Investigation into the diversity of antifungal aerobic endospore-forming bacteria associated with bulk and crop rhizosphere soil.

Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.Members of the genus Bacillus are mainly Gram positive, aerobic rod shaped, endospore-forming bacteria that are increasingly being recognised for their ability to promote plant growth and antagonise fungal pathogens. From a biological control perspective, Bacillus spp. strains that produce antifungal compounds are of particular interest. In this study, aerobic endospore-formers were isolated from an undisturbed indigenous grassland soil and screened for antifungal activity and other plant growth promoting traits. Endospore-formers were also isolated from rhizosphere soil associated with the roots of maize, wheat and kale grown in pots containing soil from the same grassland site. Microbial diversity amongst isolates showing antifungal activity was investigated using different molecular fingerprinting methods, namely, intergenic transcribed spacer–PCR (ITS-PCR), random amplified polymorphic DNA-PCR (RAPD-PCR) and 16S rRNA gene amplification and sequencing. Characterization of the active antimicrobial compound(s) associated with selected isolates was also attempted. Prior to isolating from bulk and rhizosphere soils, samples were pre-heated to eliminate heat sensitive vegetative cells. Mean endospore counts were; wheat rhizosphere, Log 6.03 c.f.u g-1 soil; maize rhizosphere, Log 5.88 c.f.u g-1 soil; kale rhizosphere Log 5.90 c.f.u g-1 soil; and bulk soil Log 5.67 c.f.u g-1soil. A total of three hundred and eighty-four isolates were screened for antagonism towards Rhizoctonia solani using dual-culture plate bioassays. Thirty four of the isolates (~9%) mostly isolated from the bulk soil inhibited R. solani at varying degrees. Differences in antimicrobial interactions were apparent in in vitro bioassay; supposedly due to different concentrations and/or types of antimicrobial compounds. Biochemical tests for amylase, cellulase, chitinase, and proteinase activity, siderophore production and inorganic phosphate solubilisation were conducted. None of the isolates possessed all of these attributes and only a few showed multiple traits. Ninety-one percent of the isolates exhibited proteinase activity, 76% were able to hydrolyze starch whereas only four displayed cellulase activity. Only four isolates from the bulk-soil were capable of solubilising inorganic phosphate. ITS-PCR and 16S rRNA gene sequence analysis showed high levels of genetic homology amongst isolates and the majority were closely associated with representatives of the B. cereus group. Isolate C76 was the exception, being closely matched with B. subtilis. ITS-PCR banding profile was useful for distinguishing between species but did not distinguish within species. RAPD-PCR distinguished finer levels of genetic diversity between and within sample sets, with primer OPG-11 showing the greatest levels of heterogeneity. DNA extraction methods and the influence of template DNA dilution were investigated to determine their influence on RAPD-PCR analysis reproducibility. Prominent bands were comparable for crude template- and kit-extracted DNA but slight changes in band intensity and in some instances, additional faint bands were observed. At the highest DNA concentrations tested (7 μg/ml), further bands with molecular weights above 2.5 kbp were apparent. Strict standardization of PCR conditions greatly reduced variability of the RAPD-PCR analysis. Isolates from the different sample sets were screened for the presence of genetic markers associated with the biosynthesis of zwittermicin A, an aminopolyol antibiotic produced by some members of the B. cereus group. In an initial screen only one isolate, W96, yielded PCR amplicons consistent with those previously reported in the literature for the zwittermicin A genes. Later a further sixteen isolates grouped with W96 on the basis of the RAPD-PCR fingerprinting profiles, were screened for the presence of these genes. Of these, only six showed PCR amplification products similar to W96. Sequence homology testing against the GenBank database confirmed the presence of the zwittermicin A genes in these isolates. Isolate W96 was selected for further extraction and characterization of its antifungal compound(s). However, after culturing in various broth media cell free supernatants of W96 failed to show antifungal activity in vitro even when the supernatants were concentrated 20-fold. These findings provide a general overview of the diversity of aerobic endospore-forming bacteria present in an undisturbed indigenous grassland soil that exhibited antifungal activity in vitro and the limited influence tested crop rhizospheres have on this diversity. Combined use of ITS-PCR, 16S rRNA sequencing and RAPD-PCR techniques served as a rapid and effective means of grouping isolates for further investigations of their potential use as biocontrol agents and plant growth promoting rhizobacteria

