Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Detection of Enterococcus cecorum to identify persistently contaminated locations using faecal and environmental samples in broiler houses of clinically healthy flocks.

Worldwide outbreaks make infections with pathogenic strains of Enterococcus cecorum (EC) one of the most important diseases in the broiler industry. Although research has increased knowledge about the pathogen, the transmission is not fully understood. Samples from different locations were collected from two broiler farms in Germany over a total of six production cycles. Samples were collected at days 1, 5, 10, 15, 21, 27, 34, 41 post-hatch and after cleaning and disinfection (C&D). A total of 1017 samples were collected from 25 different locations on the farms. Samples were analysed in the laboratory for EC by quantitative real-time PCR. Overall, 7.5% of the samples were positive. The probabilities for positive and negative samples did not differ between the farms. The number of findings differed significantly between the cycles. Compared to other samples, the chances of detecting EC in faecal samples were significantly higher. Most positive samples were found in the last week of the production periods, indicating an accumulation of EC in the barn environment. After C&D, positive PCR results were obtained in four out of 14 locations. A re-introduction from contaminated environment seemed possible. However, one pooled faecal sample was positive 1 day post-hatch. The locations that showed positive results after C&D and the positive faecal sample 1 day post-hatch indicated the persistence of EC in broiler houses of clinically healthy flocks that could lead to potential horizontal transmission routes. The present study detected potential EC sources and may help to improve hygienic measures to avoid transmissions.RESEARCH HIGHLIGHTSMethodology is suitable to detect EC during production and after C&D.Locations were detected that may serve as a reservoir for EC.Cycles with fewer positive samples were observed.Cleaning and disinfection had a major impact on the detection of EC.

Genomic Characterization and Phylogenetic Analysis of Linezolid-Resistant Enterococcus from the Nostrils of Healthy Hosts Identifies Zoonotic Transmission.

Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic contents and dissemination patterns. Here, we used whole-genome sequencing to characterize the resistome, virulome and mobile genetic elements of nine linezolid-resistant (LZDR) enterococci (seven optrA-E. faecalis, one poxtA-E. faecium and one optrA-E. casseliflavus) previously obtained from the nares of healthy dogs, pigs, pig farmers and tracheal samples of nestling storks in Spain. Also, the relatedness of the isolates with publicly available genomes was accessed by core-genome single nucleotide polymorphism (SNP) analysis. The optrA gene of the E. faecalis and E. casseliflavus isolates was located downstream of the fexA gene. The optrA gene in the E. casseliflavus isolate was carried in a plasmid (pURX4962), while those in the seven E. faecalis isolates were chromosomally located. The OptrA proteins were mostly variants of wild type (DP-2: Y176D/T481P; RDK: I104R/Y176D/E256K; DD-3: Y176D/G393D; and EDD: K3E/Y176D/G393D), except two that were wild type (one E. faecalis and one E. casseliflavus). The poxtA gene in the E. faecium isolate was found alone within its contig. The cfrD was upstream of ermB gene in the E. casseliflavus isolate and flanked by ISNCY and IS1216. All the LZDR enterococci carried plasmid rep genes (2-3) containing tetracycline, chloramphenicol and aminoglycoside resistance genes. All isolates except E. casseliflavus carried at least one intact prophage, of which E. faecalis-ST330 (X4957) from a pig carried the highest (n = 5). Tn6260 was associated with lnuG in E. faecalis-ST330 while Tn554 was with fexA in E. feaecalis-ST59 isolates. All except E. casseliflavus (n = 0) carried at least two metal resistance genes (MRGs), of which poxtA-carrying E. faecium-ST1739 isolate contained the most (arsA, copA, fief, ziaA, znuA, zosA, zupT, and zur). SNP-based analyses identified closely related optrA-E. faecalis isolates from a pig and a pig farmer on the same farm (SNP = 4). Moreover, optrA- carrying E. faecalis-ST32, -ST59, and -ST474 isolates from pigs were related to those previously described from humans (sick and healthy) and cattle in Spain, Belgium, and Switzerland (SNP range 43-86). These findings strongly suggest the transmission of LZDR-E. faecalis between a pig and a pig farmer and potential inter-country dissemination. These highlight the need to strengthen molecular surveillance of LZDR enterococci in all ecological niches and body parts to direct appropriate control strategies.

