Water reuse has become a promising alternative to alleviate stress on conventional freshwater resources in the face of population growth, sea level rise, source water depletion, eutrophication of water bodies, and climate change. Potable water reuse intentionally looks to purify wastewater effluent to drinking water quality or better through the development and implementation of advanced treatment trains. While membrane-based treatment has become a widely-adopted treatment step to meet this purpose, there is growing interest in implementing treatment trains that harness microorganisms as a more sustainable and less energy-intensive means of removing contaminants of emerging concern (CECs), through biological degradation or transformation. In this dissertation, various aspects of the operation of a microbially-active carbon-based advanced treatment train producing water intended for potable reuse are examined, including fate of dissolved organic carbon, underlying microbial populations, and functional genes are explored. Further, dynamics associated with antibiotic resistance genes (ARGs), identified as a microbially-relevant CECs, are also assessed. Overall, this dissertation advances understanding associated with the interplay between and within treatment processes as they relate to removal of various organic carbon fractions, microbially community dynamics, functional genes, and ARGs. Further, when relevant, these insights are contextualized to operational conditions, process upsets, water quality parameters, and other intended water uses within the water industry with the goal of broadening the application of advanced molecular tools beyond the scope of academic research. Specifically, this dissertation illuminates relationships among organic carbon fractions and molecular markers within an advanced treatment train employing flocculation, coagulation, and sedimentation (FlocSed), ozonation, biologically active carbon (BAC) filtration, granular active carbon (GAC) contacting, and UV disinfection. Biodegradable dissolved organic carbon (BDOC) analysis was adapted specifically as an assay relevant to assessing dissolved organic carbon biodegradability by BAC/GAC-biofilms and applied to profile biodegradable/non-biodegradable organic carbon as wastewater effluent passed through each of these treatment stages. Of particular interest was the role of ozonation in producing bioavailable organic carbon that can be effectively removed by BAC filtration. In addition to understanding the removal of fractionalized organic carbon, next generation DNA sequencing technologies (NGS) were utilized to better understand the microbial dynamics characteristic of complex microbial communities during disinfection and biological treatment. Specifically, this analysis was focused on succession and colonization of taxa, genes related to a wide range of functional interests (e.g. metabolic processes, horizontal gene transfer, DNA repair, and nitrogen cycling), and microbial CECs. Finally, NGS technologies were employed to assess the differences between a wide range of water use categories, including conventional drinking water, potable reuse, and non-potable reuse effluent's microbiomes to identify core and discriminatory taxa associated with intended water usage. The outcomes of this dissertation provide valuable information for optimizing carbon-based treatment trains as an alternative to membrane-based treatment for sustainable water reuse and also advance the application of NGS as a diagnostic tool for assessing the efficacy of various water treatment technologies for achieving treatment goals. / Doctor of Philosophy / Several factors have led to increased stress on conventional drinking water sources and widespread global water scarcity. Projections indicate that continued population growth, increased water demand, and degradation of current freshwater resources will negatively contribute to water needs and underscore the need to secure new potable (i.e. fit for human consumption) sources. Water reuse is a promising alternative to offset the growing demands on traditional potable sources and ameliorate negative consequences associated with water scarcity. Discharge of treated wastewater to marine environments is especially a lost opportunity, as the water will no longer be of value to freshwater habitats or as a drinking water source. Water reuse challenges the conventional wastewater treatment paradigm by providing advanced treatment of wastewater effluent to produce a valuable resource that can be safely used directly for either non-potable (e.g., irrigation, firefighting) or