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Microbes live within complex communities of interacting populations, either free-living in waters and soils or symbionts of animals and plants. Their interactions include the production of antimicrobial peptides (bacteriocins) to antagonize competitors, and these producers must carry their own immunity gene for self-protection. Whether other coexisting populations are sensitive or resistant to the bacteriocin producer will be key for the population dynamics within the microbial community. The immunity gene frequently consists of an ABC transporter to repel its own bacteriocin but rarely protects against a nonrelated bacteriocin. A case where this cross-resistance occurs mediated by a shared ABC transporter has been shown between enterocins MR10A/B and AS-48. The first is an L50-like leaderless enterocin, while AS-48 is a circular enterocin. In addition, L50-like enterocins such as MR10A/B have been found in E. faecalis and E. faecium, but AS-48 appears only in E. faecalis. Thus, using the ABC transporter of the enterocin MR10A/B gene cluster of Enterococcus faecalis MRR10-3 as a cross-resistance model, we aimed to unravel to what extent a particular ABC transporter can be shared across multiple bacteriocinogenic bacterial populations. To this end, we screened the MR10A/B-ABC transporters in available microbial genomes and analyzed their sequence homologies and distribution. Overall, our main findings are as follows: (i) the MR10A/B-ABC transporter is associated with multiple enterocin gene clusters; (ii) the different enterocins associated with this transporter have a saposin-like fold in common; (iii) the Mr10E component of the transporter is more conserved within its associated enterocin, while the Mr10FGH components are more conserved within the carrying species. This is the least known component of the transporter, but it has shown the greatest specificity to its corresponding enterocin. Bacteriocins are now being investigated as an alternative to antibiotics; h

The overall theme of this dissertation is the application of mass spectrometry approaches to investigate infectious diseases. Chapter 1 contains background information regarding host-pathogen interactions, importance of clinical samples, and advances in mass spectrometry sample preparation, database curation, and data analysis techniques that familiarize the reader with the tools used during these studies. The following chapters describe primary author works completed by the author of this dissertation.Chapter 2 describes the discovery and characterization of microproteins produced by Staphylococcus aureus. The microproteins were discovered using a peptidogenomic approach and further validated with in-vitro and in-vivo assays. One of the novel microproteins is chemically and structurally similar to previously described virulence factors but appears to have a distinct mechanism of action. The other novel microprotein drives a dissociation phenotype reminiscent of exfoliative toxins. Elucidating the structural and biophysical mechanisms driving the interplay of these microproteins may provide a deeper understanding of host-pathogen interaction. Chapter 3 describes the application of multi-omics approaches to differentiate two similarly presenting enterococcal bacteremia subtypes and provides a global level view of the host immune and metabolic dysregulation. Using high resolution proteomics and metabolomics profiling of clinical serum samples we define a panel of over twenty biomarkers for further validation while also providing a number of targets of clinical importance.

Microbes live within complex communities of interacting populations, either free-living in waters and soils or symbionts of animals and plants. Their interactions include the production of antimicrobial peptides (bacteriocins) to antagonize competitors, and these producers must carry their own immunity gene for self-protection. Whether other coexisting populations are sensitive or resistant to the bacteriocin producer will be key for the population dynamics within the microbial community. The immunity gene frequently consists of an ABC transporter to repel its own bacteriocin but rarely protects against a nonrelated bacteriocin. A case where this cross-resistance occurs mediated by a shared ABC transporter has been shown between enterocins MR10A/B and AS-48. The first is an L50-like leaderless enterocin, while AS-48 is a circular enterocin. In addition, L50-like enterocins such as MR10A/B have been found in E. faecalis and E. faecium, but AS-48 appears only in E. faecalis. Thus, using the ABC transporter of the enterocin MR10A/B gene cluster of Enterococcus faecalis MRR10-3 as a cross-resistance model, we aimed to unravel to what extent a particular ABC transporter can be shared across multiple bacteriocinogenic bacterial populations. To this end, we screened the MR10A/B-ABC transporters in available microbial genomes and analyzed their sequence homologies and distribution. Overall, our main findings are as follows: (i) the MR10A/B-ABC transporter is associated with multiple enterocin gene clusters; (ii) the different enterocins associated with this transporter have a saposin-like fold in common; (iii) the Mr10E component of the transporter is more conserved within its associated enterocin, while the Mr10FGH components are more conserved within the carrying species. This is the least known component of the transporter, but it has shown the greatest specificity to its corresponding enterocin. Bacteriocins are now being investigated as an alternative to antibiotics; h

Shrimp is one of the most consumed seafood products globally. Antimicrobial drugs play an integral role in disease mitigation in shrimp and other aquaculture settings to meet production demand, but their prevalent use in these industries raises public health concerns about the emergence and spread of antimicrobial resistant microorganisms. Vibrio spp., as the most common causative agents of seafood-borne infections in humans and illness in shrimp, and Enterococcus spp., as an indicator organism, are focal bacteria of interest for the monitoring of antimicrobial resistance (AMR) in seafood. In this study, 400 samples of retail shrimp meat were collected from randomly selected grocery stores in the Greater Sacramento, California, area between September 2019 and June 2020, and were tested for Vibrio spp. and Enterococcus spp. The prevalence of Vibrio and Enterococcus in these samples were 60.25% (241/400) and 89.75% (359/400), respectively. Subsamples of confirmed Vibrio and Enterococcus isolates (n = 110 each) were subjected to antimicrobial susceptibility testing (AST) using the NARMS Gram-negative and Gram-positive drug panels, respectively. Results from AST indicated that Vibrio isolates had high phenotypic resistance to ampicillin (52/110, 47.27%) and cefoxitin (39/110, 35.45%). Enterococcus were most frequently resistant to lincomycin (106/110, 96.36%), quinupristin-dalfopristin (96/110, 87.27%), ciprofloxacin (93/110, 84.55%), linezolid (86/110, 78.18%), erythromycin (58/110, 52.73%), and chloramphenicol (43/110, 39.09%). Multidrug resistance (resistance to ≥3 drug classes) was observed in 8.18% of Vibrio isolates (9/110) and 93.64% of Enterococcus isolates (103/110). No significant differences in the prevalence of AMR (P > 0.05) were found between isolates from farm raised and wild caught shrimp (P = 1.0), nor between isolates from shrimp of domestic and imported origin (P = 1.0) in Vibrio isolates. Similarly, Enterococcus isolates from wild caught shrimp sa

The antioxidant activity of lactic acid bacteria (LAB) has been studied in depth in strains isolated from terrestrial organisms and fermented or unprocessed foods; however, information on isolates of marine origin is still being determined. This feature is of particular interest due to its potential use in functional foods in this work. The antioxidant capacity of 16 bacteriocinogenic LAB isolated from marine organisms from the Chubut coast (2010-2022 period) was studied with in vivo and in vitro assays. The in vitro antioxidant capacity was assessed by reduction of 2,2-diphenyl-1-picrylhydrazyl (DPPH), cupric ion-reducing antioxidant capacity (CUPRAC), and hydroxyl radical scavenging in unlysed cells. Saccharomyces cerevisiae was used as a eukaryotic biological model to evaluate the protective effect in vivo. The antioxidant capacity results from the DPPH radical scavenging test exhibited values between 15-37.5%, while hydroxyl radical scavenging showed values between 13.3-61.9%, and, for the CUPRAC method, values varied between 0.223-0.551, equivalent to mmol of ascorbic acid per ml of cellular suspension. The LAB protective effect on the survival rate of S. cerevisiae displayed results between 36-90%. No relationship was established between the values obtained with the different methods or between members of the same genus or species; consequently, the antioxidant behavior could be considered strain-depend. Results suggest that the antioxidant capacity of bacteriocinogenic LAB of marine origin can be used as a metabolic selection trait with biotechnological potential in the development of functional foods., A atividade antioxidante de bactérias ácido láticas (BAL) tem sido extensivamente estudada em cepas isoladas de organismos terrestres, alimentos fermentados e não processados. No entanto, esta informação sobre isolamentos de origem marinha ainda está sendo determinada. Tal característica é de particular interesse, devido ao seu potencial uso em alimentos funcionais. Neste trabalho, a capacidade antioxidante de 16 BAL bacteriocinogênicas de organismos marinhos da costa de Chubut (período 2010-2022) foi estudada através de ensaios in vitro e in vivo. A capacidade antioxidante in vitro foi avaliada pela redução de 2,2-difenil-1-picrilhidrazil (DPPH), atividade antioxidante redutora de íons cúpricos (CUPRAC) e eliminação de radicais hidroxila, usando células não lisadas. A Saccharomyces cerevisiae foi usado como modelo biológico eucariótico para avaliar o efeito protetor in vivo. Os resultados obtidos pelo sequestro dos radicais DPPH mostraram valores entre 15-37.5%; o ensaio de sequestro de radicais hidroxila apresentou valores entre 13.3-61.9% e o método CUPRAC os valores variaram entre 0.223-0.551, equivalente a mmol de ácido ascórbico por ml de suspensão celular. O efeito protetor do BAL na sobrevivência de S. cerevisiae apresentou valores entre 36-90%. Não foi possível estabelecer relações entre os valores obtidos por diferentes métodos ou entre membros do mesmo gênero ou espécie; portanto, a capacidade antioxidante pode ser considerada uma característica dependente da cepa. Os resultados sugerem que a capacidade antioxidante de BAL bacteriocinogênica de origem marinha pode ser utilizada como uma característica de seleção metabólica com potencial biotecnológico no desenvolvimento de alimentos funcionais., La actividad antioxidante de las bacterias ácido lácticas (BAL) ha sido estudiada en profundidad en cepas aisladas de organismos terrestres, alimentos fermentados y sin procesar. Sin embargo, esta información en aislamientos de origen marino aún está siendo determinada. Tal característica presenta un interés particular, debido a su uso potencial en alimentos funcionales. En este trabajo, se estudió la capacidad antioxidante de 16 BAL bacteriocinogénicas provenientes de organismos marinos de la costa del Chubut (periodo 2010-2022), mediante ensayos in vitro e in vivo. Se evaluó la capacidad antioxidante in vitro por medio de la reducción de 2,2-difenil-1-picrihidrazilo (DPPH), actividad antioxidante reductora de iones cúpricos (CUPRAC) y barrido de radicales oxidrilos, empleando células sin lisar. Se utilizó Saccharomyces cerevisiae como modelo biológico eucariota para valorar el efecto protector in vivo. Los resultados obtenidos por secuestro de radicales DPPH exhibieron valores entre 15-37.5%; el ensayo de secuestro de radicales oxidrilos mostró valores entre 13.3-61.9% y el método CUPRAC los valores variaron entre 0.223-0.551, equivalentes a mmol de ácido ascórbico por ml de suspensión celular. El efecto protector de las BAL en la supervivencia de S. cerevisiae presentó valores entre 36-90%. No fue posible establecer relaciones entre los valores obtenidos por distintos métodos o entre miembros del mismo género o especie; por ello, la capacidad antioxidante podría considerarse una característica dependiente de la cepa. Los resultados sugieren que la capacidad antioxidante de BAL bacteriocinogénicas de origen marino podría usarse como un carácter metabólico de selección con potencial biotecnológico en el desarrollo de alimentos funcionales.