Protective effect of SARS-CoV-2 preventive measures against ESKAPE and Escherichia coli infections.

BACKGROUND/OBJECTIVES: We investigated whether behavioral precautions adopted during Coronavirus disease (COVID-19) pandemic also influenced the spreading and multidrug resistance (MDR) of ESKAPEEc (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii [AB], Pseudomonas aeruginosa, Enterobacter spp and Escherichia Coli, [EC]) among Intensive Care Unit (ICU) patients. SUBJECTS/METHODS: We performed a single-center retrospective study in adult patients admitted to our COVID-19-free surgical ICU. Only patients staying in ICU for more than 48 hours were included. The ESKAPEEc infections recorded during the COVID-19 period (June 1, 2020 - February 28, 2021) and in the corresponding pre-pandemic period (June 1, 2019 - February 28, 2020) were compared. An interrupted time series analysis was performed to rule out possible confounders. RESULTS: Overall, 173 patients in the COVID-19 period and 132 in the pre-COVID-19 period were investigated. The ESKAPEEc infections were documented in 23 (13.3%) and 35 (26.5%) patients in the pandemic and the pre-pandemic periods, respectively (p = 0.005). Demographics, diagnosis, comorbidities, type of surgery, Simplified Acute Physiology Score II, length of mechanical ventilation, hospital and ICU length of stay, ICU death rate, and 28-day hospital mortality were similar in the two groups. In comparison with the pre-pandemic period, no AB was recorded during COVID-19 period, (p = 0.017), while extended-spectrum beta-lactamase-producing EC infections significantly decreased (p = 0.017). Overall, the ESKAPEEc isolates during pandemic less frequently exhibited multidrug-resistant (p = 0.014). CONCLUSIONS: These findings suggest that a robust adherence to hygiene measures together with human contact restrictions in a COVID-19 free ICU might also restrain the transmission of ESKAPEEc pathogens.

Gardnerella vaginalis in urinary tract infections, are men spared?

Gardnerella vaginalis in association with anaerobes has been linked to bacterial vaginosis in women, while urinary tract infections (UTIs) in men have rarely been reported. The aim of the review was to reveal the significance of G. vaginalis UTIs in men. Prevalence of G. vaginalis UTIs in men varied from 0.5 to >27% according to patients' groups. Most patients had comorbidity such as urolithiasis or stents, transplants, tumors and diabetes, however, infections can also affect immunocompetent patients. We observed G. vaginalis-associated bacteriuria and leukocyturia in a kidney transplant man. Complications of the UTIs such as bacteremia (in 9/11 cases), hydronephrosis (4/11) and abscesses or septic emboli have been reported. Bacterial vaginosis in female partners has been a risk factor for UTIs in males. In women, biofilm Gardnerella phenotype, stabilized by Atopobium vaginae and Prevotella bivia was linked to ≥6-fold higher antibiotic resistance rates compared with the planktonic phenotype. Non-susceptibility to metronidazole and levofloxacin was found also in males. Therefore, if aerobic urine cultures are negative, urine and blood samples from male patients with predisposing factors and clinical signs of UTIs and bacteremia, can be taken. Plates should be incubated for 2-4 days in capnophilic/microaerophilic conditions, however only anaerobic incubation can help with detecting G. vaginalis strains which grow only anaerobically. Susceptibility testing of the isolates is highly important. Briefly, adherent G. vaginalis phenotype can be sexually transmissible. Despite the infrequency of G. vaginalis UTIs in men, the infections should be considered since they are often linked to severe complications.

Vagococcus salmoninarum II-qPCR, tropism and egg-associated transmission.

Vagococcus salmoninarum was identified as the causative agent of a chronic epizootic in broodstock "coaster" brook trout (Salvelinus fontinalis) at the Iron River National Fish Hatchery. The epizootic spanned more than a year, was unresponsive to multiple florfenicol treatments, and resulted in >50% mortality of the affected fish. The decision was made to cull the remaining fish during spawning, which presented an opportunity to more thoroughly examine V. salmoninarum sampling methods, organ tropism and vertical transmission. A newly developed qPCR targeting the pheS gene was used in concert with bacterial culture to show that V. salmoninarum indeed disproportionately affects females and has a tropism for female reproductive tissues. The study demonstrates that some female reproductive tissues (e.g. ovarian fluid, unfertilized eggs) are also an effective option for non-lethal detection. Despite the widespread presence of V. salmoninarum in ovarian fluid and on egg surfaces, we found no evidence of intra-ova transmission.

WGS of 1058 Enterococcus faecium from Copenhagen, Denmark, reveals rapid clonal expansion of vancomycin-resistant clone ST80 combined with widespread dissemination of a vanA-containing plasmid and acquisition of a heterogeneous accessory genome.

OBJECTIVES: From 2012 to 2015, a sudden significant increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. Clonal relatedness of VREfm and vancomycin-susceptible E. faecium (VSEfm) was investigated, transmission events between hospitals were identified and the pan-genome and plasmids from the largest VREfm clonal group were characterized. METHODS: WGS of 1058 E. faecium isolates was carried out on the Illumina platform to perform SNP analysis and to identify the pan-genome. One isolate was also sequenced on the PacBio platform to close the genome. Epidemiological data were collected from laboratory information systems. RESULTS: Phylogeny of 892 VREfm and 166 VSEfm revealed a polyclonal structure, with a single clonal group (ST80) accounting for 40% of the VREfm isolates. VREfm and VSEfm co-occurred within many clonal groups; however, no VSEfm were related to the dominant VREfm group. A similar vanA plasmid was identified in ≥99% of isolates belonging to the dominant group and 69% of the remaining VREfm. Ten plasmids were identified in the completed genome, and ∼29% of this genome consisted of dispensable accessory genes. The size of the pan-genome among isolates in the dominant group was 5905 genes. CONCLUSIONS: Most probably, VREfm emerged owing to importation of a successful VREfm clone which rapidly transmitted to the majority of hospitals in the region whilst simultaneously disseminating a vanA plasmid to pre-existing VSEfm. Acquisition of a heterogeneous accessory genome may account for the success of this clone by facilitating adaptation to new environmental challenges.

Chicken Meat-Associated Enterococci: Influence of Agricultural Antibiotic Use and Connection to the Clinic.

Industrial farms are unique, human-created ecosystems that provide the perfect setting for the development and dissemination of antibiotic resistance. Agricultural antibiotic use amplifies naturally occurring resistance mechanisms from soil ecologies, promoting their spread and sharing with other bacteria, including those poised to become endemic within hospital environments. To better understand the role of enterococci in the movement of antibiotic resistance from farm to table to clinic, we characterized over 300 isolates of Enterococcus cultured from raw chicken meat purchased at U.S. supermarkets by the Consumers Union in 2013. Enterococcus faecalis and Enterococcus faecium were the predominant species found, and antimicrobial susceptibility testing uncovered striking levels of resistance to medically important antibiotic classes, particularly from classes approved by the FDA for use in animal production. While nearly all isolates were resistant to at least one drug, bacteria from meat labeled as raised without antibiotics had fewer resistances, particularly for E. faecium Whole-genome sequencing of 92 isolates revealed that both commensal- and clinical-isolate-like enterococcal strains were associated with chicken meat, including isolates bearing important resistance-conferring elements and virulence factors. The ability of enterococci to persist in the food system positions them as vehicles to move resistance genes from the industrial farm ecosystem into more human-proximal ecologies.IMPORTANCE Bacteria that contaminate food can serve as a conduit for moving drug resistance genes from farm to table to clinic. Our results show that chicken meat-associated isolates of Enterococcus are often multidrug resistant, closely related to pathogenic lineages, and harbor worrisome virulence factors. These drug-resistant agricultural isolates could thus represent important stepping stones in the evolution of enterococci into drug-resistant human pathogens. Although significant efforts have been made over the past few years to reduce the agricultural use of antibiotics, continued assessment of agricultural practices, including the roles of processing plants, shared breeding flocks, and probiotics as sources for resistance spread, is needed in order to slow the evolution of antibiotic resistance. Because antibiotic resistance is a global problem, global policies are needed to address this threat. Additional measures must be taken to mitigate the development and spread of antibiotic resistance elements from farms to clinics throughout the world.

Vancomycin-resistant enterococci (VRE): a reason to isolate?

In recent years, an increase in invasive VRE infections has been reported worldwide, including Germany. The most common gene encoding resistance to glycopeptides is VanA, but predominant VanB clones are emerging. Although neither the incidence rates nor the exact routes of nosocomial transmission of VRE are well established, screening and strict infection control measures, e.g. single room contact isolation, use of personal protective clothing by hospital staff and intensified surface disinfection for colonized individuals, are implemented in many hospitals. At the same time, the impact of VRE infection on mortality remains unclear, with current evidence being weak and contradictory. In this short review, we aim to give an overview on the current basis of evidence on the clinical effectiveness of infection control measures intended to prevent transmission of VRE and to put these findings into a larger perspective that takes further factors, e.g. VRE-associated mortality and impact on patient care, into account.

An epidemiological and molecular study regarding the spread of vancomycin-resistant Enterococcus faecium in a teaching hospital in Bogotá, Colombia 2016.

BACKGROUND: Enterococcus faecium is ranked worldwide as one of the top ten pathogens identified in healthcare-associated infections (HAI) and is classified as one of the high priority pathogens for research and development of new antibiotics worldwide. Due to molecular biology techniques' higher costs, the approach for identifying and controlling infectious diseases in developing countries has been based on clinical and epidemiological perspectives. Nevertheless, after an abrupt vancomycin-resistant Enterococcus faecium dissemination in the Méderi teaching hospital, ending up in an outbreak, further measures needed to be taken into consideration. The present study describes the vancomycin-resistant Enterococcus faecium pattern within Colombian's largest installed-bed capacity hospital in 2016. METHODS: Thirty-three vancomycin-resistant Enterococcus faecium isolates were recovered during a 5-month period in 2016. Multilocus variable-number tandem-repeat analysis was used for molecular typing to determine clonality amongst strains. A modified time-place-sequence algorithm was used to trace VREfm spread patterns during the outbreak period and estimate transmission routes. RESULTS: Four clonal profiles were identified. Chronological clonal profile follow-up suggested a transitional spread from profile "A" to profile "B", returning to a higher prevalence of "A" by the end of the study. Antibiotic susceptibility indicated high-level vancomycin-resistance in most isolates frequently matching vanA gene identification. DISCUSSION: Transmission analysis suggested cross-contamination via healthcare workers. Despite epidemiological control of the outbreak, post-outbreak isolates were still being identified as having outbreak-related clonal profile (A), indicating reduction but not eradication of this clonality. This study supports the use of combined molecular and epidemiological strategies in an approach to controlling infectious diseases. It contributes towards a more accurate evaluation of the effectiveness of the epidemiological measures taken regarding outbreak control and estimates the main cause related to the spread of this microorganism.

Pathogenicity and Virulence of Trueperella pyogenes: A Review.

Bacteria from the species Trueperella pyogenes are a part of the biota of skin and mucous membranes of the upper respiratory, gastrointestinal, or urogenital tracts of animals, but also, opportunistic pathogens. T. pyogenes causes a variety of purulent infections, such as metritis, mastitis, pneumonia, and abscesses, which, in livestock breeding, generate significant economic losses. Although this species has been known for a long time, many questions concerning the mechanisms of infection pathogenesis, as well as reservoirs and routes of transmission of bacteria, remain poorly understood. Pyolysin is a major known virulence factor of T. pyogenes that belongs to the family of cholesterol-dependent cytolysins. Its cytolytic activity is associated with transmembrane pore formation. Other putative virulence factors, including neuraminidases, extracellular matrix-binding proteins, fimbriae, and biofilm formation ability, contribute to the adhesion and colonization of the host tissues. However, data about the pathogen-host interactions that may be involved in the development of T. pyogenes infection are still limited. The aim of this review is to present the current knowledge about the pathogenic potential and virulence of T. pyogenes.

Environmental Panels as a Proxy for Nursing Facility Patients With Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococcus Colonization.

Background: Most nursing facilities (NFs) lack methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) surveillance programs due to limited resources and high costs. We investigated the utility of environmental screening of high-touch surfaces in patient rooms as a way to circumvent these challenges. Methods: We compared MRSA and VRE culture data from high-touch surfaces in patients' rooms (14450 samples from 6 NFs) and ranked each site's performance in predicting patient colonization (7413 samples). The best-performing sites were included in a MRSA- and a VRE-specific panel that functioned as a proxy for patient colonization. Molecular typing was performed to confirm available concordant patient-environment pairs. Results: We identified and validated a MRSA panel that consisted of the bed controls, nurse call button, bed rail, and TV remote control. The VRE panel included the toilet seat, bed controls, bed rail, TV remote control, and top of the side table. Panel colonization data tracked patient colonization. Negative predictive values were 89%-92% for MRSA and 82%-84% for VRE. Molecular typing confirmed a strong clonal type relationship in available concordant patient-environment pairs (98% for MRSA, 91% for VRE), pointing to common epidemiological patterns for environmental and patient isolates. Conclusions: Environmental panels used as a proxy for patient colonization and incorporated into facility surveillance protocols can guide decolonization strategies, improve awareness of MRSA and VRE burden, and inform efforts to reduce transmission. Targeted environmental screening may be a viable surveillance strategy for MRSA and VRE detection in NFs.

Relentless spread and adaptation of non-typeable vanA vancomycin-resistant Enterococcus faecium: a genome-wide investigation.

Background: VRE are prevalent among patients in ICUs. Non-typeable vanA VRE, due to loss of one of the genes used for MLST (pstS), have increased in Australia, suggestive of a new, hospital-acquired lineage. Objectives: To understand the significance of this lineage and its transmission using WGS of strains isolated from patients in ICUs across New South Wales, Australia. Methods: A total of 240 Enterococcus faecium isolates collected between February and May 2016, and identified by conventional PCR as vanA positive, were sequenced. Isolates originated from 12 ICUs in New South Wales, grouped according to six local health districts, and represented both rectal screening swab (n = 229) and clinical (n = 11) isolates. Results: ST analysis revealed the absence of the pstS gene in 84.2% (202 of 240) of vanA isolates. Two different non-typeable STs were present based on different allelic backbone patterns. Loss of the pstS gene appeared to be the result of multiple recombination events across this region. Evidence for pstS-negative lineage spread across all six local health districts was observed suggestive of inter-hospital transmission. In addition, multiple outbreaks were detected, some of which were protracted and lasted for the duration of the study. Conclusions: These findings confirmed the evolution, emergence and dissemination of non-typeable vanA E. faecium. This study has highlighted the utility of WGS when attempting to describe accurately the hospital-based pathogen epidemiology, which in turn will continue to inform optimal infection control measures necessary to halt the spread of this important nosocomial organism.

Elucidating vancomycin-resistant Enterococcus faecium outbreaks: the role of clonal spread and movement of mobile genetic elements.

Background: Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as a nosocomial pathogen worldwide. The dissemination of VREfm is due to both clonal spread and spread of mobile genetic elements (MGEs) such as transposons. Objectives: We aimed to combine vanB-carrying transposon data with core-genome MLST (cgMLST) typing and epidemiological data to understand the pathways of transmission in nosocomial outbreaks. Methods: Retrospectively, 36 VREfm isolates obtained from 34 patients from seven VREfm outbreak investigations in 2014 were analysed. Isolates were sequenced on a MiSeq and a MinION instrument. De novo assembly was performed in CLC Genomics Workbench and the hybrid assemblies were obtained through Unicycler v0.4.1. Ridom SeqSphere+ was used to extract MLST and cgMLST data. Detailed analysis of each transposon and their integration points was performed using the Artemis Comparison Tool (ACT) and multiple blast analyses. Results: Four different vanB transposons were found among the isolates. cgMLST divided ST80 isolates into three cluster types (CTs); CT16, CT104 and CT106. ST117 isolates were divided into CT24, CT103 and CT105. Within VREfm isolates belonging to CT103, two different vanB transposons were found. In contrast, VREfm isolates belonging to CT104 and CT106 harboured an identical vanB transposon. Conclusions: cgMLST provides a high discriminatory power for the epidemiological analysis of VREfm. However, additional transposon analysis is needed to detect horizontal gene transfer. Combining these two methods allows investigation of both clonal spread as well as the spread of MGEs. This leads to new insights and thereby better understanding of the complex transmission routes in VREfm outbreaks.

Mathematical modelling of vancomycin-resistant enterococci transmission during passive surveillance and active surveillance with contact isolation highlights the need to identify and address the source of acquisition.

