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Vibrio vulnificus is a marine pathogen of human health concern, capable of causing potentially fatal wound infections in a select group of the population. Previous studies have indicated this species’ strong negative correlation with salinity, not typically found above 30 ppt. This study assessed the ability of V. vulnificus to become Viable But Nonculturable in response to elevated salinity (35 ppt) as well as investigated novel methods for confirming their entrance into this state. Results showed a complete loss of culturability in both Environmental and Clinical strains of this bacterium by 9 days after inoculation. Using a High Content Imager, it was determined that these pathogens were not dying (< 10%) in response to the treatment and were partially becoming cocci (≈35%). This study indicates the importance of understanding the impact environmental parameters have on this human pathogen, and what it means for reliably detecting them. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Please read the abstract on p 79-80 of this document / Dissertation (MSc Agric (Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

M. Tech. / Background: Waterborne disease contributes significantly to the total global disease burden. Populations in rural areas of South Africa depend on untreated waters for consumption and sanitation. Contamination of public water supplies by harmful bacteria such as pathogenic E. coli poses a major risk for public health. Ingestion of these pathogenic microorganisms present in the contaminated and untreated waters could cause infection, leading to systemic inflammatory responses manifested by the production of various proinflammatory cytokines. To date, there is no human system test available to detect whether water, following ingestion, would cause disease (i.e. whether the water is infectious). The current water testing methods only test for the presence of indicator organisms, such as faecal coliforms, total coliforms, and Escherichia coli. A reliable in-vitro bioassay that could assess whether the water would cause an inflammatory response was investigated in this study. Objectives: Pro-inflammatory cytokines and whole-blood have been used in similar studies to detect the inflammatory responses following exposure to specific stimulants such as dust, lipopolysaccharide (LPS), E. coli and various others. It has been reported that larger numbers of these contaminants induced higher levels of pro-inflammatory cytokine expression. This implies that the pro-inflammatory cytokine expression could be used as a marker of infection since, inflammation occurs in response to infection. Successful infection is thus necessary for inflammation to occur, and high levels of pro-inflammatory cytokine expression confirm that infection has occurred. Thus if pro-inflammatory cytokines could serve as indicators for infection, these cytokines could be used as indicators for bacterial pathogenicity of water.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The increased consumption of fresh produce has been shown to be related to increases in foodborne disease outbreaks and these have in many cases been ascribed directly to carry-over of pathogens from contaminated irrigation water. In South Africa, rivers are the main source of irrigation water but many have been found to be unsuitable for irrigation of fresh produce because of the unacceptably high levels of faecal contamination. The main aim of this study was to do a baseline evaluation of the microbiological quality of the Plankenburg and Eerste Rivers and to determine which bacterial contaminants are present. Two sampling sites were selected for the Plankenburg (Plank-1 and -3) and one for the Eerste River (Eerste-1). The microbiological analysis included aerobic colony count (ACC), aerobic and anaerobic sporeformers, Staphylococcus, Salmonella, Listeria, enterococci, coliforms, faecal coliforms and E. coli using standard methods. The faecal contamination levels for both rivers exceeded the DWAF and WHO guidelines of <1 000 E. coli per 100 mL water for irrigation of fresh produce intended to be consumed raw. The Plankenburg River sites always had higher coliform contamination levels (1 200 - 13 000 000 MPN per 100 mL water) than the Eerste River site (230 - 79 000 MPN per 100 mL water). There was also a high incidence of index organisms including Salmonella, Staphylococcus, Listeria and endosporeformers. The isolation of intestinal enterococci suggested the presence of potential pathogens that can cause disease outbreaks. The baseline data also showed large variations in microbial loads over the 15 month study with the faecal coliform counts ranging for Plank-1 from 1 200 to 7 000 000 MPN.100mL-1, Plank-3 from 10 to 460 000 MPN.100mL-1 and Eerste-1 from 28 to 79 000 MPN.100mL-1. The water temperatures at all three sites ranged from 12.1° to 21.7°C with COD values in most cases