potable (i.e., drinking water) applications. The means of achieving advanced treatment of wastewater effluents can take many forms, commonly relying on the utilization of membrane filtration. However, membrane filtration is an intensive process and suffers from high initial costs, high operational costs, membrane fouling with time, and the production of a salty and difficult to dispose of waste stream. These drawbacks have motivated the water reuse industry to explore more sustainable approaches to achieving high quality effluents. One such alternative relies on the utilization of microorganisms to provide biological degradation and transformation of contaminants through a process known as biologically active filtration (BAF). Comparatively to membrane systems, BAF is more cost effective and produces significantly fewer byproducts while still producing high quality treated water for reuse. However, the range in quality of the resulting treated water has not yet been fully established, in part due to the lack of understanding of the complex microbial communities responsible for biological treatment. As water and wastewater treatment technologies have evolved over the past century, many biological treatments have remained largely 'black box' due to the lack of effective tools to identify the tens of thousands of species of microbes that inhabit a typical system and to track their dynamics with time. Instead, analysis has largely focused on basic water quality indicators. This dissertation takes important steps in advancing the implementation of the study of DNA and biodegradable organic carbon (BDOC) analysis to improve understanding of the mechanisms that drive different water reuse treatment technologies and to identify potential vulnerabilities. Insights gained through application of these tools are contextualized to observed operational conditions, process upsets, and water quality measurements. This helped to advance the use of DNA-based tools to better inform water treatment engineering practice. Specifically, this dissertation dives into the relationships between organic carbon and DNA-based markers within an advanced treatment train employing flocculation, coagulation, and sedimentation (FlocSed), ozonation, biologically active carbon (BAC) filtration, granular active carbon (GAC) contacting, and UV disinfection. Development and application of the BDOC test revealed that the bulk of organic carbon entering the treatment train is dissolved. Further, BDOC analysis served to characterize the impact of specific treatment processes and changes in operational conditions on both biodegradable and non-biodegradable organic carbon fractions. Such information can help to inform continued process optimization. Utilization of DNA-based technologies shed light on the functional capacity of microbial communities present within each stage of treatment and the fate of antibiotic resistance genes (ARGs). ARGs are of concern because, when present in human pathogens, they can result in the failure of antibiotics to cure deadly infections. Other functional genes of interest were also examined using the DNA-based analysis, including genes driving metabolic processes and nitrogen cycling that are critical to water purification during BAF treatment. Also, the DNA-based analyses made it possible to better understand the effects of disinfectants on microbes. Interestingly, some ARG types increased in relative abundance (a measure analogous to percent composition) response to treatments, such as disinfection, and others decreased. Characterization of the microbial communities and their dynamic response to changing operation conditions were also observed. For example, it was possible to characterize how the profiles of microbes changed with time, an ecological process called succession, during BAC filtration and GAC contacting. Generally, this analysis, coupled with the functional analysis, shed light on the important, divergent roles of bacterial communities on organic degradation during both BAC and GAC treatment. Finally, a study was conducted that compared the microbiome (i.e. entire microbial community) between a wide range of conventional drinking water, potable reuse water, and non-potable reuse waters. Here it was found that significant differences existed between the microbial communities of water intended for potable or non-potable usage. This work also looked to expand the application of NGS technologies beyond strictly academic research by developing the application of more advanced DNA-based tools for treatment train assessment and monitoring.