Characteristics of tuberculosis patients and the evaluation of compliance to the national TB management guidelines at clinics in a rural community from Mpumalanga province, South Africa

This study serves as baseline investigation into tuberculosis (TB) patient population characteristics and the compliance of clinics in rural settings to the national TB guidelines in terms of diagnosing the disease. A total of 62 TB positive patients’ files were reviewed. Patients were diagnosed using: smear microscopy (41.9%); chest radiography (37.1%); Xpert MTB/RIF (9.7%); symptoms (3.2%); abdomen sonar (1.6%); and, no record (6.5%). Lack of complete compliance was identified, including large dependencies on chest X-ray as the first line of diagnosis and inadequate diagnosis of extra-pulmonary TB. These findings could assist identifying health system gaps for provincial and national control programs.The Institute of Tropical Medicine in Antwerp, Belgium (ITM) through a framework agreement with the Belgian Development Cooperation (DGD) and South African National Parks.http://www.tandfonline.com/loi/ojid20am2016Veterinary Tropical Disease

Prevalence and occupational exposure to zoonotic diseases in high-risk populations in the Free State Province, South Africa

IntroductionZoonotic diseases are responsible for 2.5 billion human cases globally and approximately 2.7 million deaths annually. Surveillance of animal handlers and livestock for zoonotic pathogens contributes to understanding the true disease burden and risk factors within a community. This study investigated the prevalence of selected zoonoses in cattle, farm workers and occupational exposure to endemic zoonotic diseases and their associated risk factors.MethodsSputum samples from farmworkers were screened for Mycobacterium bovis. Blood specimens from farmworkers and archived sera were tested for serological evidence of Brucella sp., hantaviruses, and Leptospira sp. Communal and commercial cattle herds were tested for bovine tuberculosis and brucellosis.ResultsMycobacterium bovis was not isolated from human samples. A total of 327 human sera were screened, and 35/327 (10.7%) were Brucella sp. IgG positive, 17/327 (5.2%) Leptospira sp. IgM positive, and 38/327 (11.6%) hantavirus IgG positive (95% CI). A higher proportion of Brucella sp. IgG-positive samples were detected among veterinarians (value of p = 0.0006). Additionally, two cattle from a commercial dairy farm were bovine tuberculosis (bTB) positive using the bTB skin test and confirmatory interferon-gamma assay. A higher percentage of confirmed brucellosis-positive animals were from communal herds (8.7%) compared to commercial herds (1.1%).DiscussionThese findings highlight the brucellosis and M. bovis prevalence in commercial and communal herds, the zoonotic disease risk in commercial and subsistence farming in developing countries, and the occupational and rural exposure risk to zoonotic pathogens

Patterns of antibiotic use, pathogens, and prediction of mortality in hospitalized neonates and young infants with sepsis: A global neonatal sepsis observational cohort study (NeoOBS)