below 100 mg.L-1. As the baseline study showed large variations in microbial loads over the 15 month study period an assessment using the Colilert-18 system of the weekly, daily and hourly variations, for 6 weeks over a period of 4 months was conducted at site Plank-2. This site was specifically used as it is an irrigation source point for nearby fresh produce farmers and is about 2 km further downstream from an informal settlement. The weekly variation trend for total coliforms (TC) showed a decrease over the entire sampling period with the highest count of 3 200 000 MPN.100 mL-1 during the warmer period. The E.coli (Ec) counts showed a similar trend with the highest count of 440 000 MPN.100 mL-1 also in March. The daily variation trends were the same for both the TC and Ec and counts found to increase from Monday to Thursday followed by a decrease to Sunday. The highest counts were on Thursday with average TC and Ec counts of 1 900 000 and 160 000 MPN.100 mL-1, respectively. The hourly variation trends were similar for both TC and Ec with counts increasing from 06h00 to 12h00 followed by a decrease to 18h00. The increases in TC and Ec counts found during the weekly, daily and hourly variation trend studies clearly suggests that the 15 month sampling that was done once a month on Mondays at 08h00 could be considered an underestimation of the contamination levels of the Plankenburg and Eerste Rivers. The overall weekly variation trend for the water temperature showed a decrease over the sampling period while the daily and hourly variation trends showed an increase from 06h00 to 18h00. The overall weekly trend for pH differed from that of the temperature with an increase over the sampling period. The analysis of covariance showed no correlation (p < 0.05) between the physico-chemical (temperature and pH) and the microbial variables (TC and Ec). Therefore it was concluded that temperature and pH had no direct impact on either the total coliform or E. coli counts. Both the Plankenburg and Eerste Rivers were found to be unsuitable for the irrigation of fresh produce intended to be consumed raw due to the high levels of faecal contamination that exceeded DWAF and WHO guidelines. Irrigation with such water could pose a health risk because of presence of potential pathogens that could be carried-over to fresh produce. / AFRIKAANSE OPSOMMING: Die toenemende gebruik van vars produkte hou direk verband met die toename in voedseloordraagbare siektes. Alte dikwels kan dit toegeskryf word aan die teenwoordigheid van patogene in besproeiingswater. In Suid Afrika is riviere die hoofbron van besproeiingswater maar dit is al gevind dat meeste ongeskik is vir gebruik as besproeïngsbron as gevolg van die onaanvaarbare hoe vlakke van fekale besmetting. Die hoofdoel van hierdie studie was om ‘n basislyn evaluasie van die mikrobiologiese kwaliteit van die Plankenburg en Eerste Riviere te doen en ook vas te stel watter bakteriese kontaminante teenwoordig is. Twee bemonsteringpunte is geselekteer vir die Plankenburg (Plank- 1 en -3) en een vir die Eerste Rivier (Eerste-1). Mikrobiologiese analises met standaard metodes het die volgende ingesluit: aërobe kolonie telings (AKT), aërobe en anaërobe spoorevormers, Staphylococcus, Salmonella, Listeria, enterococci, koliforms, fekale koliforms en E. coli met gebruik van standaard metode. Die fekale besmettingsvlakke vir beide riviere het die DWAF en WHO leistreep van <1 000 E. coli per 100 mL water vir besproeiing van vars produkte wat rou geëet kan word oorskry. Die Plankenburg Rivier bemonsteringspunte het in alle gevalle ‘n hoër kolivorm besmettingsvlak (1 200 - 13 000 000 MPN per 100 mL water) as die Eerste Rivier punt (230 - 79 000 MPN per 100 mL water) gehad. Daar was ook ‘n hoër voorkoms van indeksorganismes insluitend Salmonella, Staphylococcus, Listeria en endosporevormers. Die voorkoms van ingewand enterococci was ‘n addisionele aanduiding van die voorkoms van patogene wat ernstige gesondheidsrisikos vir die verbruiker kan inhou. Die basislyn data het groot variasies in die mikrobe vlakke oor die 15 maand van studie getoon. Die faecal koliforms vir Plank- 1 het gewissel van 1 200 tot 7 000 000 MPN.100mL-1, vir Plank-3 van 10 tot 460 000 MPN.100mL-1 en vir Eerste-1 van 28 tot 79 000 MPN.100mL-1. Die water temperature het gewissel van 12.1° tot 21.7°C met die CSB waardes in meeste gevalle minder as 100 mg.L-1. Aangesien daar sulke groot variasies in mikrobe ladings oor die 15 maande tydperk voorgekom het, is die Colilert-18 sisteem gebruik om die weeklikse, daaglikse en uurlikse variasies vas te stel vir 6 weke oor ‘n periode van 4 maande by die Plank-2 bemonsteringspunt. Daar is spesifiek op die bemonsteringspunt gefokus omdat dit as ‘n besproeiingsbron gebruik word deur groente produsente. Dit is ook gelee ongeveer 2 km stroomaf van ‘n informele nedersetting. Die weeklikse variasies in totaal koliforms (TC) het ‘n afname oor die hele bemonsteringsperiode getoon, met