Tillförsel av växtnäring på åkermark är nödvändig för matproduktionen. En återförsel av mänskliga fekalier och urin via källsorterat toalettavfall (klosettavloppsvatten) ökar cirkulationen av näring, bidrar till förbättrad jordhälsa och minskar risken för övergödning i vattendrag. Antibiotikaresistenta bakterier och andra patogener förekommer i mänsklig avföring då människor är sjuka. Därför finns en potentiell risk med användningen av klosettavloppsvatten som gödningsmedel, då det skulle kunna innebära att antibiotikaresistenta bakterier och andra patogener sprids i naturen. Inom Enterococcus spp. finns arter som bär på resistens mot bland annat antibiotikumet Vankomycin. Ett sätt att förhindra spridning av bland annat antibiotikaresistenta bakterier är om klosettvattnet hygieniseras innan det används som gödsel. Enterokocker är ett släkte av bakterier som används som indikatororganismer för att kontrollera hygieniseringsmetoders effekt. En hygieniseringsmetod är att tillsätta urea till klosettvatten, vilket via enzymet ureas bryts ned till NH3 som är toxiskt för mikroorganismer. Enterococcus spp. värde som indikatororganismer vid ureahygienisering har diskuterats, då det förekommer arter med hög tålighet mot NH3. I detta arbete undersöktes enterokockers förekomst i avloppsvatten och klosettvatten, hur många av dem som bar på vankomycinresistens, om skillnad i avdödningshastighet föreligger mellan olika enterokocker vid ureahygienisering 1% i 14oC, samt så diskuterades enterokockers värde som indikatororganismer. Odlingsmediet som användes främst var VRE-agar med och utan tillsatt Vankomycin, men det gjordes även en jämförelse med SlaBa. Vid ureahygieniseringen följdes isolat av kända stammar och stammar isolerade ur avloppsvatten vilka tillsattes i autoklaverat klosettvatten, samt naturligt förekommande stammar av enterokocker i klosettvatten och avloppsvatten. Resultaten visade att enterokocker förekommer med 45 838 cfu/ml avloppsvatten, varav 0,24% bar på resist, Fertilizing arable land is necessary for food production. Using source separated toilet waste (blackwater) as fertilizer increases the circulation of nutrients, it contributes to improved soil health and reduces the risk of eutrophication. Antibiotic-resistant bacteria and other pathogens occurs in human feces when people are sick. Thus, there is a potential risk with re-circulating blackwater as fertilizer, as it could lead to spreading of antibiotic-resistant bacteria and other pathogens in nature. Within Enterococcus spp. there are species that are resistant to the antibiotic Vancomycin. One way to prevent the spread of antibiotic-resistant bacteria and pathogens in nature is if the blackwater is sanitized before it is used as fertilizer. Enterococci is a genus of bacteria that is often used as an indicator organism to control the effect of sanitation methods. One sanitizing method is to add urea to blackwater. Urea breaks down by the enzyme urease into NH3, which is toxic to microorganisms. The value of Enterococcus spp. as indicator organisms in urea sanitation has been discussed, as there are species with a high tolerance to NH3. In this study the presence of enterococci in sewar water and blackwater was investigated, how many of them that had vancomycin resistance, whether there is a difference in the killing rate between enterococci with urea sanitization 1% in 14oC. Also, the value of enterococci as indicator organisms was discussed. The culture medium used in this study was mainly VRE agar with and without added Vancomycin, but a comparison was also made with SlaBa. The inactivation of enterococci in following solutions was analyzed during the urea sanitation: isolates of known strains of enterococci, and strains isolated from sewar water were added to autoclaved blackwater, as well as naturally occurring strains of enterococci in untreated blackwater and sewar water. The results showed that enterococci occur with 45,838 cfu/ml of sewar water, of which 0.2

The pathogenic microorganism Salmonella enterica has been associated with several outbreaks and recalls of spices, herbs, and seeds. To control these pathogens additional treatment methods, such as fumigation with chlorine dioxide (ClO2) or hydrogen peroxide (H2O2) gas and recovery methods are needed. Recovery methods should accurately quantify all viable cells, even those injured, to prevent overestimation of treatment effectiveness. This study was performed to determine the effect of different recovery media and supplements on the recovery of multiple strains of S. enterica and Enterococcus faecium NRRL B2354, from chlorine dioxide or hydrogen peroxide treated low moisture foods (LMF) black peppercorns, dried basil leaves, and chia seeds. Also, this study aimed to compare the log reduction of these two microorganisms to evaluate E. faecium NRRL B2354 as a surrogate for S. enterica. On average, recovery of S. enterica was 3.43 log and 4.77 log CFU/g from ClO2 and H2O2 treated LMFs, respectively on the selective media Xylose Lysine Deoxycholate agar, while the average recovery on non-selective media was 4.50 log CFU/g and 5.74 log CFU/g from ClO2 and H2O2 treated LMFs, respectively. The use of non-selective media was correlated with increased recovery compared to selective media. In further studies, addition of sodium pyruvate, ferrous sulfate, or 3'3'-thiodiproionate supplements to MTSAYE did not show increased recovery (P>0.05). On each treatment and LMF combination tested, there was no significant difference between the log reduction of S. enterica and E. faecium NRRL B2354, indicating its suitability as a surrogate under the test conditions.

Antibiotic resistance (AR) is a critical and looming threat to human health that requires action across the One Health continuum (humans, animals, environment). Coordinated surveillance within the environmental sector is largely underdeveloped in current National Action Plans to combat the spread of AR, and a lack of effective study approaches and standard analytical methods have led to a dearth of impactful environmental monitoring data on the prevalence and risk of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquatic environments. In this dissertation, integrated surveillance approaches of surface water and wastewater systems are demonstrated, and efforts are made towards standardizing both metagenomic- and culture-based techniques for globally comparable environmental monitoring. A field study of differentially-impacted watersheds on the island of Puerto Rico post-Hurricane Maria demonstrated the effectiveness of metagenomics in defining direct impact of anthropogenic stress and human fecal contamination on the proliferation of ARGs in riverine systems. The contribution of treated wastewater effluents to the dissemination of highly mobile and clinically-relevant ARGs and their connection to local clinical settings was also revealed. At the international scale, a transect of conventional activated sludge wastewater treatment plants (WWTPs), representing both US/European and Asian regions, were found to significantly attenuate ARG abundance through the removal of total bacterial load and human fecal indicators, regardless of influent ARG compositions. Strong structural symmetry between microbiome and ARG compositions through successional treatment stages suggested that horizontal gene transfer plays a relatively minor role in actively shaping resistomes during treatment. Risk assessment models, however, indicated high-priority plasmid-borne ARGs in final treated effluents discharged around the world, indicating potentially increased

Aims: Our objective was to determine how injectable antimicrobials affected populations of Salmonella enterica, Escherichia coli and Enterococcus spp. in feedlot cattle. Methods and Results: Two arrival date blocks of high-risk crossbred beef cattle (n = 249; mean BW = 244 kg) were randomly assigned one of four antimicrobial treatments administered on day 0: sterile saline control (CON), tulathromycin (TUL), ceftiofur (CEF) or florfenicol (FLR). Faecal samples were collected on days 0, 28, 56, 112, 182 and study end (day 252 for block 1 and day 242 for block 2). Hide swabs and subiliac lymph nodes were collected the day before and the day of harvest. Samples were cultured for antimicrobial-resistant Salmonella, Escherichia coli and Enterococcus spp. The effect of treatment varied by day across all targeted bacterial populations (p ≤ 0.01) except total E. coli. Total E. coli counts were greatest on days 112, 182 and study end (p ≤ 0.01). Tulathromycin resulted in greater counts and prevalence of Salmonella from faeces than CON at study end (p ≤ 0.01). Tulathromycin and CEF yielded greater Salmonella hide prevalence and greater counts of 128ERYR E. coli at study end than CON (p ≤ 0.01). No faecal Salmonella resistant to tetracyclines or third-generation cephalosporins were detected. Ceftiofur was associated with greater counts of 8ERYR Enterococcus spp. at study end (p ≤ 0.03). By the day before harvest, antimicrobial use did not increase prevalence or counts for all other bacterial populations compared with CON (p ≥ 0.13). Conclusions: Antimicrobial resistance (AMR) in feedlot cattle is not caused solely by using a metaphylactic antimicrobial on arrival, but more likely a multitude of environmental and management factors.

Periprostheteic joint infection (PJI) is a rare complication of arthroplasty with severe consequences for the affected patients. PJI most often necessitates additional surgery and prolonged courses of antibiotic treatment, leading to worse functional results and increased societal costs. Evaluating treatment of PJI as well as preventive efforts are essential to increase our understanding of PJI and to enable improved approaches in the future.In this thesis four studies covering different aspects of PJI are included. PJIs caused by streptococci and enterococci were investigated in papers I and II, respectively. Patients in Skåne with growth of either streptococci or enterococci in sterile cultures from prosthetic joints were included and data from the medical records were reviewed retrospectively. The aim was to describe the affected populations, surgical and antimicrobial treatments and treatment outcome. Paper I showed that streptococcal PJIs were often acute hematogenous infections treated with surgical debridement. Successful outcome was achieved in 89% of cases. Paper II showed that enterococcal PJI were often early postoperative infections in elderly fragile patients, where enterococci were found as a part of a polymicrobial flora. Overall cure was reached in 67% of cases. However, when complete cure, defined as preservation of a functional joint and eradication of infection, was the treament intention, this was achieved in 80% of cases. In paper III the effect of a national infection control programme on the incidence of PJI after primary total knee arthroplasty (TKA) was evaluated. Through matching of 45,438 primary TKAs from the Swedish Knee Arthroplasty Register to the Swedish Prescribed Drug Register, 2505 TKAs were identified as having received ≥28 days of continuous antibiotic treatment within 2 years of TKA. Subsequent review of medical records identified 644 PJIs, giving a cumulative 2 year incidence of 1.45%. PJI incidence rates were similar during bo

Fecal contamination of surface water has been associated with multiple enteric disease outbreaks and food recalls. Thus, it is important to understand factors associated with fecal contamination of agricultural water sources. Since fecal indicator bacteria (FIB) were used to monitor surface water for potential fecal contamination, the purpose of the present study was to characterize associations between environmental factors, and (i) FIB (E. coli, Enterococcus, and coliform) levels, and (ii) host-specific fecal marker detection. This study used data collected from 224 sites along 3 waterways, which spanned an urban-rural gradient around Syracuse, New York. Between 2008 and 2017, 2,816 water samples were collected, and E. coli, Enterococcus, and/or coliform concentrations were enumerated. Thirty-one samples were also tested for human and ruminant microbial source-tracking markers. Water quality (e.g., turbidity, nitrate) and weather data were also collected for each site. Univariable Bayesian regression was used to characterize the relationship between each microbial target and land use, water quality, and weather factor. For eachmodel, probability of direction and region of practical equivalence overlap (ROPE) were calculated to characterize the association’s direction and strength, respectively. While levels of different FIB were not correlated with each other, FIB levels were associated with environmental conditions. Specifically, FIB levels were also positively associated with temperature, nutrient and sediment levels. Log10 E. coli levels increased by 0.20 (CI = 0.11, 0.31) and log10 Enterococcus levels increased by 0.68 (CI=0.08, 1.24) for each log10 increase in salinity and nitrate, respectively. These findings may indicate that similar processes drove microbial, sediment, and nutrient contamination of the sampled watersheds. While fecal contamination was strongly associated with land use, the direction of association varied between FIBs and the buffer distanc

Antibiotic resistance is a major 21st century One Health (humans, animals, environment) challenge whose spread limits options to treat bacterial infections. There is growing interest in monitoring water environments, including surface water and wastewater, which have been identified as key recipients, pathways, and sources of antibiotic resistant bacteria (ARB). Aquatic environments also facilitate the transmission and amplification of ARB. Enterococcus spp. often carry clinically-important antibiotic resistance genes and are of interest as environmental monitoring targets. Enterococcus spp. are Gram-positive bacteria that are typically of fecal origin; however, they are also found in relevant environmental niches, with various species and strains that are opportunistic human pathogens. Although the value of environmental monitoring of antibiotic-resistant Enterococcus has been recognized by both national and international organizations, lack of procedural standardization has hindered generation of comparable data needed to implement integrated surveillance programs. Here we provide a comprehensive methodological review to assess the techniques used for the culturing and characterization of antibiotic-resistant Enterococcus across water matrices for the purpose of environmental monitoring. We analyzed 117 peer-reviewed articles from 33 countries across six continents. The goal of this review is to provide a critical analysis of (i) the various methods applied globally for isolation, confirmation, and speciation of Enterococcus isolates, (ii) the different methods for profiling antibiotic resistance among enterococci, and (iii) the current prevalence of resistance to clinically-relevant antibiotics among Enterococcus spp. isolated from various environments. Finally, we provide advice regarding a path forward for standardizing culturing of Enterococcus spp. for the purpose of antibiotic resistance monitoring in wastewater and wastewater-influenced waters within a glob

BackgroundChildren may be at higher risk for swimming-associated illness following exposure to fecally-contaminated recreational waters. We analyzed a pooled data set of over 80,000 beachgoers from 13 beach sites across the United States to compare risks associated with the fecal indicator bacteria Enterococcus spp. (measured by colony forming units, CFU and quantitative polymerase chain reaction cell equivalents, qPCR CE) for different age groups across different exposures, sites and health endpoints.MethodsSites were categorized according to the predominant type of fecal contamination (human or non-human). Swimming exposures of varying intensity were considered according to degree of contact and time spent in the water. Health endpoints included gastrointestinal and respiratory symptoms and skin rashes. Logistic regression models were used to analyze the risk of illness as a function of fecal contamination in water as measured by Enterococcus spp. among the exposed groups. Non-swimmers (those who did not enter the water) were excluded from the models to reduce bias and facilitate comparison across groups.ResultsGastrointestinal symptoms were the most sensitive health endpoint and strongest associations were observed with Enterococcus qPCR CE at sites impacted by human fecal contamination. Under several exposure scenarios, associations between illness and Enterococcus spp. levels were significantly higher among children compared to adolescents and adults. Respiratory symptoms were also associated with Enterococcus spp. exposures among young children at sites affected by human fecal sources, although small sample sizes resulted in imprecise estimates for these associations.ConclusionUnder many exposure scenarios, children were at higher risk of illness associated with exposure to fecal contamination as measured by the indicator bacteria Enterococcus spp. The source of fecal contamination and the intensity of swimming exposure were also important factors affecting th

ObjectiveWe compared the rates of hospital-onset secondary bacterial infections in patients with coronavirus disease 2019 (COVID-19) with rates in patients with influenza and controls, and we investigated reports of increased incidence of Enterococcus infections in patients with COVID-19.DesignRetrospective cohort study.SettingAn academic quaternary-care hospital in San Francisco, California.PatientsPatients admitted between October 1, 2019, and October 1, 2020, with a positive SARS-CoV-2 PCR (N = 314) or influenza PCR (N = 82) within 2 weeks of admission were compared with inpatients without positive SARS-CoV-2 or influenza tests during the study period (N = 14,332).MethodsNational Healthcare Safety Network definitions were used to identify infection-related ventilator-associated complications (IVACs), probable ventilator-associated pneumonia (PVAP), bloodstream infections (BSIs), and catheter-associated urinary tract infections (CAUTIs). A multiple logistic regression model was used to control for likely confounders.ResultsCOVID-19 patients had significantly higher rates of IVAC and PVAP compared to controls, with adjusted odds ratios of 4.7 (95% confidence interval [CI], 1.7-13.9) and 10.4 (95 % CI, 2.1-52.1), respectively. COVID-19 patients had higher incidence of BSI due to Enterococcus but not BSI generally, and whole-genome sequencing of Enterococcus isolates demonstrated that nosocomial transmission did not explain the increased rate. Subanalyses of patients admitted to the intensive care unit and patients who required mechanical ventilation revealed similar findings.ConclusionsCOVID-19 is associated with an increased risk of IVAC, PVAP, and Enterococcus BSI compared with hospitalized controls, which is not fully explained by factors such as immunosuppressive treatments and duration of mechanical ventilation. The mechanism underlying increased rates of Enterococcus BSI in COVID-19 patients requires further investigation.

Multidrug-resistant (MDR) Enterococcus faecalis are major causes of hospital-acquired infections. Numerous clinical strains of E. faecalis harbor a large pathogenicity island that encodes enterococcal surface protein (Esp), which is suggested to promote biofilm production and virulence, but this remains controversial. To resolve this issue, we characterized the Esp N-terminal region, the portion implicated in biofilm production. Small angle X-ray scattering indicated that the N-terminal region had a globular head, which consisted of two DEv-Ig domains as visualized by X-ray crystallography, followed by an extended tail. The N-terminal region was not required for biofilm production but instead significantly strengthened biofilms against mechanical or degradative disruption, greatly increasing retention of Enterococcus within biofilms. Biofilm strengthening required low pH, which resulted in Esp unfolding, aggregating, and forming amyloid-like structures. The pH threshold for biofilm strengthening depended on protein stability. A truncated fragment of the first DEv-Ig domain, plausibly generated by a host protease, was the least stable and sufficient to strengthen biofilms at pH ≤ 5.0, while the entire N-terminal region and intact Esp on the enterococcal surface was more stable and required a pH ≤ 4.3. These results suggested a virulence role of Esp in strengthening enterococcal biofilms in acidic abiotic or host environments.

Conjugative transfer is the most important means for spreading antibiotic resistance genes. It is used by Gram-positive and Gram-negative bacteria, and archaea as well. Conjugative transfer is mediated by molecular membrane-spanning nanomachines, so called Type 4 Secretion Systems (T4SS). The T4SS of the broad-host-range inc18-plasmid pIP501 is organized in a single operon encoding 15 putative transfer proteins. pIP501 was originally isolated from a clinical Streptococcus agalactiae strain but is mainly found in Enterococci. In this study, we demonstrate that the small transmembrane protein TraB is essential for pIP501 transfer. Complementation of a markerless pIP501∆traB knockout by traB lacking its secretion signal sequence did not fully restore conjugative transfer. Pull-downs with Strep-tagged TraB demonstrated interactions of TraB with the putative mating pair formation proteins, TraF, TraH, TraK, TraM, and with the lytic transglycosylase TraG. As TraB is the only putative mating pair formation complex protein containing a secretion signal sequence, we speculate on its role as T4SS recruitment factor. Moreover, structural features of TraB and TraB orthologs are presented, making an essential role of TraB-like proteins in antibiotic resistance transfer among Firmicutes likely., Version of record

Fecal contamination of surface water has been associated with multiple enteric disease outbreaks and food recalls. Thus, it is important to understand factors associated with fecal contamination of agricultural water sources. Since fecal indicator bacteria (FIB) were used to monitor surface water for potential fecal contamination, the purpose of the present study was to characterize associations between environmental factors, and (i) FIB (E. coli, Enterococcus, and coliform) levels, and (ii) host-specific fecal marker detection. This study used data collected from 224 sites along 3 waterways, which spanned an urban-rural gradient around Syracuse, New York. Between 2008 and 2017, 2,816 water samples were collected, and E. coli, Enterococcus, and/or coliform concentrations were enumerated. Thirty-one samples were also tested for human and ruminant microbial source-tracking markers. Water quality (e.g., turbidity, nitrate) and weather data were also collected for each site. Univariable Bayesian regression was used to characterize the relationship between each microbial target and land use, water quality, and weather factor. For eachmodel, probability of direction and region of practical equivalence overlap (ROPE) were calculated to characterize the association’s direction and strength, respectively. While levels of different FIB were not correlated with each other, FIB levels were associated with environmental conditions. Specifically, FIB levels were also positively associated with temperature, nutrient and sediment levels. Log10 E. coli levels increased by 0.20 (CI = 0.11, 0.31) and log10 Enterococcus levels increased by 0.68 (CI=0.08, 1.24) for each log10 increase in salinity and nitrate, respectively. These findings may indicate that similar processes drove microbial, sediment, and nutrient contamination of the sampled watersheds. While fecal contamination was strongly associated with land use, the direction of association varied between FIBs and the buffer distanc

Fecal contamination of surface water has been associated with multiple enteric disease outbreaks and food recalls. Thus, it is important to understand factors associated with fecal contamination of agricultural water sources. Since fecal indicator bacteria (FIB) were used to monitor surface water for potential fecal contamination, the purpose of the present study was to characterize associations between environmental factors, and (i) FIB (E. coli, Enterococcus, and coliform) levels, and (ii) host-specific fecal marker detection. This study used data collected from 224 sites along 3 waterways, which spanned an urban-rural gradient around Syracuse, New York. Between 2008 and 2017, 2,816 water samples were collected, and E. coli, Enterococcus, and/or coliform concentrations were enumerated. Thirty-one samples were also tested for human and ruminant microbial source-tracking markers. Water quality (e.g., turbidity, nitrate) and weather data were also collected for each site. Univariable Bayesian regression was used to characterize the relationship between each microbial target and land use, water quality, and weather factor. For eachmodel, probability of direction and region of practical equivalence overlap (ROPE) were calculated to characterize the association’s direction and strength, respectively. While levels of different FIB were not correlated with each other, FIB levels were associated with environmental conditions. Specifically, FIB levels were also positively associated with temperature, nutrient and sediment levels. Log10 E. coli levels increased by 0.20 (CI = 0.11, 0.31) and log10 Enterococcus levels increased by 0.68 (CI=0.08, 1.24) for each log10 increase in salinity and nitrate, respectively. These findings may indicate that similar processes drove microbial, sediment, and nutrient contamination of the sampled watersheds. While fecal contamination was strongly associated with land use, the direction of association varied between FIBs and the buffer distanc

The pathogenic microorganism Salmonella enterica has been associated with several outbreaks and recalls of spices, herbs, and seeds. To control these pathogens additional treatment methods, such as fumigation with chlorine dioxide (ClO2) or hydrogen peroxide (H2O2) gas and recovery methods are needed. Recovery methods should accurately quantify all viable cells, even those injured, to prevent overestimation of treatment effectiveness. This study was performed to determine the effect of different recovery media and supplements on the recovery of multiple strains of S. enterica and Enterococcus faecium NRRL B2354, from chlorine dioxide or hydrogen peroxide treated low moisture foods (LMF) black peppercorns, dried basil leaves, and chia seeds. Also, this study aimed to compare the log reduction of these two microorganisms to evaluate E. faecium NRRL B2354 as a surrogate for S. enterica. On average, recovery of S. enterica was 3.43 log and 4.77 log CFU/g from ClO2 and H2O2 treated LMFs, respectively on the selective media Xylose Lysine Deoxycholate agar, while the average recovery on non-selective media was 4.50 log CFU/g and 5.74 log CFU/g from ClO2 and H2O2 treated LMFs, respectively. The use of non-selective media was correlated with increased recovery compared to selective media. In further studies, addition of sodium pyruvate, ferrous sulfate, or 3'3'-thiodiproionate supplements to MTSAYE did not show increased recovery (P>0.05). On each treatment and LMF combination tested, there was no significant difference between the log reduction of S. enterica and E. faecium NRRL B2354, indicating its suitability as a surrogate under the test conditions.

Antibiotic resistance (AR) is a critical and looming threat to human health that requires action across the One Health continuum (humans, animals, environment). Coordinated surveillance within the environmental sector is largely underdeveloped in current National Action Plans to combat the spread of AR, and a lack of effective study approaches and standard analytical methods have led to a dearth of impactful environmental monitoring data on the prevalence and risk of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in aquatic environments. In this dissertation, integrated surveillance approaches of surface water and wastewater systems are demonstrated, and efforts are made towards standardizing both metagenomic- and culture-based techniques for globally comparable environmental monitoring. A field study of differentially-impacted watersheds on the island of Puerto Rico post-Hurricane Maria demonstrated the effectiveness of metagenomics in defining direct impact of anthropogenic stress and human fecal contamination on the proliferation of ARGs in riverine systems. The contribution of treated wastewater effluents to the dissemination of highly mobile and clinically-relevant ARGs and their connection to local clinical settings was also revealed. At the international scale, a transect of conventional activated sludge wastewater treatment plants (WWTPs), representing both US/European and Asian regions, were found to significantly attenuate ARG abundance through the removal of total bacterial load and human fecal indicators, regardless of influent ARG compositions. Strong structural symmetry between microbiome and ARG compositions through successional treatment stages suggested that horizontal gene transfer plays a relatively minor role in actively shaping resistomes during treatment. Risk assessment models, however, indicated high-priority plasmid-borne ARGs in final treated effluents discharged around the world, indicating potentially increased

This study focuses on the impact of infiltration rates on colloidal transport and reactive processes associated with E. faecalis JH2-2 using water-saturated sediment columns. The infiltration rates influence the physical transport of bacteria by controlling the mean flow velocity. This, in turn, impacts biological processes in pore water owing to the higher or lower residence time of the bacteria in the column. In the present study, continuous injection of E. faecalis (suspended in saline water with varying conditions of dissolved oxygen and nutrient concentrations) into a lab-scale sediment column was performed at flow velocities of 0.02 cm min−1 and 0.078 cm min−1, i.e., at residence times of 1–5 hours. The impact of residence times on reactive processes is significant for field scale setups. A process-based model with a first-order rate coefficient for each biological process was fitted for each obtained condition-specific dataset from the experimental observations (breakthrough curves). The coefficients were converted to a dimensionless form to facilitate the comparison of biological processes. These results indicate that the processes of attachment and growth were flow-dependent. The growth process in the absence of dissolved oxygen was the most dominant process, with a Damkoehler number of approximately 48.

Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless single-stranded DNA recombineering to generate specific gene-editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic-selectable markers to generate gene deletion mutants. As a further proof of principle, we use CRISPR-Cas9-mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. IMPORTANCE Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to

The antitumor efficacy of cancer immunotherapy can correlate with the presence of certain bacterial species within the gut microbiome. However, many of the molecular mechanisms that influence host response to immunotherapy remain elusive. In this study, we show that members of the bacterial genus Enterococcus improve checkpoint inhibitor immunotherapy in mouse tumor models. Active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in nonprotective E. faecalis was sufficient to promote immunotherapy response, and its activity required the peptidoglycan sensor NOD2. Notably, SagA-engineered probiotics or synthetic muropeptides also augmented anti-PD-L1 antitumor efficacy. Taken together, our data suggest that microbiota species with specialized peptidoglycan remodeling activity and muropeptide-based therapeutics may enhance cancer immunotherapy and could be leveraged as next-generation adjuvants.

Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyze the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus species. MRSE (n = 17) and Enterococcus species (Enterococcus faecalis n = 8 and Enterococcus faecium n = 10) strains were used. MIC of the mixture of DTAM, HTAM, and ENDX and that of vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and of vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays. MIC90 of tamoxifen metabolites was 1 mg/liter for MRSE strains and 2 mg/liter for E. faecalis and E. faecium strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 4× MIC for MRSE (SE385 and SE742) and at 2× MIC and 4× MIC for E. faecalis (EVR1 and EVR2) strains, respectively. SE385 and EVR2 strains treated with the tamoxifen metabolites mixture presented higher membrane permeabilization. Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drug treatments against these bacterial pathogens.

The objectives of this study were to characterize overall genomic antibiotic resistance profiles of fecal Escherichia coli and Enterococcus spp. from dairy cattle at different production stages using whole-genome sequencing and to determine the association between antimicrobial resistance (AMR) phenotypes and their corresponding genotypes. The Comprehensive Antibiotic Resistance Database (CARD) and ResFinder, two publicly available databases of antimicrobial resistance genes, were used to annotate isolates. Based on the ResFinder database, 27.5% and 20.0% of tested E. coli isolates (n = 40) harbored single and ≥3 antimicrobial resistance genes, respectively; for Enterococcus spp., we observed 87.8% and 8.2%, respectively. The highest prevalence of AMR genes in E. coli was for resistance to tetracycline (27.5%), followed by sulphonamide (22.5%) and aminoglycoside (20.0%); the predominant antimicrobial resistance genes in Enterococcus spp. targeted macrolide drugs (77.6%). Based on the CARD database, resistance to ≥3 antimicrobial classes was observed in all E. coli and 77.6% in Enterococcus spp. isolates. A high degree of agreement existed between the resistance phenotype and the presence of resistance genes for various antimicrobial classes for E. coli but much less so for isolates of Enterococcus. Consistent with prior work, fecal E. coli and Enterococcus spp. isolates from calves harbored a wide spectrum of resistance genes, compared to those from cattle at other production stages, based on the cross-sectional samples from the studied farm.

BackgroundUnderstanding the effects of herd management practices on the prevalence of multidrug-resistant pathogenic Salmonella and commensals Enterococcus spp. and Escherichia coli in dairy cattle is key in reducing antibacterial resistant infections in humans originating from food animals. Our objective was to explore the herd and cow level features associated with the multi-drug resistant, and resistance phenotypes shared between Salmonella, E. coli and Enterococcus spp. using machine learning algorithms.MethodsRandomly collected fecal samples from cull dairy cows from six dairy farms in central California were tested for multi-drug resistance phenotypes of Salmonella, E. coli and Enterococcus spp. Using data on herd management practices collected from a questionnaire, we built three machine learning algorithms (decision tree classifier, random forest, and gradient boosting decision trees) to predict the cows shedding multidrug-resistant Salmonella and commensal bacteria.ResultsThe decision tree classifier identified rolling herd average milk production as an important feature for predicting fecal shedding of multi-drug resistance in Salmonella or commensal bacteria. The number of culled animals, monthly culling frequency and percentage, herd size, and proportion of Holstein cows in the herd were found to be influential herd characteristics predicting fecal shedding of multidrug-resistant phenotypes based on random forest models for Salmonella and commensal bacteria. Gradient boosting models showed that higher culling frequency and monthly culling percentages were associated with fecal shedding of multidrug resistant Salmonella or commensal bacteria. In contrast, an overall increase in the number of culled animals on a culling day showed a negative trend with classifying a cow as shedding multidrug-resistant bacteria. Increasing rolling herd average milk production and spring season were positively associated with fecal shedding of multidrug- resistant Salmonella

Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyze the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus species. MRSE (n = 17) and Enterococcus species (Enterococcus faecalis n = 8 and Enterococcus faecium n = 10) strains were used. MIC of the mixture of DTAM, HTAM, and ENDX and that of vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and of vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays. MIC90 of tamoxifen metabolites was 1 mg/liter for MRSE strains and 2 mg/liter for E. faecalis and E. faecium strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 4× MIC for MRSE (SE385 and SE742) and at 2× MIC and 4× MIC for E. faecalis (EVR1 and EVR2) strains, respectively. SE385 and EVR2 strains treated with the tamoxifen metabolites mixture presented higher membrane permeabilization. Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drug treatments against these bacterial pathogens.

Enterolert-E is an easy-to-use method for the enumeration of enterococci in water samples as an indicator of fecal pollution. This most probable number technique replaced the laborious and more time-consuming MEA-BEA plating method, and it is used extensively in ballast water testing and monitoring. In spring 2018, the Control Union Water ballast water test facility measured high enterococci concentrations in Wadden Sea water without any correlation with polluted freshwater input. By isolating bacteria from samples incubated in Enterolert-E culture medium, followed by analyses of colony morphology and DNA, it is shown that these erroneously high concentrations were caused by Bacillus licheniformis, a gram-positive rod-shaped chlorine-resistant bacterium. It is concluded that control analyses or the MEA-BEA method or dilution to reduce salinity must be performed when high enterococci concentrations are measured in water samples that are not suspected to be polluted.

Introduction: The optimal duration of systemic antimicrobial treatment for catheter-related bloodstream infections (CRBSI) is unknown. In this systematic review, we aimed to assess the efficacy of short-course treatment for CRBSI due to Gram-negative bacteria, coagulase-negative staphylococci and enterococci. Methods: We systematically searched the electronic bibliographic databases MEDLINE, EMBASE and Cochrane Library for studies published before February 2021. All studies that investigated the duration of adequate systemic antibiotic treatment in adult patients with uncomplicated intravascular catheter infections due to Gram-negative bacteria, coagulase-negative staphylococci or enterococci were eligible for inclusion. Studies including concomitant treatment with antibiotic lock therapy were excluded. The primary outcomes were clinical failure/cure, mortality and microbiologic-confirmed relapse. Results: Seven retrospective cohort studies and one case-cohort study met the inclusion criteria. No randomized controlled studies met inclusion criteria. The quality of the included studies was low (n = 7) to moderate (n = 1). No significant differences were observed regarding mortality and microbiological relapse between short-course and long-course systemic antibiotic treatment in patients with CRBSI due to coagulase-negative staphylococci or Gram-negative bacteria. No association was found between mortality and treatment duration in the two studies assessing enterococcal CRBSI. Conclusion: The limited data available suggests that shorter systemic antibiotic treatment duration may be sufficient for uncomplicated CRBSI. Further well-designed prospective studies are needed to confirm these findings. Trial Registration Number: CRD42021224946 (PROSPERO).

Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyze the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus species. MRSE (n = 17) and Enterococcus species (Enterococcus faecalis n = 8 and Enterococcus faecium n = 10) strains were used. MIC of the mixture of DTAM, HTAM, and ENDX and that of vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and of vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays. MIC90 of tamoxifen metabolites was 1 mg/liter for MRSE strains and 2 mg/liter for E. faecalis and E. faecium strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 4× MIC for MRSE (SE385 and SE742) and at 2× MIC and 4× MIC for E. faecalis (EVR1 and EVR2) strains, respectively. SE385 and EVR2 strains treated with the tamoxifen metabolites mixture presented higher membrane permeabilization. Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drug treatments against these bacterial pathogens.

Enterolert-E is an easy-to-use method for the enumeration of enterococci in water samples as an indicator of fecal pollution. This most probable number technique replaced the laborious and more time-consuming MEA-BEA plating method, and it is used extensively in ballast water testing and monitoring. In spring 2018, the Control Union Water ballast water test facility measured high enterococci concentrations in Wadden Sea water without any correlation with polluted freshwater input. By isolating bacteria from samples incubated in Enterolert-E culture medium, followed by analyses of colony morphology and DNA, it is shown that these erroneously high concentrations were caused by Bacillus licheniformis, a gram-positive rod-shaped chlorine-resistant bacterium. It is concluded that control analyses or the MEA-BEA method or dilution to reduce salinity must be performed when high enterococci concentrations are measured in water samples that are not suspected to be polluted.

To evaluate the resistance to fluoroquinolones in clinical isolates of Gram-positive cocci records of processed culture al the Bacteriological Reference Center of the Autonomous Service University Hospital of Maracaibo during the period January 2011-December 2015, were reviewed. The data was analyzed using WHONETTM software (version 5.6) and IBM® SPSS® Statistics for Windows (version 24). A prevalence of 29.70% was found for Gram-positive cocci (9,292 strains), with 76.18% of isolates corresponding to the genus Staphylococcus (7,072); 15.30% to Enterococcus (1422) and 8.59% to Streptococcus (798). For Staphylococcus, resistance was higher in methicillin-resistant strains. The highest resistance rates were detected in enterococci, especially vancomycin-resistant Enterococcus faecium. No resistance to fluoroquinolones was detected in vancomycin-resistant Enterococcus faecalis strains. Streptococci, including Streptococcus pneumoniae, were mostly sensitive to fluoroquinolones. Most strains of staphylococci and enterococci; presented cross resistance to all tested fluoroquinolones. The distribution of resistance to fluoroquinolones per year of study in each bacterial genus was different. Resistance to fluoroquinolones shows an increasing trend in the three genera of Gram-positive cocci evaluated, Para evaluar la resistencia a fluoroquinolonas en aislamientos clínicos de cocos grampositivos se revisaron los resultados de los cultivos procesados en el Centro de Referencia Bacteriológica del Servicio Autónomo Hospital Universitario de Maracaibo, durante el periodo enero 2011-diciembre 2015. Los datos fueron analizados mediante el software WHONETTM, (versión 5,6) y el IBM® SPSS® Statistics para Windows, (versión 25). Se encontró una prevalencia de 29,70% para los cocos grampositivos (9.292 cepas), correspondiendo el 76,18% de los aislamientos al género Staphylococcus (7.072); 15,30% a Enterococcus (1.422) y 8,59% a Streptococcus (798). Para Staphylococcus, la resistencia fue mayor en cepas resistentes a meticilina. Las tasas de resistencia más elevadas se detectaron en los enterococos, especialmente en Enterococcus faecium resistente a vancomicina. No se detectó resistencia a fluoroquinolonas en las cepas de Enterococcus faecalis resistentes a vancomicina. Los estreptococos, incluyendo Streptococcus pneumoniae, se mostraron, mayormente, sensibles a las fluoroquinolonas. La mayoría de las cepas de estafilococos y enterococos; presentó, resistencia cruzada a todas las fluoroquinolonas probadas. La distribución de la resistencia a las fluoroquinolonas por año de estudio en cada género bacteriano fue diferente. La resistencia a las fluoroquinolonas muestra una tendencia creciente en los tres géneros de cocos grampositivos evaluados

In this study, the antimicrobial effect of exopolysaccharide (EPS) extracted from Enterococcus strains [E. durans K48 (MT437,248), E. faecium R114 (MT437,249) and E. faecium T52 (MT437,250)] isolated from Kishk was applied against some foodborne pathogenic bacteria using well diffusion and microdilution methods. The antioxidant activity of EPS was also evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay and ferric reducing antioxidant power (FRAP) method. The cytotoxicity effect of EPS on human Gingival Fibroblast (HGF) cell line was also assessed. The results obtained by antimicrobial test showed that the most resistant bacteria to the examined EPS was Listeria monocytogenes, and the most susceptible were Staphylococcus aureus and E. faecalis. The results showed that the DPPH inhibitory percentage of EPS (25 mg/mL) from E. durans K48, E. faecium R114, and E. faecium T52 was 53%, 58% and 64%, respectively. EPS from E. faecium T52 displayed the highest reducing power, but statistically, there was no significant difference between the reducing power of EPS T52 and EPS R114 (P ≥ 0.05). The lowest toxicity percentage of EPS k48, EPS T52, and EPS R114 on normal human cell line at a concentration of 0.2 mg/mL was 10%, 15%, and 13%, respectively, which was statistically significant (P < 0.05). The obtained results in the present study indicate that EPS from the examined LAB strains with no in vitro cytotoxicity can be a potential source of natural antioxidant and antibacterial agent to be used in food and pharmaceutical industries.

Background. Periprosthetic joint infection (PJI) is a devastating complication of joint replacement surgery. Most observational studies of PJI are retrospective or single-center, and reported management approaches and outcomes vary widely. We hypothesized that there would be substantial heterogeneity in PJI management and that most PJIs would present as late acute infections occurring as a consequence of bloodstream infections. Methods. The Prosthetic joint Infection in Australia and New Zealand, Observational (PIANO) study is a prospective study at 27 hospitals. From July 2014 through December 2017, we enrolled all adults with a newly diagnosed PJI of a large joint. We collected data on demographics, microbiology, and surgical and antibiotic management over the first 3 months postpresentation. Results. We enrolled 783 patients (427 knee, 323 hip, 25 shoulder, 6 elbow, and 2 ankle). The mode of presentation was late acute (30 days postimplantation and 7 days of symptoms; 351, 45%), followed by early (30 days postimplantation; 196, 25%) and chronic (30 days postimplantation with 30 days of symptoms; 148, 19%). Debridement, antibiotics, irrigation, and implant retention constituted the commonest initial management approach (565, 72%), but debridement was moderate or less in 142 (25%) and the polyethylene liner was not exchanged in 104 (23%). Conclusions. In contrast to most studies, late acute infection was the most common mode of presentation, likely reflecting hematogenous seeding. Management was heterogeneous, reflecting the poor evidence base and the need for randomized controlled trials.Copyright © 2020 Oxford University Press. All rights reserved.

Background. Periprosthetic joint infection (PJI) is a devastating complication of joint replacement surgery. Most observational studies of PJI are retrospective or single-center, and reported management approaches and outcomes vary widely. We hypothesized that there would be substantial heterogeneity in PJI management and that most PJIs would present as late acute infections occurring as a consequence of bloodstream infections. Methods. The Prosthetic joint Infection in Australia and New Zealand, Observational (PIANO) study is a prospective study at 27 hospitals. From July 2014 through December 2017, we enrolled all adults with a newly diagnosed PJI of a large joint. We collected data on demographics, microbiology, and surgical and antibiotic management over the first 3 months postpresentation. Results. We enrolled 783 patients (427 knee, 323 hip, 25 shoulder, 6 elbow, and 2 ankle). The mode of presentation was late acute (30 days postimplantation and 7 days of symptoms; 351, 45%), followed by early (30 days postimplantation; 196, 25%) and chronic (30 days postimplantation with 30 days of symptoms; 148, 19%). Debridement, antibiotics, irrigation, and implant retention constituted the commonest initial management approach (565, 72%), but debridement was moderate or less in 142 (25%) and the polyethylene liner was not exchanged in 104 (23%). Conclusions. In contrast to most studies, late acute infection was the most common mode of presentation, likely reflecting hematogenous seeding. Management was heterogeneous, reflecting the poor evidence base and the need for randomized controlled trials.Copyright © 2020 Oxford University Press. All rights reserved.

The early life gut microbiota has been reported to be involved in neonatal weight gain and later infant growth. Therefore, this early microbiota may constitute a target for the promotion of healthy neonatal growth and development with potential consequences for later life. Unfortunately, we are still far from understanding the association between neonatal microbiota and weight gain and growth. In this context, we evaluated the relationship between early microbiota and weight in a cohort of full-term infants. The absolute levels of specific fecal microorganisms were determined in 88 vaginally delivered and 36 C-section-delivered full-term newborns at 1 month of age and their growth up to 12 months of age. We observed statistically significant associations between the levels of some early life gut microbes and infant weight gain during the first year of life. Classifying the infants into tertiles according to their Staphylococcus levels at 1 month of age allowed us to observe a significantly lower weight at 12 months of life in the C-section-delivered infants from the highest tertile. Univariate and multivariate models pointed out associations between the levels of some fecal microorganisms at 1 month of age and weight gain at 6 and 12 months. Interestingly, these associations were different in vaginally and C-section-delivered babies. A significant direct association between Staphylococcus and weight gain at 1 month of life was observed in vaginally delivered babies, whereas in C-section-delivered infants, lower Bacteroides levels at 1 month were associated with higher later weight gain (at 6 and 12 months). Our results indicate an association between the gut microbiota and weight gain in early life and highlight potential microbial predictors for later weight gain.

The objectives of this study were to characterize overall genomic antibiotic resistance profiles of fecal Escherichia coli and Enterococcus spp. from dairy cattle at different production stages using whole-genome sequencing and to determine the association between antimicrobial resistance (AMR) phenotypes and their corresponding genotypes. The Comprehensive Antibiotic Resistance Database (CARD) and ResFinder, two publicly available databases of antimicrobial resistance genes, were used to annotate isolates. Based on the ResFinder database, 27.5% and 20.0% of tested E. coli isolates (n = 40) harbored single and ≥3 antimicrobial resistance genes, respectively; for Enterococcus spp., we observed 87.8% and 8.2%, respectively. The highest prevalence of AMR genes in E. coli was for resistance to tetracycline (27.5%), followed by sulphonamide (22.5%) and aminoglycoside (20.0%); the predominant antimicrobial resistance genes in Enterococcus spp. targeted macrolide drugs (77.6%). Based on the CARD database, resistance to ≥3 antimicrobial classes was observed in all E. coli and 77.6% in Enterococcus spp. isolates. A high degree of agreement existed between the resistance phenotype and the presence of resistance genes for various antimicrobial classes for E. coli but much less so for isolates of Enterococcus. Consistent with prior work, fecal E. coli and Enterococcus spp. isolates from calves harbored a wide spectrum of resistance genes, compared to those from cattle at other production stages, based on the cross-sectional samples from the studied farm.

BackgroundUnderstanding the effects of herd management practices on the prevalence of multidrug-resistant pathogenic Salmonella and commensals Enterococcus spp. and Escherichia coli in dairy cattle is key in reducing antibacterial resistant infections in humans originating from food animals. Our objective was to explore the herd and cow level features associated with the multi-drug resistant, and resistance phenotypes shared between Salmonella, E. coli and Enterococcus spp. using machine learning algorithms.MethodsRandomly collected fecal samples from cull dairy cows from six dairy farms in central California were tested for multi-drug resistance phenotypes of Salmonella, E. coli and Enterococcus spp. Using data on herd management practices collected from a questionnaire, we built three machine learning algorithms (decision tree classifier, random forest, and gradient boosting decision trees) to predict the cows shedding multidrug-resistant Salmonella and commensal bacteria.ResultsThe decision tree classifier identified rolling herd average milk production as an important feature for predicting fecal shedding of multi-drug resistance in Salmonella or commensal bacteria. The number of culled animals, monthly culling frequency and percentage, herd size, and proportion of Holstein cows in the herd were found to be influential herd characteristics predicting fecal shedding of multidrug-resistant phenotypes based on random forest models for Salmonella and commensal bacteria. Gradient boosting models showed that higher culling frequency and monthly culling percentages were associated with fecal shedding of multidrug resistant Salmonella or commensal bacteria. In contrast, an overall increase in the number of culled animals on a culling day showed a negative trend with classifying a cow as shedding multidrug-resistant bacteria. Increasing rolling herd average milk production and spring season were positively associated with fecal shedding of multidrug- resistant Salmonella

Enterococcus lactis and the heterotypic synonym Enterococcus xinjiangensis from dairy origin have recently been identified as a novel species based on 16S rRNA gene sequence analysis. Enterococcus faecium type strain NCTC 7171T was used as the reference genome for determining E. lactis and E. faecium to be separate species. However, this taxonomic classification did not consider the diverse lineages of E. faecium, and the double nature of hospital-associated (clade A) and community-associated (clade B) isolates. Here, we investigated the taxonomic relationship among isolates of E. faecium of different origins and E. lactis, using a genome-based approach. Additional to 16S rRNA gene sequence analysis, we estimated the relatedness among strains and species using phylogenomics based on the core pangenome, multilocus sequence typing, the average nucleotide identity and digital DNA–DNA hybridization. Moreover, following the available safety assessment schemes, we evaluated the virulence profile and the ampicillin resistance of E. lactis and E. faecium clade B strains. Our results confirmed the genetic and evolution-ary differences between clade A and the intertwined clade B and E. lactis group. We also confirmed the absence in these strains of virulence gene markers IS16, hylEfm and esp and the lack of the PBP5 allelic profile associated with ampicillin resistance. Taken together, our findings support the reassignment of the strains of E. faecium clade B as E. lactis.

The objectives of this study were to characterize overall genomic antibiotic resistance profiles of fecal Escherichia coli and Enterococcus spp. from dairy cattle at different production stages using whole-genome sequencing and to determine the association between antimicrobial resistance (AMR) phenotypes and their corresponding genotypes. The Comprehensive Antibiotic Resistance Database (CARD) and ResFinder, two publicly available databases of antimicrobial resistance genes, were used to annotate isolates. Based on the ResFinder database, 27.5% and 20.0% of tested E. coli isolates (n = 40) harbored single and ≥3 antimicrobial resistance genes, respectively; for Enterococcus spp., we observed 87.8% and 8.2%, respectively. The highest prevalence of AMR genes in E. coli was for resistance to tetracycline (27.5%), followed by sulphonamide (22.5%) and aminoglycoside (20.0%); the predominant antimicrobial resistance genes in Enterococcus spp. targeted macrolide drugs (77.6%). Based on the CARD database, resistance to ≥3 antimicrobial classes was observed in all E. coli and 77.6% in Enterococcus spp. isolates. A high degree of agreement existed between the resistance phenotype and the presence of resistance genes for various antimicrobial classes for E. coli but much less so for isolates of Enterococcus. Consistent with prior work, fecal E. coli and Enterococcus spp. isolates from calves harbored a wide spectrum of resistance genes, compared to those from cattle at other production stages, based on the cross-sectional samples from the studied farm.

BackgroundHigh doses of ampicillin are often used to achieve therapeutic drug concentrations in infants. A paradoxical antibiotic effect, often called the Eagle effect, occurs when increasing concentrations of antibiotic above a threshold results in decreased efficacy. It is unknown if infants treated with ampicillin are at risk for this paradoxical effect.MethodsWe identified infants <28 days of age with Escherichia coli, Enterococcus or Streptococcus agalactiae (group B streptococcus) bloodstream infections from 1997 to 2012 and previously included in an ampicillin pharmacokinetic (PK) modeling study. We compared the odds of death for ampicillin dose, estimated time above the minimum inhibitory concentration (T > MIC) and PK parameters using separate logistic regression models. Adjusted logistic regression and Poisson models were used to calculate the odds of prolonged bacteremia ≥3 days and the duration of bacteremia, respectively, for dose, T > MIC and multiple PK parameters.ResultsAmong 1272 infants meeting inclusion criteria, odds of death 7 or 30 days after the positive blood culture were not consistent with a paradoxical effect across any of the dosing regimens or PK parameters evaluated. The odds of prolonged bacteremia was lowest at the lowest dose category and the lowest daily dose category but not associated with the area-under-the-concentration time curve from 0 to 24 hours, or the maximum or minimum concentrations at steady state. T > MIC of ≥50% of the dosing interval was associated with decreased duration of bacteremia and odds of prolonged bacteremia.ConclusionsIt is unlikely that a paradoxical antibiotic effect will have a clinical correlate when ampicillin is used for neonatal bacteremia. A T > MIC ≥50% decreased both duration of bacteremia and odds of prolonged bacteremia.

As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (nontraditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination, and crop contamination risk (Escherichia coli, total coliforms [TC], Enterococcus, and Aeromonas). Sites included nontidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW), and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. E. coli, TC, enterococci (∼86%, 98%, and 90% positive, respectively; n = 333), and Aeromonas (∼98% positive; n = 133) were widespread in water samples tested. Highest E. coli counts were in rivers, TC counts in TB, and enterococci in rivers and ponds (P < 0.001 in all cases) compared to other water types. Aeromonas counts were consistent across sites. Seasonal dynamics were detected in NF and PW samples only. E. coli counts were higher in the vegetable crop-growing (May-October) than nongrowing (November-April) season in all water types (P < 0.05). Only one RW and both PW sites met the U.S. Food Safety Modernization Act water standards. However, implementation of recommended mitigation measures of allowing time for microbial die-off between irrigation and harvest would bring all other sites into compliance within 2 days. This study provides comprehensive microbial data on alternative irrigation water and serves as an important resource for food safety planning and policy setting.IMPORTANCE Increasing demands for fresh fruit and vegetables, a variable climate affecting agricultural water availability, and microbial food safety goals are pressing the need to identify new, safe, alternative sources of irrigation water. Our study

Introduction. The appearance of resistant bacteria is a natural phenomenon that has been potentiated by the inappropriate use of antibiotics and self-medication, which in turn has contributed to the appearance of resistant strains of Enterobacter and Enterococcus. As a result, a significant increase in health care-associated infections, morbidity and mortality rates, and health system costs has been observed. The objective was to analyze health care-associated infections, particularly bacteremia, urinary tract infection, endocarditis and meningitis, caused by resistant Enterobacter and Enterococcus strains, in Hispanic America, between the years 2002 and 2017. Materials and methods. A bibliographic search was performed in Elsevier and SciELO, using Health Sciences Descriptors (DeCS). Twenty-six review and original articles that met the inclusion and exclusion criteria were analyzed. Results. Bacteremia was the most common infection in 30.4% of the cases. It is worth noting the limited information found regarding endocarditis and meningitis. Enterococcusfaecalis and Enterococcus faecium represented 90% of the clinical isolates. Antibiotic resistance continues to rise steadily, especially by Enterococcus faecalis. Conclusion. Health care-associated infections caused by bacteria such as Enterococcus faecalis, Enterococcus faecium, Enterobacter cloacae, and Enterobacter aerogenes are common, although a moderate amount of information is available. An effective approach to this problem in the health care system is necessary for their proper control.control., Introducción. La aparición de bacterias resistentes es un fenómeno natural que ha sido potencializado por el uso inapropiado de antibióticos y por la automedicación, lo cual ha contribuido con la aparición de cepas resistentes de Enterobacter y Enterococcus, ocasionando a su vez un incremento significativo en las infecciones asociadas a la atención en salud (IAAS), a las tasas de morbimortalidad y a los costos del sistema de salud. El objetivo fue analizar las IAAS, enfatizando en bacteriemia, infección del tracto urinario, endocarditis y meningitis, causadas por cepas de Enterobacter y Enterococcus resistentes a los antimicrobianos, en Hispanoamérica, durante los años 2002 a 2017. Materiales y métodos. Se realizó una búsqueda bibliográfica en Elsevier y SciELO, empleando términos Descriptores en Ciencias de la Salud (DeCS). Se analizaron 26 artículos de revisión y originales que cumplían con los criterios de inclusión y exclusión. Resultados. La principal infección fue la bacteriemia en un 30,4%, y es de resaltar la escasa información encontrada acerca de endocarditis y meningitis. Enterococcus faecalis y Enterococcus faecium representaron el 90% de los aislamientos clínicos. La resistencia a los antibióticos continúa en ascenso constante, especialmente por parte de Enterococcus faecalis. Conclusión. Las IAAS producidas por microorganismos como Enterococcus faecalis, Enterococcus faecium, Enterobacter cloacae y Enterobacter aerogenes son frecuentes, aunque se cuenta con una cantidad moderada de información. El abordaje conceptual de esta problemática en el área de salud, puede ser una herramienta idónea para su control.

The virulent lactococcal phage P087 was isolated from a dairy environment in 1978. This phage was then recognized as the reference member for one of the ten phage groups currently known to infect Lactococcus lactis strains. The double-stranded DNA genome of this Siphoviridae phage is composed of 60,074 bp and is circularly permuted. Five tRNA and 88 orfs were found within an uncommon genome architecture. Eleven structural proteins were also identified through SDS-PAGE and LC-MS/MS analyses. Of note, 11 translated orfs from the structural module of phage P087 have identities to gene products found in a prophage located in the genome of Enterococcus faecalis V583. The alignment of both genomic sequences suggests that DNA exchanges could occur between these two phages which are infecting low G+C bacteria found in similar ecological niches.

In sub-Saharan Africa, many families travel to collect water and store it in their homes for daily use, presenting an opportunity for the introduction of fecal contamination. One stored and one source water sample were each collected from 45 households in rural Kenya. All 90 samples were analyzed for fecal indicator bacteria (E. coli and enterococci) and species-specific contamination using molecular microbial source tracking assays. Human (HF183), avian (GFD), and ruminant (BacR) contamination were detected in 52, two, and four samples, respectively. Stored water samples had elevated enterococci concentrations (p < 0.01, Wilcoxon matched pairs test) and more frequent BacR detection (89% versus 27%, p < 0.01, McNemar's exact test) relative to source water samples. fsQCA (fuzzy set qualitative comparative analysis) was conducted on the subset of households with no source water BacR contamination to highlight combinations of factors associated with the introduction of BacR contamination to stored water supplies. Three combinations were identified: (i) ruminants in the compound, safe water extraction methods, and long storage time, (ii) ruminants, unsafe water extraction methods, and no soap at the household handwashing station, and (iii) long storage time and no soap. This suggests that multiple pathways contribute to the transmission of ruminant fecal contamination in this context, which would have been missed if data were analyzed using standard regression techniques.

CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9-Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR-Cas9 inhibition.