BACKGROUND: There is limited data on antibiotic treatment in hospitalized neonates in low- and middle-income countries (LMICs). We aimed to describe patterns of antibiotic use, pathogens, and clinical outcomes, and to develop a severity score predicting mortality in neonatal sepsis to inform future clinical trial design. METHODS AND FINDINGS: Hospitalized infants <60 days with clinical sepsis were enrolled during 2018 to 2020 by 19 sites in 11 countries (mainly Asia and Africa). Prospective daily observational data was collected on clinical signs, supportive care, antibiotic treatment, microbiology, and 28-day mortality. Two prediction models were developed for (1) 28-day mortality from baseline variables (baseline NeoSep Severity Score); and (2) daily risk of death on IV antibiotics from daily updated assessments (NeoSep Recovery Score). Multivariable Cox regression models included a randomly selected 85% of infants, with 15% for validation. A total of 3,204 infants were enrolled, with median birth weight of 2,500 g (IQR 1,400 to 3,000) and postnatal age of 5 days (IQR 1 to 15). 206 different empiric antibiotic combinations were started in 3,141 infants, which were structured into 5 groups based on the World Health Organization (WHO) AWaRe classification. Approximately 25.9% (n = 814) of infants started WHO first line regimens (Group 1-Access) and 13.8% (n = 432) started WHO second-line cephalosporins (cefotaxime/ceftriaxone) (Group 2-"Low" Watch). The largest group (34.0%, n = 1,068) started a regimen providing partial extended-spectrum beta-lactamase (ESBL)/pseudomonal coverage (piperacillin-tazobactam, ceftazidime, or fluoroquinolone-based) (Group 3-"Medium" Watch), 18.0% (n = 566) started a carbapenem (Group 4-"High" Watch), and 1.8% (n = 57) a Reserve antibiotic (Group 5, largely colistin-based), and 728/2,880 (25.3%) of initial regimens in Groups 1 to 4 were escalated, mainly to carbapenems, usually for clinical deterioration (n = 480; 65.9%). A total of 564/3,195 infants (17.7%) were blood culture pathogen positive, of whom 62.9% (n = 355) had a gram-negative organism, predominantly Klebsiella pneumoniae (n = 132) or Acinetobacter spp. (n = 72). Both were commonly resistant to WHO-recommended regimens and to carbapenems in 43 (32.6%) and 50 (71.4%) of cases, respectively. MRSA accounted for 33 (61.1%) of 54 Staphylococcus aureus isolates. Overall, 350/3,204 infants died (11.3%; 95% CI 10.2% to 12.5%), 17.7% if blood cultures were positive for pathogens (95% CI 14.7% to 21.1%, n = 99/564). A baseline NeoSep Severity Score had a C-index of 0.76 (0.69 to 0.82) in the validation sample, with mortality of 1.6% (3/189; 95% CI: 0.5% to 4.6%), 11.0% (27/245; 7.7% to 15.6%), and 27.3% (12/44; 16.3% to 41.8%) in low (score 0 to 4), medium (5 to 8), and high (9 to 16) risk groups, respectively, with similar performance across subgroups. A related NeoSep Recovery Score had an area under the receiver operating curve for predicting death the next day between 0.8 and 0.9 over the first week. There was significant variation in outcomes between sites and external validation would strengthen score applicability. CONCLUSION: Antibiotic regimens used in neonatal sepsis commonly diverge from WHO guidelines, and trials of novel empiric regimens are urgently needed in the context of increasing antimicrobial resistance (AMR). The baseline NeoSep Severity Score identifies high mortality risk criteria for trial entry, while the NeoSep Recovery Score can help guide decisions on regimen change. NeoOBS data informed the NeoSep1 antibiotic trial (ISRCTN48721236), which aims to identify novel first- and second-line empiric antibiotic regimens for neonatal sepsis. TRIAL REGISTRATION: ClinicalTrials.gov, (NCT03721302)

The epidemiology of tuberculosis in cattle and humans living in the wildlife-livestock-human interface in the rural Mnisi community Mpumalanga province South Africa