die hoogstes telling van 3 200 000 MPN.100 mL-1 gedurende die warmer tydperk. Die E.coli (Ec) tellings het ‘n soortgelyke neiging getoon, met die hoogste telling van 440 000 MPN.100 mL-1 ook in Maart. Die daaglikse neigings was dieselfde vir beide die TC en Ec en die tellings het vermeerder van Maandag tot Donderdag, met ‘n afname tot Sondag. Die hoogste telling was op Donderdag met gemiddelde TC and Ec tellings van 1 900 000 and 160 000 MPN.100 mL-1, respektiewelik. Die uurlikse variasie profiel was soortgelyk vir beide TC and Ec met tellings wat vermeerder het van 06h00 tot 12h00 gevolg deur ‘n afname tot 18h00. Die toename in TC en Ec getalle soos vasgestel gedurende die weeklikse, daaglikse en uurlikse variasie het duidelik getoon dat die bemonsterings wat een maal per maand op Maandae om 08h00 gedurende die 15 maande tydperk uitgevoer is, tot ‘n erg onderskatting van die besmettings vlakke in die Plankenburg en Eerste Riviere gelei het. Die algehele weeklikse variasies vir die water temperatuur het ‘n verlaging oor die bemonsteringstydperk getoon terwyl die daaglikse en uurlikse variasie neigings ‘n verhoging van 06h00 tot 18h00 getoon het. Die weeklikse neigings vir pH het van die van die temperatuur verskil. Die analises van kovariante het geen korrelasie (p < 0.05) tussen die fisiese-chemiese (temperature en pH) parameters en die mikrobe veranderlikes (TC en Ec) getoon nie. Dus is daar afgelei dat temperatuur en pH geen direkte impak op die totale kolivorm of E. coli tellings gehad nie. Die data van die studie het duidelik getoon dat water van beide die Plankenburg en Eerste Riviere nie geskik is vir gebruik vir besproeiing van vars produkte wat rou geëet gaan word nie. In beide gevalle het die fekale besmettingsvlakke die DWAF en WHO leistreep oorskry. Besproeiing met sulke water hou ‘n gesondheidsgevaar in as gevolg van die teenwoordigheid van potensiële patogene wat oorgedra kan word na vars produkte.

Bacteria cause significant morbidity and mortality throughout the world, including deadly diseases such as tuberculosis, meningitis, cholera, and pneumonia. Timely and accurate bacterial identification is critical in areas such as clinical diagnostics, environmental monitoring, food safety, water and air quality assessment, and identification of biological threat agents. At present, there is an established need for high throughput, sensitive, selective, and rapid methods for the detection of pathogenic bacteria, as existing methods, while nominally effective, have failed to sufficiently reduce the massive impact of bacterial contamination and infection. The work presented in this thesis focuses on addressing this need and augmenting conventional microorganism research through development of mass spectrometry (MS)-based proteomic applications. MS, a well established tool for addressing biological problems, offers a broad range of laboratory procedures that can be used for taxonomic classification and identification of microorganisms. These methods provide a powerful complement to many of the widely used molecular biology approaches and play critical functions in various fields of science. While implementation of modern biomolecule-identifying instrumentation, such as MS, has long been postulated to have a role in the microbiology laboratory, it has yet to be accepted on a large scale. Described in this document are MS methods that erect strong foundations on which new bacterial diagnostics may be based. A general introduction on key aspects of this work is presented in Chapter 1, where different approaches for detection of pathogenic bacteria are reviewed, and an overview regarding MS and microbial identification is provided. Chapter 2 presents the first implementation of microbial identification via rapid, open air Direct Analysis in Real Time MS (DART MS) to generate ions directly from microbial samples, including the disease-causing bacteria, Coxiella burnetii, Streptococcus pyogenes, and Escherichia coli. Chapter 3 expands on whole cell C. burnetii MS analysis and presents a rapid differentiation method to the strain-level for C. burnetii using mass profiling/fingerprinting matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and multivariate pattern recognition. Chapter 4 presents a unique "top-down" proteomics approach using 15N-labeled bacteriophage amplification coupled with MALDI-TOF MS as a detector for the rapid and selective identification of Staphylococcus aureus. Chapter 5 extends the idea of using isotopically labeled bacteriophage amplification by implementing a "bottom-up" proteomics approach that not only identifies S. aureus in a sample, but also quantifies the bacterial concentration in the sample using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) as a detector. In conclusion, Chapter 6, summarizes and contextualizes the work presented in this dissertation, and outlines how future research can build upon the experimentation detailed in this document.