In order to assess the possible impact of the marine outfall of the city of Limón on the surrounding marine environment, six sampling stations were established, four near the vent zone and two controls. Water samples were taken in each station 0m and 10m deep during 2012- 2013 to measure nutrients, physicochemical parameters, microalgae concentration, and colony forming units (CFU) of Enterococcus. Microalgae abundance was compared between stations under outfall influence, their controls and depths using an analysis of similarities (ANOSIM). A SIMPER analysis was also conducted to determine species when significant differences were found. Phytoplankton abundance was also used to rank the dominance of species using the Olmstead-Tukey diagram and a canonical correspondence analysis. Results showed that in the area, there is a marked influence of rainfall on nutrients, silicate and orthophosphate, in addition to parameters such as dissolved oxygen and salinity. The dissolved oxygen concentration was high, reflecting that there is no significant organic load in the environment, while CFUs remained in ranges well below the maximum limits (35 UFC/100 mL) set by the United States Environmental Protection Agency (USEPA). Phytoplankton diversity was high and composed mostly of cosmopolitan species. Due to the low nutrient concentration and CFU counts, together with the high microalgae diversity, it is concluded that there is no direct evidence of a negative impact of the marine outfall on the studied area., Com o objetivo de avaliar o possível impacto do emissário submarino na cidade de Limón em relação ao ambiente marítimo circundante, foram estabelecidas 6 estações de amostragem, 4 delas próximas à zona de desague e 2 controles; em cada uma foram coletadas amostras de água a 0 m e 10 m durante os anos 2012-2013 para medir nutrimentos, parâmetros físico-químicos, concentração de microalgas e quantidade de unidades formadoras de colônias (UFC) de Enterococcus. Foram contrastadas: a abundância de algas, as variáveis físico-químicas e ambientais entre as estações sob a influência do emissário, seus controles e entre as profundidades, com uma análise de semelhanças (ANOSIM); nos casos de significativa discrepância, foi realizada, adicionalmente, uma análise SIMPER para determinar as variáveis responsáveis. A abundância fitoplanctônica foi usada para hierarquizar a dominância de espécies por meio do diagrama de Olmstead e Tukey, e incorporá-las em uma análise de correspondência canônica. Determinou-se que na zona existe uma marcada influência das chuvas sobre a concentração de nutrimentos, silicato e ortofosfato, além de parâmetros como o oxigênio dissolvido e a salinidade. As UFC mantiveram-se abaixo do limite máximo de 35 UFC/100 mL estabelecidos pela Agência de Proteção Ambiental dos Estados Unidos (USEPA). A diversidade fitoplanctônica foi alta e esteve composta, principalmente, por espécies cosmopolitas. Devido à concentração de nutrientes e UFC serem baixas, junto com uma alta diversidade de algas, conclui-se que não existe evidência direta de um impacto negativo do emissário na zona estudada., Con el objetivo de evaluar la posible afectación del emisario submarino en la ciudad de Limón, sobre el medio marino circundante, se establecieron 6 estaciones de muestreo, 4 de ellas cercanas a la zona de desfogue y 2 controles; en cada una se tomaron muestras de agua a 0 m y 10 m durante el 2012-2013 para medir nutrimentos, parámetros fisicoquímicos, concentración de microalgas y cantidad de unidades formadoras de colonias (UFC) de Enterococcus. Se contrastó la abundancia algal entre las estaciones bajo influencia del emisario, sus controles y entre profundidades, con un análisis de similitud (ANOSIM); en los casos en que hubo diferencia significativa se procedió a realizar un análisis SIMPER para determinar las especies responsables. La abundancia fitoplanctónica se utilizó para jerarquizar la dominancia de especies mediante el diagrama de Olmstead y Tukey e incorporarlas en un análisis de correspondencia canónica. Se determinó que en la zona existe una marcada influencia de las lluvias sobre la concentración de los nutrimentos, silicato y ortofosfato, además de parámetros como el oxígeno disuelto y la salinidad. Las UFC se mantuvieron por debajo del límite máximo de 35 UFC/100 mL establecidos por la Agencia de Protección Ambiental de los Estados Unidos (USEPA). La diversidad fitoplanctónica fue alta y estuvo compuesta, principalmente, por especies cosmopolitas. Debido a que la concentración de nutrientes y UFC son bajas, junto con una alta diversidad algal, se concluye que no existe evidencia directa de un impacto negativo del emisario sobre la zona estudiada.

Unsafe sanitation systems poses a risk for pathogen transmission, wherefore it is important to both inactivate pathogens present in human excreta and conduct safe sanitation systems from use to end-use. The Peepoo toilet, using ammonia sanitisation, have been suggested as a low-cost sanitation solution and is implemented in schools in Kibera, an urban slum in Kenya. This master thesis aim to study the inactivation efficiency of ammonia sanitisation when treating human excreta with urea, and to quantify the risks of exposure to microbial hazards from the Peepoo sanitation system using faecal indicator bacteria. Excreta was collected from four schools in Kibera. After adding urea to mimic the inactivation of the Peepoo in the laboratory, the inactivation rate was correlated to temperature and free ammonia concentration for Campylobacter spp., Escherichia coli and Enterococcus spp.. Campylobacter spp. and E. coli both had a high inactivation rate even at low temperature and low addition of urea. Inactivation rate of Enterococcus spp. was lower and close to zero when 1.87 % urea was added for 15 °C. For Enterococcus spp. a lag-phase was observed, which was not affected by temperature but by concentration of free ammonia. For investigated bacteria, inactivation rate increased with increased temperature and free ammonia concentration. Along the Peepoo management chain, several hazardous events were identified such ascontamination during usage, handling and transportation. Sampling showed a higher contamination of Enterococcus spp. than of E. coli. Enterococcus spp. was used as a faecal indicator for Ascaris and E. coli was used as an indicator of E. coli O157:H7 in a quantitative microbial risk assessment (QMRA). Through the QMRA, the risk of infection of Ascaris and E. coli O157:H7 for one exposure event was simulated based on a Exponential and a Beta-Poisson dose-response model respectively. The risk of infection of Ascaris was around 12 % regardless of where exposure occ

Background: The optimal treatment duration for patients with bloodstream infection is understudied. The Bacteremia Antibiotic Length Actually Needed for Clinical Effectiveness (BALANCE) pilot randomized clinical trial (RCT) determined that it was feasible to enroll and randomize intensive care unit (ICU) patients with bloodstream infection to 7 versus 14 days of treatment, and served as the vanguard for the ongoing BALANCE main RCT. We performed this BALANCE-Ward pilot RCT to examine the feasibility and impact of potentially extending the BALANCE main RCT to include patients hospitalized on non-ICU wards. Method(s): We conducted an open pilot RCT among a subset of six sites participating in the ongoing BALANCE RCT, randomizing patients with positive non-Staphylococcus aureus blood cultures on non-ICU wards to 7 versus 14 days of antibiotic treatment. The co-primary feasibility outcomes were recruitment rate and adherence to treatment duration protocol. We compared feasibility outcomes, patient/pathogen characteristics, and overall outcomes among those enrolled in this BALANCE-Ward and prior BALANCE-ICU pilot RCTs. We estimated the sample size and non-inferiority margin impacts of expanding the BALANCE main RCT to include non-ICU patients. Result(s): A total of 134 patients were recruited over 47 site-months (mean 2.9 patients/site-month, median 1.0, range 0.1-4.4 patients/site-month). The overall recruitment rate exceeded the BALANCE-ICU pilot RCT (mean 1.10 patients/site-month, p < 0.0001). Overall protocol adherence also exceeded the adherence in the BALANCE-ICU pilot RCT (125/134, 93% vs 89/115, 77%, p = 0.0003). BALANCE-Ward patients were older, with lower Sequential Organ Failure Assessment scores, and higher proportions of infections caused by Escherichia coli and genito-urinary sources of bloodstream infection. The BALANCE-Ward pilot RCT patients had an overall 90-day mortality rate of 17/133 (12.8%), which was comparable to the 90-day mortality rate in the I

Background: The optimal treatment duration for patients with bloodstream infection is understudied. The Bacteremia Antibiotic Length Actually Needed for Clinical Effectiveness (BALANCE) pilot randomized clinical trial (RCT) determined that it was feasible to enroll and randomize intensive care unit (ICU) patients with bloodstream infection to 7 versus 14 days of treatment, and served as the vanguard for the ongoing BALANCE main RCT. We performed this BALANCE-Ward pilot RCT to examine the feasibility and impact of potentially extending the BALANCE main RCT to include patients hospitalized on non-ICU wards. Method(s): We conducted an open pilot RCT among a subset of six sites participating in the ongoing BALANCE RCT, randomizing patients with positive non-Staphylococcus aureus blood cultures on non-ICU wards to 7 versus 14 days of antibiotic treatment. The co-primary feasibility outcomes were recruitment rate and adherence to treatment duration protocol. We compared feasibility outcomes, patient/pathogen characteristics, and overall outcomes among those enrolled in this BALANCE-Ward and prior BALANCE-ICU pilot RCTs. We estimated the sample size and non-inferiority margin impacts of expanding the BALANCE main RCT to include non-ICU patients. Result(s): A total of 134 patients were recruited over 47 site-months (mean 2.9 patients/site-month, median 1.0, range 0.1-4.4 patients/site-month). The overall recruitment rate exceeded the BALANCE-ICU pilot RCT (mean 1.10 patients/site-month, p < 0.0001). Overall protocol adherence also exceeded the adherence in the BALANCE-ICU pilot RCT (125/134, 93% vs 89/115, 77%, p = 0.0003). BALANCE-Ward patients were older, with lower Sequential Organ Failure Assessment scores, and higher proportions of infections caused by Escherichia coli and genito-urinary sources of bloodstream infection. The BALANCE-Ward pilot RCT patients had an overall 90-day mortality rate of 17/133 (12.8%), which was comparable to the 90-day mortality rate in the I