The aim of this study was to investigate the prevalence and epidemiological significance of tuberculosis (TB) in bovine and humans living at a wildlife/livestock/human interface, as well as the risk factors associated with TB transmission at that interface. The Mnisi community was chosen as it is located at the western border of the Kruger National park (KNP) and enables research at the wildlife/livestock/human interface. The first objective of the study entailed investigating the presence of bovine tuberculosis (BTB) in 10% of Mnisi?s livestock, using the comparative intradermal skin test. A low individual prevalence of 0.33 % (95% CI.0.14 ? -0.79) was detected. Further investigations into the causative agent in livestock, using genotyping techniques identified the KNP parental strain, M. bovis KNP VNTR -1 strain.1 Supporting records from the provisional Mpumalanga Veterinary Services and the physical location of dip-tanks where BTB was detected, it was established the infection was a result of spillback infection from wildlife in the neighbouring KNP. The epidemiological significance of BTB in human TB was investigated through the isolation and genetic characterisation of the Mycobacterium tuberculosis complex (MTBC) strain population in the Mnisi community. Mycobacterium bovis was not detected in the human population. However, a high genetic diversity of M. tuberculosis was observed among the 13 isolates obtained.The M. tuberculosis isolates were identified as the following eight families: T; Beijing; LAM 11_ZWE; EAI5; MANU1; X1; X2; and S families. The predominant lineage was as T family, sub-lineage ST53. Based on the high diversity (8 clusters/13isolates) and the predominance of the T family, it was concluded that the TB population structure within the Mnisi community was largely impacted by human migration from urban towns and neighbouring Mozambique. A questionnaire was administered to investigate BTB transmission risk factors at the livestock/human interface. It was established that there were low risk levels of BTB transmission at the human/livestock interface mainly based on the fact that the majority of the households in the community obtained pasteurised milk commercially, and although undercooked/raw meat and organs were preferred, the majority of respondents reported that they discarded the meat if changes in meat quality were observed.Thesis (PhD)--University of Pretoria, 2016.tm2016Veterinary Tropical DiseasesPh

The prevalence of bovine tuberculosis in cattle at the wildlife/livestock interface in the Mnisi community, South Africa

Bovine tuberculosis (BTB) is caused by Mycobacterium bovis (M. bovis), of the Mycobacterium tuberculosis complex. The pathogen has a wide host range including humans, wildlife, livestock as well as domestic animals. The disease has great implications in livestock productivity, conservation of endangered wildlife and public health due to its zoonotic potential (Firdessa et al., 2012). Reports have mentioned the endemic levels and potential spread of BTB in South African game reserves, particularly the Kruger National Park (Michel et al., 2006). However, limited to no research has been conducted to investigate the potential spill-back of BTB into livestock, especially in communities' bordering the game reserves at the wildlife/livestock/human interface.Includes bibliographical referencesThis study was funded by the Institute of Tropical Medicine in Antwerp, Belgium (ITM) and University of Pretoria (UP)ab202

Risk factors for zoonotic tuberculosis at the wildlife-livestock-human interface in South Africa

A cross-sectional study was conducted to investigate the risk factors associated with zoonotic tuberculosis in humans and its transmission to people living at the wildlife-livestock-human interface. A questionnaire was administered to collect information on food consumption habits, food handling practices, and knowledge of zoonotic TB. Sputum samples were also collected from 150 individuals that belonged to households of cattle farmers with or without a bTB infected herd. In addition, 30 milk samples and 99 nasal swabs were randomly collected from cattle in bTB infected herds for isolation of Mycobacterium bovis (M. bovis). The sputum samples were screened for TB using the GeneXpert test and this was followed by mycobacterial culture and speciation using molecular techniques. No M. bovis was isolated from TB positive sputum samples and only one sample was confirmed as Mycobacterium tuberculosis (M. tuberculosis). M. bovis was isolated from 6.6% (n = 2/30) milk samples and 9% (n = 9/99) of nasal swabs. Ownership of a bTB infected herd and consumption of milk were recognized as highly significant risk factors associated with a history of TB in the household using multiple correspondence analysis (MCA) and logistic regression. The findings from this study have confirmed the potential for zoonotic TB transmission via both unpasteurized milk and aerosol thus, the role of M. bovis in human TB remains a concern for vulnerable communities

Prevalence and immediate outcome of candida colonized preterm neonates admitted to Special Care Unit of Mulago Hospital, Kampala Uganda

BACKGROUND: Candida species is the third commonest cause of sepsis among neonates. Colonization by Candida is a predictor for candidemia among preterm neonates. OBJECTIVES: To determine prevalence of early Candida colonization and early outcome among colonized preterm neonates admitted to Mulago hospital Special Care Unit. METHODS: A prospective observational cohort was conducted between December 2008 and April 2009. Preterm neonates aged >72 hours and less than one week were screened for Candida colonization of the groin, oral pharynx and rectum using CHROMagar. Colonized neonates were followed up for 14 days. Blood cultures were done for those with signs of septicaemia. The Fisher's exact tests and logistic regression were conducted for factors associated with colonization and mortality among colonized neonates. P values of < 0.05 were considered significant and confidence interval of 95% was used. RESULTS: Candida colonization occurred in 50/213 (23.5%) neonates. Gestational age ≤ 30 weeks was the only factor independently associated with colonization (p = 0.005). Of the colonized 14/46 (30.4%) died and 13/46 (28.3%) developed mucocutaneous candidiasis. No candidemia was identified. Multiple site colonization was independently associated with mortality (p=0.035). CONCLUSION: The consequence of high colonization observed in this study needs to be further elucidated in Uganda

Prevalence and immediate outcome of candida colonized preterm neonates admitted to Special Care Unit of Mulago Hospital, Kampala Uganda

Background: Candida species is the third commonest cause of sepsis among neonates. Colonization by Candida is a predictor for candidemia among preterm neonates. Objectives: To determine prevalence of early Candida colonization and early outcome among colonized preterm neonates admitted to Mulago hospital Special Care Unit. Methods: A prospective observational cohort was conducted between December 2008 and April 2009. Preterm neonates aged \u3e72 hours and less than one week were screened for Candida colonization of the groin, oral pharynx and rectum using CHROMagar. Colonized neonates were followed up for 14 days. Blood cultures were done for those with signs of septicaemia. The Fisher\u27s exact tests and logistic regression were conducted for factors associated with colonization and mortality among colonized neonates. P values of \u3c 0.05 were considered significant and confidence interval of 95% was used. Results: Candida colonization occurred in 50/213 (23.5%) neonates. Gestational age ≤ 30 weeks was the only factor independently associated with colonization (p = 0.005). Of the colonized 14/46 (30.4%) died and 13/46 (28.3%) developed mucocutaneous candidiasis. No candidemia was identified. Multiple site colonization was independently associated with mortality (p=0.035). Conclusion: The consequence of high colonization observed in this study needs to be further elucidated in Uganda

Risk factors for zoonotic tuberculosis at the wildlife-livestock-human interface in South Africa

International audienceA cross-sectional study was conducted to investigate the risk factors associated with zoonotic tuberculosis in humans and its transmission to people living at the wildlife-livestock-human interface. A questionnaire was administered to collect information on food consumption habits, food handling practices, and knowledge of zoonotic TB. Sputum samples were also collected from 150 individuals that belonged to households of cattle farmers with or without a bTB infected herd. In addition, 30 milk samples and 99 nasal swabs were randomly collected from cattle in bTB infected herds for isolation of Mycobacterium bovis (M. bovis). The sputum samples were screened for TB using the GeneXpert test and this was followed by mycobacterial culture and speciation using molecular techniques. No M. bovis was isolated from TB positive sputum samples and only one sample was confirmed as Mycobacterium tuberculosis (M. tuberculosis). M. bovis was isolated from 6.6% (n = 2/30) milk samples and 9% (n = 9/99) of nasal swabs. Ownership of a bTB infected herd and consumption of milk were recognized as highly significant risk factors associated with a history of TB in the household using multiple correspondence analysis (MCA) and logistic regression. The findings from this study have confirmed the potential for zoonotic TB transmission via both unpasteurized milk and aerosol thus, the role of M. bovis in human TB remains a concern for vulnerable communities