Landfilling is the most widely used method for the disposal of municipal solid wastes (MSW) in the United States due to its simplicity and low cost. According to the 2014 report on Advancing Sustainable Materials Management by the USEPA, only 34% of the total MSW generated in the US was recycled, while 13% was combusted for energy recovery. In 2014, 53% of the MSW generated, (i.e. 136 million tons) in the US was landfilled. The treatment of landfill leachates, generated by percolation of water through the landfill, primarily due to precipitation, has been found to be one of the major challenges associated with landfill operation and management. Currently, leachates from most landfills are discharged into wastewater treatment plants, where they get treated along with domestic sewage. Issues associated with treatment of landfill leachates due to their high nitrogen and heavy metal content have been widely studied. Recently, it has been observed that the organic carbon in landfill leachates, specifically humic and fulvic acids (together referred to as "humic substances") contain aromatic groups that can absorb large amounts of ultraviolet (UV) light, greatly reducing the UV transmissivity in wastewater plants using UV disinfection as the final treatment step. This interference with UV disinfection is observed even when landfill leachates constitute a very small fraction (of the order of 1%) of the total volumetric flow into wastewater treatment plants. Humic substances are present as dissolved organic matter (DOM) and typically show very low biodegradability. Removing these substances using chemical treatment or membrane processes is an expensive proposition. However, the concentrations of humic substances are found to be reduced in leachates from landfill cells that have aged for several years, suggesting that these substances may be degraded under the conditions of long-term landfilling. The primary objective of this research was to use a two-stage process employing thermophilic pretreatment followed by a mesophilic anaerobic membrane bioreactor (AnMBR) to mimic the conditions of long-term landfilling. The AnMBR was designed to keep biomass inside the reactor and accelerate degradation of biologically recalcitrant organic carbon such as humic substances. The treatment goal was to reduce UV absorbance in raw landfill leachates, potentially providing landfills with an innovative on-site biological treatment option prior to discharging leachates into wastewater treatment plants. The system was operated over 14 months, during which time over 50% of UV-quenching organic carbon and 45% of UV absorbance was consistently removed. To the best of our knowledge, these removal values are higher than any reported using biological treatment in the literature. Comparative studies were also performed to evaluate the performance of this system in treating young leachates versus aged leachates. Next-generation DNA sequencing and quantitative PCR (qPCR) were used to characterize the microbial community in raw landfill leachates and the bioreactors treating landfill leachate. Analysis of microbial community structure and function revealed the presence of known degraders of humic substances in raw as well as treated landfill leachates. The total number of organisms in the bioreactors were found to be higher than in raw leachate. Gene markers corresponding to pathogenic bacteria and a variety of antibiotic resistance genes (ARGs) were detected in raw landfill leachates and the also in the reactors treating leachate, which makes it necessary to compare these ARG levels with wastewater treatment in order to determine if leachates can act as sources of ARG addition into wastewater treatment plants. In addition, the high UV absorbance of leachates could hinder the removal of ARBs and ARGs by UV disinfection, allowing their release into surface water bodies and aiding their proliferation in natural and engineered systems. / Ph. D.

The rising number of antibiotic resistant bacteria (ARB) may introduce to the post antibiotic era because they cause a loss of the therapeutic potential of antibiotics. For many years the important role of the natural environment as reservoir and dissemination pathway for ARB and responsible genes has been largely overlooked. However, especially aquatic ecosystems provide optimal conditions for the antibiotic resistance (AR) evolution: first, aquatic ecosystems are frequently affected by anthropogenic activities that cause multiple pollutions for example with heavy metals, that potentially cause co-selection of antibiotic- and heavy metal resistance. Second, aquatic ecosystems feature a dissemination pathway between human populations and natural environments via the urban water cycle. Water cycles between human associated environments (e.g. house holds and clinics) via waste water through waste water treatment plants into natural ecosystems (e.g. water bodies) and back as drinking water after purification. Third, ecosystem internal biotic interactions such as competition between bacteria and predation by the natural consumers seem to impact AR evolution sustainably. The present doctoral thesis focuses on the impact of abiotic and biotic factors on the proliferation of AR and responsible genes in natural aquatic environments, with special emphasis on (i) heavy metal driven co-selection of antibiotic and heavy metal resistance and (ii) on the impact of competition and predation on the evolution of AR. In order to quantify the risk of heavy metal driven co-selection for AR spread, I provide a first risk assessment based on literature values of environmental heavy metal loadings and related AR. Additionally, I developed a limit value named minimum co-selective concentration (MCC), which is the lowest concentration of a heavy metal that can potentially cause coselection in nature. It turned out that Cu, Zn, Ni, Hg, and Cd are suspected to be the main co-selecting heavy metals in the aquatic environment. I further investigated heavy metal driven co-selection of AR in a river ecosystem, the Western Bug River (Ukraine). I found indications for co-selection of resistance to five antibiotics (ciprofloxacin, gentamicin, amikacin, tobramycin, and cefepime) and two metals (Ni and Cd) caused by Ni- and Cd-levels. Both metals exceed their MCC for water samples and Cd additionally in sediments. As a second focal point the present work emphasis on ecological interactions effecting AR evolution. Currently three possible effects of ecological interactions on AR spread are discussed. First, environmental antibiotic levels are rather low, however they might favour ARB due to a competitive advantage. The reason is that even sublethal antibiotic levels reduce the growth of sensitive bacteria while resistant cells remain unaffected by the antibiotic action. Second, predation by protozoa is believed to impact conjugation between prey bacteria (and thus the transfer of DNA and potential resistance genes) by keeping bacteria in a growing stage that favours conjugation. Third, in order to escape predation by protozoa, bacteria evolved grazing defence mechanisms such as the formation of inedible biofilms, which can feedback on the evolution of AR. With an ordinary differential equation model, I tested the effect of low antibiotic levels and losses (e.g. due to predation) on the proliferation of ARB in a modelled planktonic system. In case that the model contains the mechanism that conjugation frequencies are highest during exponential growth, I found that (i) (grazing) losses enhance conjugation frequencies between bacteria and that (ii) medium levels of antibiotics and (grazing) losses favour resistant cells in the competition to sensitive bacteria. Biofilms are thought to be \'hot spots\' for conjugation but some plasmids have lower conjugation frequencies in biofilms compared to planktonic systems. As a first step, in order to discover predation effects on plasmid spread in plankton - biofilm systems I investigated grazing resistance of bacteria in grazing experiments. Both plankton and biofilm phenotypes were consumed, when exposed to their specialized grazer (either plankton-feeder or biofilmfeeder), whereas the other phenotype remained grazing-resistant and thus became the dominant prey type. Both predators together effectively control planktonic and biofilm prey. With regards to the spread of AR-genes via conjugation, I speculate that the feeding preference of the present predator can affect the invasion success of resistance plasmids in planktonic - biofilm systems. For dynamic systems, I assume that dynamics of predator and prey traits (plankton vs. biofilm-feeder and biofilm vs. planktonic prey) will lead to dynamics of conjugation frequencies in planktonic or biofilm bacteria. I assume that conjugation events are more frequent in the dominant prey type (plankton or biofilm). However, other factors such as pili-type of the plasmid (short and rigid pili, prefers conjugation in biofilms or long and flexible pili, prefers conjugation in plankton) might additionally influence plasmid invasion success in plankton - biofilm morphotypes.

L'augmentation et la dissémination de la résistance aux antibiotiques chez les bacilles à Gram-négatif, particulièrement les Entérobactéries, les bactéries du genre Pseudomonas et Acinetobacter, représentent un problème majeur de santé publique. Les infections nosocomiales causées par les bactéries multi-résistantes ont conduit non seulement à une augmentation de la mortalité, de la morbidité et du coût de traitement, mais aussi continuent à mettre en danger la vie des patients surtout immunodéprimés. L'utilisation abusive et non contrôlée des antibiotiques a grandement contribué à la large diffusion de la résistance aux antibiotiques. Cependant, des études récentes ont démontré que cette résistance pouvait émerger à partir de sources anciennes et/ou environnementales. Ainsi, face à cette préoccupation mondiale et suite à de nombreuses recommandations, plusieurs études épidémiologiques et moléculaires ont été rapportées afin de contrôler et surveiller la diffusion et la dissémination de la résistance aux antibiotiques. Il est cependant prioritaire de développer des nouveaux outils de surveillance de la résistance aux antibiotiques. C'est dans cette optique que ce projet de thèse s'articule avec comme objectifs :- Le développement et la mise en place de nouveaux outils et logiciels de surveillance et de diagnostic des bactéries multi-résistantes, - La réalisation des études d'épidémiologie moléculaire sur les isolats cliniques de bactéries multi-résistantes responsables d'épidémies. / The increase and spread of multidrug-resistant (MDR) gram-negative bacteria especially Enterobacteriaceae, Pseudomonas, and Acinetobacter (E.P.A) species have become a major concern worldwide. The hospital-acquired infections caused by MDR bacteria have led not only to an increase in mortality, morbidity, and cost of treatment, but also continue to endanger the life of patients, especially those immunocompromised. Although, the frequent misuse of antibiotic drug has greatly contributed to worldwide dissemination of antibiotics resistance. Recent studies have shown that these resistance determinants could emerge from ancient or environmental sources. Front of this worldwide concern, and various recommendations, several epidemiological and molecular studies have been reported in order to control the spread and the dissemination of the antibiotic resistance. However, it is a priority to develop new tools for monitoring antibiotic resistance. Therefore, it is in this context that the project of this thesis was conducted with two essential objectives: -The development and implementation of news tools and software for monitoring and diagnosis of potential MDR bacteria. -The achievement of molecular epidemiology studies from clinical MDR bacteria responsible of outbreak.