Quality control procedures during food processing may involve either lengthy enrichment steps, precluding enumeration of bacteria in contaminated food, or direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow on selective media, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured Escherichia coli B-41560, originally an isolate from ground beef. Bacteria were propagated in tryptic soy broth (TSB), ground beef, or infant milk formula (IMF) to a density of 106-108 CFU/mL, and stressed for six minutes either in lactic acid (pH of 4.5) or heat-shocked for 3 min. at 60°C. Samples were pour- plated in basal layers of either tryptic soy agar (TSA), Sorbitol MacConkey (SMAC), or Violet Red Bile (VRB) agar and resuscitated for 4h prior to addition of agar overlays. Other stressed bacteria were plated directly onto the commercial media Petrifilm and Easygel. Our results indicate that the use of selective and nonselective agar overlays for sensitive recovery and accurate enumeration of E. coli B-41560 is dependent on the stress treatment and food system. These data underscore the need to implement food safety measures that address sublethally- injured bacteria such as E. coli O157:H7, without the use of enrichment steps, in order to avoid underestimation of true densities for target pathogens. / Department of Biology

Cryptosporidiosis is an infection caused by Cryptosporidium; a protozoan parasite that infects the gastrointestinal tract. The infection is of major public health concern in both developed and developing countries. Faecal samples were collected from 160 in-patient adults, with complaint of diarrhoea, admitted at Victoria hospital in Alice, Nkonkobe Municipality. Twenty apparently healthy subjects were included as controls. All diarrhoea positive patients were interviewed to record socio-demographic information, water supply and animal contact. Initial screening was carried out by microscopy and ELISA to detect positive Cryptosporidium. Genomic DNA was extracted from microscopically positive samples and a PCR reaction was perform to amplify the (18S) SSUrRNA gene for further identification and epidemiology of Cryptosporidium. Data were analysed using Pearson‘s χ2 and Fisher‘s exact test to assess the univariate association between Cryptosporidium infection and the possible risk factors. Of the 180 subjects screened for cryptosporidial infection, Cryptosporidium antigen was detected in 122 giving an overall prevalence of 67.8 percent. In HIV-positive diarrhoea patients, prevalence increased with ages; between 31-43 (mean age 36.5 yr) and 70-82 (mean age 75.8 yr) had a higher prevalence (100 percent) of the antigen than 18-30 (mean age 23.2 yr) and 83-95 (mean age 88.8 yr) (50.0 percent) in HIV-positive diarrhoea patients (P > 0.05). In HIV-negative diarrhoea patients, prevalence was highest in the 18-30 (mean age 23.2 yr) (87.5 percent) and least (35.7 percent) in those aged 83-95 (mean age 88.8 yr) (P > 0.05). Cryptosporidium antigen was higher in females than in males. Of 115 females (mean age 46.7yr) who participated in the study, antigen was detected in 90 (78.2 percent) against 32 (71.1 percent) of 45 males (mean age 42.6yr). None of the 20 apparently healthy control subjects was found to be infected with Cryptosporidium. Cryptosporidium was detected in 27 HIV-positive and 97 HIV-negative diarrhoea patients by any one of the techniques. Antigen detection by ELISA 14 showed the highest positivity 96 (76.8 percent) in HIV- negative and 26 (74.3 percent) in HIV- positive diarrhoea patients. PCR detected eighty-nine (71.2 percent) cases in HIV-negative and 23 (65.7 percent) in HIV-positive patients with diarrhoea. Only 13 (37.1 percent) HIV-positive and 34 (27.2 percent) HIV-negative diarrhoea patients were found positive for Cryptosporidium by modified ZN. No significant difference was observed in sensitivity of antigen detection by ELISA and PCR (96.9 percent) in HIV-negative diarrhoea patients, respectively. Specificity of the staining technique was 88.9 percent in HIV-positive and 96.6 percent in HIV-negative diarrhoea patients. No significant difference was found in specificity of antigen detection by ELISA and PCR in HIV-positive and HIV-negative diarrhoea patients, respectively. Positive predictive value of ZN staining in both HIV-positive and HIV-negative diarrhoea patients (92.3 and 96.9 percent) was statistically higher than ELISA and PCR. No significant difference was observed in negative predictive value of ZN technique for detection of Cryptosporidium between HIV-positive and HIV- negative diarrhoea patients. Differences found in prevalence rates due to water source, suggest that the high infection rates of specific groups are associated with their exposure to the contaminated water supply. The results indicate that Cryptosporidium infection is highly prevalent in adult faecal specimens in the Nkonkobe Municipality, an indication of active infection that is likely to emerge as major human pathogen in this location due to socioeconomic changes which favour transmission. However, sequencing analysis is required to differentiate between Cryptosporidium genotypes in the various outbreaks

Sampling data of many surface water bodies in Broward County tend to reveal abnormal levels of bacterial concentrations for total coliform, Escherichia coli and Enterococcus. Given the presence of septic systems in geohydrologically sensitive areas throughout the County, several studies (Morin 2005 and Bocca 2007) suggested a correlation in the high bacterial densities and the septic sites. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection