which could be used to populate user system databases thereby ensuring the accurate identification of isolates in future. The availability of improved diagnostics will in turn facilitate studies to elucidate the epidemiology, pathogenicity and true prevalence of this species in dairy herds.The research presented herein is funded, in part, by the University of Pretoria, National Health Laboratory Services, RESCOM, the National Research Foundation (NRF) Research Technology Fund and the KZN Department of Agriculture and Rural Development. The MALDI-TOF MS work is supported in part by the National Research Foundation (NRF) of South Africa (Grant specific unique reference number, UID 74426).https://bmcvetres.biomedcentral.comMedical Microbiolog

Disruption in the Regulation of Immune Responses in the Placental Subtype of Preeclampsia

Preeclampsia is a pregnancy-specific disorder, of which one of its major subtypes, the placental subtype is considered a response to an ischemic placental environment, impacting fetal growth and pregnancy outcome. Inflammatory immune responses have been linked to metabolic and inflammatory disorders as well as reproductive failures. In healthy pregnancy, immune regulatory mechanisms prevent excessive systemic inflammation. However, in preeclampsia, the regulation of immune responses is disrupted as a result of aberrant activation of innate immune cells and imbalanced differentiation of T-helper cell subsets creating a cytotoxic environment in utero. Recognition events that facilitate immune interaction between maternal decidual T cells, NK cells, and cytotrophoblasts are considered an indirect cause of the incomplete remodeling of spiral arteries in preeclampsia. The mechanisms involved include the activation of immune cells and the subsequent secretion of cytokines and placental growth factors affecting trophoblast invasion, angiogenesis, and eventually placentation. In this review, we focus on the role of excessive systemic inflammation as the result of a dysregulated immune system in the development of preeclampsia. These include insufficient control of inflammation, failure of tolerance toward paternal antigens at the fetal–maternal interface, and subsequent over- or insufficient activation of immune mediators. It is also possible that external stimuli, such as bacterial endotoxin, may contribute to the excessive systemic inflammation in preeclampsia by stimulating the release of pro-inflammatory cytokines. In conclusion, a disrupted immune system might be a predisposing factor or result of placental oxidative stress or excessive inflammation in preeclampsia. Preeclampsia can thus be considered a hyperinflammatory state associated with defective regulation of the immune system proposed as a key element in the pathological events of the placental subtype of this disorder

Prevalence of carbapenem resistance genes in Acinetobacter baumannii isolated from clinical specimens obtained from an academic hospital in South Africa

Acinetobacter baumannii is an important cause of hospital-acquired infections. The occurrence of carbapenem resistance that is caused by the carbapenem-hydrolysing class D β-lactamases and the metallo-β-lactamases (MBLs) limits the range of therapeutic alternatives in treating A. baumannii infections. In this study, two multiplex polymerase chain reactions were performed to screen for both carbapenem-hydrolysing class D β-lactamases and MBL genes in 97 clinical isolates of A. baumannii. Oxacillinase (OXA)-51 had a prevalence of 83% (81/97), and OXA-23 had a prevalence of 59% (57/97). One isolate was positive for an MBL [Verona integron-encoded metallo β-lactamases (VIM)]. Therefore, continuous surveillance and monitoring of A. baumannii is crucial because of the high prevalence of antibiotic resistance genes.http://www.sajei.co.za/index.php/SAJEIam2013ay201

Prevalence of Clostridium difficile toxin genes in Pretoria

The hypervirulent polymerase chain reaction (PCR) ribotype 027 strain of Clostridium difficile produces toxins A, B and a binary toxin. Toxin detection kits are commonly used in diagnostic laboratories, but have been unsuccessful in detecting all of the relevant C. difficile strains, and the toxins produced. In this study, conventional PCR was used to detect the presence of the genes of toxin A, toxin B and the binary toxin of C. difficile. Eighty-four frozen (collected between 2006-2007) and 13 fresh (collected in 2010) stool specimens, obtained in Pretoria, were analysed. The genes for toxin A, toxin B and the binary toxin were detected in one of the fresh stool specimens. This may have implications for healthcare facilities, and suggests the possible emergence of the highly virulent PCR ribotype 027 strain of C. difficile in Pretoria. This emphasises the importance of continuous surveillance and monitoring of C. difficile outbreaks.http://www.sajei.co.za/index.php/SAJEIay201

Characteristics and outcomes of patients admitted to a tertiary academic hospital in Pretoria with HIV and severe pneumonia : a retrospective cohort study

BACKGROUND : Human immunodeficiency virus (HIV) contributes significantly to morbidity and mortality in South Africa. Pneumonia and opportunistic infections remain a major cause for hospital admission among those living with HIV, even in the era of the widespread availability of antiretroviral therapy. METHODS : In this retrospective cohort study, the records of patients admitted with HIV and severe pneumonia, requiring high care/intensive care admission, during a period of 12 months (February 2018 to January 2019) were reviewed. Demographic details, antiretroviral use, HIV viral load, CD4 count, sputum culture results and radiological imaging of patients were recorded. Data was analysed to determine variables associated with mortality. RESULTS : One hundred and seventeen patient records were reviewed for this study. The patients were young (mean age 38.3 years), had advanced disease with low CD4 counts (mean 120.2 cells/mm3) and high HIV viral loads (mean 594,973.7 copies/mL). Only 36.9% (42/117) were on highly active antiretroviral therapy (HAART) on presentation to the hospital. Mycobacterium tuberculosis (M. tuberculosis) was found to be the cause for pneumonia in 35% (41/117), whilst Pneumocystis jirovecii (P. jirovecii) was found in 21.4% (25/117). Bacterial pneumonia was the cause in 17.1% (20/117) of patients while no specific aetiology was found in 26.6% (31/117) of patients in the cohort. Mortality among the cohort studied was high (40.1%) and the average length of stay in hospital in excess of two weeks. The need for ICU admission, ventilation and CMV viremia was associated with increased mortality. Chest X-ray findings did not correlate with the aetiology of pneumonia, but multiple B-lines on lung ultrasound correlated with P. jirovecii as an aetiology and there was a signal that pleural effusion with fibrin stranding predicts tuberculosis. CONCLUSIONS : Patients studied presented with advanced HIV and were often naïve to antiretroviral therapy. Mortality in this cohort of young patients was high, which emphasis the need for earlier diagnosis and treatment of HIV at a primary care level. Lung ultrasound may have clinical utility in the management of patients with HIV and pneumonia, particularly to diagnose P. jirovecii as an aetiology.http://www.biomedcentral.com/bmcinfectdisam2023Internal MedicineMedical Microbiolog

High prevalence of oxacillinases in clinical multidrug-resistant Acinetobacterbaumannii isolates from the Tshwane region, South Africa - an update

BACKGROUND : Acinetobacter baumannii is an important hospital-acquired pathogen in healthcare facilities that frequently causes bacteraemia and ventilator-associated pneumonia in intensive care units. Acinetobacter baumannii can be isolated from various sites in the hospital environment like medical equipment, bed linen, medical personnel and indwelling catheters. It is difficult to treat A. baumannii infections because of their highly resistant antimicrobial profiles. The purpose of this study was to determine the prevalence of β-lactamase genes in multidrug-resistant (MDR) clinical A. baumannii isolates using Multiplex-PCR (M-PCR) assays. METHODS : One hundred MDR A. baumannii isolates were collected from the diagnostic division of the Department of Medical Microbiology after routine analysis of the submitted specimens. All collected isolates were identified and tested for susceptibility using the VITEK 2® system (bioMérieux, France). Six isolates were excluded from this study because the isolates were incorrectly identified as A. baumannii with the VITEK 2® system (bioMérieux, France). Molecular tests, namely M-PCR assays, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed. MLST analyses were performed on representative isolates from the four major pulsotypes (≥5 isolates with 80 % similarity) and selective isolates from each minor pulsotype. RESULTS : All the A. baumannii isolates showed 100 % resistance to ampicillin, amoxicillin, cefuroxime, cefuroximine axetil, cefoxitin, cefotaxime and nitrofurantoin. Seven percent of the isolates were resistant to amikacin. Two percent of the isolates were classified as having intermediate susceptibility to tigecycline. A. baumannii isolates showed an antibiotic resistance profile of 67 % and higher to antibiotics, such as ceftazidime, cefepime, imipenem, meropenem, gentamicin, ciprofloxacin and trimethoprim/sulfamethoxazole. None of the isolates were resistant to colistin. The M-PCR assays showed that 99 % of the isolates contained the OXA-51 gene and 77 % contained the OXA-23 gene. None of the isolates contained the GES, GIM, IMP, KPC, NDM, OXA-24, OXA-58, PER, SIM, SPM, VEB and VIM genes. Representative A. baumannii isolates were grouped into five existing sequence types (ST): ST106, ST258, ST339, ST502, ST758 and ST848. Isolates belonging to the pan-European clonal lineages I and II (EUI and EUII) were identified. CONCLUSION : The high prevalence of MDR A. baumannii isolates has a severe impact on available treatment choices and this in return impacts on treatment outcomes in the studied healthcare facilities. The most dominant ST among the collected isolates was ST758, member of the EUI group. The presence of the OXA-23 gene was not restricted to a specific ST. Continuous research and surveillance is necessary to monitor the circulating β-lactamase genes in clinical settings to guide infection control policies in order to try and curb the spread of this bacterium.ML was supported by a National Research Foundation (NRF) grant. The MALDI-TOF analysis is based on research supported in part by the National Research Foundation (NRF) of South Africa (Grant specific unique reference number (UID) 74426).http://www.biomedcentral.com/bmcinfectdis/am201

Normal flora and bacterial vaginosis in pregnancy : an overview

The female genital tract is an intricate, yet balanced ecosystem that hosts a variety of different residential microflora. The physiological changes that occur during pregnancy may disrupt this balanced ecosystem and predispose women to a potentially pathogenic microbiota. Bacteria that are associated with bacterial vaginosis (BV) are opportunistic pathogens that frequently form part of this microbiota. The overgrowth of and infections with these bacteria are linked to poor obstetric outcomes and increased transmission of other reproductive tract infections (RTIs). These infections increase women’s susceptibility of acquiring HIV, the rates of HIV shedding and the development of Acquired Immune Deficiency Syndrome (AIDS) in HIV infected patients. It is unknown how the plethora of bacterial species associated with BV contributes to the dynamics of this condition. The use of high-throughput methods have led to the in-depth investigation of different BV-related bacterial species and the functional capabilities of these species. However, the pathogenesis of BV is still poorly defined and the role of individual BV-related bacterial species in specific pregnancy complications is unclear and controversial. The majority of BV infections are asymptomatic and successful diagnosis is complicated by the lack of reliable and standardized diagnostic tests.University of Pretoria, the Medical Research Council (South Africa) and the National Health Laboratory Service (NHLS).http://www.tandfonline.com/loi/imby202017-05-31hb2016Medical Microbiolog

Assessment of Atopobium vaginae and Gardnerella vaginalis concentrations in a cohort of pregnant South African women

OBJECTIVES : The purpose of this cross-sectional study was to assess Atopobium vaginae and Gardnerella vaginalis concentrations in pregnant women of different age groups, gestational age groups, vaginal flora categories and HIV status, and also to determine which DNA concentrations best discriminated between bacterial vaginosis (BV)-positive and non-BV categories. METHODS : Self-collected vaginal swabs were obtained from 220 pregnant women attending an antenatal clinic in Pretoria, Gauteng, South Africa, from July 2012 to December 2012. BV was detected with the Nugent scoring system, and A. vaginae and G. vaginalis DNA was quantified with a multiplex quantitative real-time PCR assay. RESULTS : Median concentrations of A. vaginae and G. vaginalis were not significantly different among various age groups (A. vaginae p=0.98 and G. vaginalis p=0.18) or different trimesters (A. vaginae p=0.31 and G. vaginalis p=0.19), but differed significantly among the vaginal flora categories (A. vaginae p<0.001 and G. vaginalis p<0.001) and HIV status (A. vaginae p<0.001 and G. vaginalis p=0.004). The presence of A. vaginae (OR=5.8; 95% CI 1.34 to 25.21 and p value=0.02) but not that of G. vaginalis (OR=1.90; 95% CI 0.81 to 4.43 and p value=0.14) was associated with HIV infection. An A. vaginae DNA concentration of ≥107 copies/mL together with a positive G. vaginalis result (≥100 copies/mL) best discriminated between BV-positive (39/220) and non-BV categories (181/220) with a sensitivity of 85% (95% CI 0.70 to 0.94) and a specificity of 82% (95% CI 0.76 to 0.88). CONCLUSION : A. vaginae and G. vaginalis were present in high numbers and concentrations in this pregnant cohort. Threshold concentrations should be established for specific populations to ensure sensitive molecular assays for BV detection.The University of Pretoria and the Medical Research Council (South Africa).http://sti.bmj.com2018-09-30hj2017Medical Microbiolog