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M.Sc. (Life Sciences (Microbiology))

A membrane protein VanZTei which is encoded by the gene vanZ from the vanA glycopeptide resistance gene cluster is a part of the large family of VanZ proteins. VanZTei confers resistance to teicoplanin in Enterococcus faecalis without the presence of other proteins encoded by the cluster. The aim of my work was to compare the ability of two orthologous proteins VanZTei and VanZg (from the genome of Enterococcus faecium) to confer resistance to glycopeptides in Staphylococcus aureus RN4220 and Enterococcus faecium. We have shown that VanZg increases resistance to teicoplanin (Tei) 8 to 16 times the and also to dalbavancin (Dalb) 8 times. VanZTei also confers resistance to Tei and Dalb, but the increase is only twofold. Conversely VanZTei confers resistance to newly synthetized glycopeptides more effectively than VanZg (fourfold increase of resistance confered by VanZTei and two to fourfold increase of resistance confered by VanZg). It suggests that both proteins have different specificity to antibiotics. In despite the mutants of S. aureus RN4220 VanZTei pRMC2 with increased resistance to teicoplanin (MICTei> 8 µg/ml) in which the resistance is dependent on vanZTei expression were selected. These resistant mutants do not carry mutation in a gene vanZTei or in its ribosomal binding site. Neither of the...

Durch den exzessiven Gebrauch von Antibiotika haben sich in den letzten Jahren zunehmend Resistenzen herausgebildet. Eine potentielle Alternative zu konventionellen Antibiotika sind prädatorische Bakterien. Das Bakterium Bdellovibrio bacteriovorus hat einen zweiphasigen Lebenszyklus bestehend aus einer Angriffsphase, in der es andere gram-negative Bakterien jagt, und einer Wachstumsphase, in der es das Zytoplasma eines Wirtes für die eigene Reproduktion nutzt. Für einen künftigen Einsatz von B. bacteriovorus als Antibiotikum müssen die Prozesse des Lebenszyklus verstanden werden. Das Proteom von B. bacteriovorus wurde bisher jedoch nur sehr wenig untersucht. Daher wurden in dieser Arbeit mithilfe der Massenspektrometrie Proteine von verschiedenen Zeitpunkten des Lebenszyklus von B. bacteriovorus relativ quantifiziert. Es konnten zahlreiche Proteine identifiziert werden, die zu spezifischen Zeitpunkten des Lebenszyklus hoch- oder herabreguliert werden. Die größten Unterschiede im Proteinmuster konnten zwischen der Angriffs- und der Wachstumsphase beobachtet werden. In der Angriffsphase sind einige Proteine herabreguliert, die mit der Proteinexpression im Zusammenhang stehen. Weiterhin wurde bestätigt, dass sich junge und gealterte Zellen der Angriffsphase deutlich voneinander unterscheiden, womit die Angriffsphase eigentlich aus zwei Phasen besteht. Auf Grundlage der Ergebnisse und eines Vergleiches mit Transkriptionsdaten wurde die Vermutung aufgestellt, dass B. bacteriovorus Proteine, welche spezifisch für die Angriffsphase sind, bereits während der Wachstumsphase synthetisiert. Im Zusammenhang mit der Forschung an B. bacteriovorus konnten auch neue Impulse bezüglich der MeCAT-basierten massenspektrometrischen Proteinquantifizierung angestoßen werden. In dieser Arbeit wurde unter anderem ein MeCAT-Reagenz mit Acrylamidfunktionalität entwickelt, welches erfolgreich als interner Standard für die Laserablation-ICP-MS von Polyacrylamidgelen verwendet werden kann. / Due to the excessive use of antibiotics, antibiotic resistance has increased over the last years. A potential alternative to conventional antibiotics are predatory bacteria. The predatory bacterium Bdellovibrio bacteriovorus has a biphasic life cycle consisting of an attack phase in which it hunts other gram-negative bacteria, and a growth phase in which it uses the cytoplasm of a prey cell as a substrate for its own reproduction. For future application of B. bacteriovorus as an antibiotic, it is necessary to understand the processes that occur during the life cycle. However, almost no information has been obtained regarding the proteome of B. bacteriovorus yet. Using mass spectrometry and an isotopic labelling strategy, proteins from different time points in the life cycle of B. bacteriovorus were quantified relatively to each other in this work. Numerous proteins were identified that are up- or down-regulated at specific time points in the life cycle. The largest differences in protein pattern existed between the attack phase and the growth phase, whereas only minor differences occurred within the growth phase. For instance, several proteins that appear to be down-regulated during the attack phase are related to protein expression. Furthermore, it was confirmed that there is a significant difference between young and aged cells of the attack phase. Therefore, the attack phase actually consists of two phases. Based on the results and on a comparison with transcription data, it was suggested that attack phase specific proteins of B. bacteriovorus are already synthesized during the growth phase. In connection with the research on B. bacteriovorus, new impulses regarding the MeCAT based protein quantification with mass spectrometry could be initiated. In this work, a MeCAT reagent with acrylamide functionality was developed, which can be used successfully as an internal standard for laser ablation ICP-MS of polyacrylamide gels.

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M. Sc. (Life Sciences (Microbiology))

No description available.

The present study aimed to evaluate bacterial isolates and the microbiome of dairies. The specific aims were: to characterize Staphylococcus spp. isolated from mastitic milk, to evaluate the presence of Lactococcus in mastitic milk as a potential causative agent of mastitis, to evaluate the association between microbiome and milk quality parameters, and to characterize Staphylococcus spp. isolated from production lines of Minas Frescal cheese. The detection of genes encoding virulence factors (enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, and selx), hemolysins (hla, hlb, hld, hlg, and hlgv), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin (tst)), genes encoding antibiotic resistance (resistance to tetracycline (tetK, tetL, and tetM), erythromycin (ermA, ermB, and ermC), methicillin (mecA and mecC), and tobramycin (ant(4\')-Ia)), molecular typing (spa, SCCmec, and agr types), and phenotyping regarding antibiotic resistance were performed in staphylococci isolates from mastitic milk, and from cheese processing plant samples. Staphylococcus aureus was identified in the majority of isolates from both origins. Several virulence factor genes were detected. The distribution of genes encoding staphylococcal enterotoxins (85.0% - 85.7% of isolates were positive for one or more enterotoxin gene) was highlighted and the gene related to H toxin was the most prevalent. Methicillin-resistant Staphylococcus aureus were identified in isolates from mastitic milk (4.1%) and cheese processing (6.0%); the genotyping and phenotyping of these isolates were described. t605 had the highest frequency in the S. aureus population studied. In mastitic milk, Lactococcus was suggested as the causative agent of an outbreak of mastitis in a dairy farm. Using next generation sequencing, the abundance of Lactococcus was observed in microbiome samples. Bacterial isolation and DNA sequencing confirmed the presence of Lactococcus lactis and Lactococcus garvieae. The microbiome of environmental samples and bulk tank milk from the dairy farm showed the Lactococcus genus among the most common bacterial taxa, suggesting other sources of this genus. Regarding milk quality parameters, the microbiome of bulk tank milk from several dairy farms was associated with somatic cell count and bacterial count. The core microbiome was described and many genera of importance were identified. Among the associations performed between microbiome and milk quality parameters, the identification of Streptococcus in samples classified with high somatic cell count and high bacterial count was highlighted. Several bacterial taxa with relative abundance significantly higher in samples classified as high and low cell count and bacterial count were shown. Real-time polymerase chain reaction was also performed associated with bacterial diversity, bacterial taxa, and bacterial count. These findings highlight the need to control and prevent bacterial contamination in the dairy industry, from herd to consumers. / O presente estudo apresentou como objetivo avaliar isolados bacterianos e microbioma de lácteos. Os objetivos específicos foram: caracterizar Staphylococcus spp. isolados de leite de vacas com mastite, avaliar a presença de Lactococcus em leite de vacas com mastite como um potencial agente causador de mastite, avaliar a associação entre microbioma de leite de tanque e parâmetros da qualidade de leite, e caracterizar Staphylococcus spp. isolados de linhas de processamento de queijo Minas frescal. A detecção de genes codificadores de fatores de virulência (enterotoxinas (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, e selx), hemolisinas (hla, hlb, hld, hlg, e hlgv), toxinas exfoliativas (eta, etb e etd), leucocidina de Panton-Valentine (pvl), toxina da síndrome do choque tóxico (tst)), genes codificadores de resistência a antibióticos (resistência a tetraciclina (tetK, tetL e tetM), eritromicina (ermA, ermB e ermC), meticilina (mecA e mecC) e tobramicina (ant(4\')-Ia)), tipagem molecular (spa, SCCmec e agr types), e fenotipagem quanto à resistência a antibióticos foram realizadas em estafilococos isolados de leite de vacas com mastite e de amostras de planta de processamento de queijo. Staphylococcus aureus foi identificado na maioria dos isolados de ambas as origens. Diversos genes de fatores de virulência foram detectados, com destaque para a distribuição de genes codificadores de enterotoxinas estafilocócicas (85,0%-85,7% dos isolados foram positivos para um ou mais genes codificadores de enterotoxinas), sendo o gene relacionado com a toxina H o mais frequente. Staphylococcus aureus meticilina resistente foram identificados em isolados de leite de vacas com mastite (4.1%) e em processamento de queijo (6.0%); o perfil genotípico e fenotípico destes isolados foram descritos. t605 foi o mais freqüente na população de S. aureus estudada. Em leite de vacas com mastite, Lactococcus foi sugerido como o agente causador de um surto de mastite numa fazenda leiteira. Usando sequenciamento de nova geração, a abundância de Lactococcus foi observada no microbioma das amostras. O isolamento e sequenciamento de DNA confirmaram a presença de Lactococcus lactis e Lactococcus garvieae. O microbioma de amostras ambientais e de leite de tanque da fazenda mostrou o gênero Lactococcus entre os mais comuns, sugerindo outras fontes deste gênero. Contemplando parâmetros da qualidade de leite, o microbioma de leite de tanque de várias fazendas leiteiras foi relacionado com contagem de células somáticas e contagem bacteriana. O core microbiome foi descrito e muitos gêneros bacterianos de importância foram identificados. Dentre as análises realizadas associando microbioma com parâmetros da qualidade de leite, foi destacada a identificação de Streptococcus em amostras classificadas com alta contagem de células somáticas e alta contagem bacteriana. Diversos táxons bacterianos com abundância relativa significativamente maior em amostras classificadas com alta e baixa contagem de células somáticas e contagem bacteriana foram mostrados. Reação em cadeia da polimerase em tempo real também foi realizada e associada com diversidade bacteriana, táxons bacterianos e contagem bacteriana. Estes levantamentos confirmam a necessidade de controlar e prevenir a contaminação bacteriana na indústria de lácteos, do rebanho leiteiro até os consumidores.