BACKGROUND: Clinical studies and mathematical simulation suggest that active surveillance with contact isolation is associated with reduced vancomycin-resistant enterococci (VRE) prevalence compared to passive surveillance. Models using pre- and post-intervention data that account for the imperfect observation and serial dependence of VRE transmission events can better estimate the effectiveness of active surveillance and subsequent contact isolation; however, such analyses have not been performed. METHODS: A mathematical model was fitted to surveillance data collected pre- and post-implementation of active surveillance with contact isolation in the haematology-oncology ward. We developed a Hidden Markov Model to describe undetected and observed VRE colonisation/infection status based on the detection activities in the ward. Bayesian inference was used to estimate transmission rates. The effectiveness of active surveillance was assumed to be via increased detection and subsequent contact isolation of VRE positive patients. RESULTS: We estimated that 31% (95% credible interval: 0.33-85%) of the VRE transmissions were due to cross-transmission between patients. The ratio of transmission rates from patients with contact isolation versus those without contact isolation was 0.33 (95% credible interval: 0.050-1.22). CONCLUSIONS: The majority of the VRE acquisitions in the haematology-oncology ward was estimated to be due to background rates of VRE, rather than within ward patient to patient acquisition. The credible interval for cross-transmission was wide which results in a large degree of uncertainty in the estimates. Factors that could account for background VRE acquisition include endogenous acquisition from antibiotic selection pressure and VRE in the environment. Contact isolation was not significantly associated with reduced VRE transmission in settings where the majority of VRE acquisition was due to background acquisition, emphasising the need to identify and address the source of acquisition. As the credible interval for the ratio of VRE transmission in contact isolated versus non-contact isolated patients crossed 1, there is a probability that the transmission rate in contact isolation was not lower. Our finding highlights the need to optimise infection control measures other than active surveillance for VRE and subsequent contact isolation to reduce VRE transmission. Such measures could include antimicrobial stewardship, environmental cleaning, and hand hygiene.

Complex Routes of Nosocomial Vancomycin-Resistant Enterococcus faecium Transmission Revealed by Genome Sequencing.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of nosocomial infection. Here, we describe the utility of whole-genome sequencing in defining nosocomial VREfm transmission. METHODS: A retrospective study at a single hospital in the United Kingdom identified 342 patients with E. faecium bloodstream infection over 7 years. Of these, 293 patients had a stored isolate and formed the basis for the study. The first stored isolate from each case was sequenced (200 VREfm [197 vanA, 2 vanB, and 1 isolate containing both vanA and vanB], 93 vancomycin-susceptible E. faecium) and epidemiological data were collected. Genomes were also available for E. faecium associated with bloodstream infections in 15 patients in neighboring hospitals, and 456 patients across the United Kingdom and Ireland. RESULTS: The majority of infections in the 293 patients were hospital-acquired (n = 249) or healthcare-associated (n = 42). Phylogenetic analysis showed that 291 of 293 isolates resided in a hospital-associated clade that contained numerous discrete clusters of closely related isolates, indicative of multiple introductions into the hospital followed by clonal expansion associated with transmission. Fine-scale analysis of 6 exemplar phylogenetic clusters containing isolates from 93 patients (32%) identified complex transmission routes that spanned numerous wards and years, extending beyond the detection of conventional infection control. These contained both vancomycin-resistant and -susceptible isolates. We also identified closely related isolates from patients at Cambridge University Hospitals NHS Foundation Trust and regional and national hospitals, suggesting interhospital transmission. CONCLUSIONS: These findings provide important insights for infection control practice and signpost areas for interventions. We conclude that sequencing represents a powerful tool for the enhanced surveillance and control of nosocomial E. faecium transmission and infection.

Experimental challenges with Renibacterium salmoninarum in Arctic charr Salvelinus alpinus.

Arctic charr Salvelinus alpinus L. is an important species in Icelandic aquaculture and the most common wild salmonid in Iceland. A study on the course of infection with the bacterium Renibacterium salmoninarum was conducted using 3 different challenge methods in brackish and fresh water. Bacterial isolation, ELISA and PCR tests were used for detection of the bacterium in multiple organ samples. In an experiment, run for 34 wk in brackish water, infection was established by intraperitoneal injection with 5 × 106 colony forming units (CFU) fish-1. There were external and internal symptoms of bacterial kidney disease (BKD) and mortalities between 6 and 13 wk after injection. A cohabitation trial was run simultaneously and infection was well established after 4 wk, as demonstrated by the detection methods applied. Symptoms of BKD were not seen and all but 1 cohabitant survived. In a separate experiment, infection was established by pumping a fixed amount of water from a tank with fingerlings infected by intraperitoneal injection into tanks with naïve fish, in fresh or brackish water, for 6 wk. Fish in the inflow tanks were reared for an additional 3 wk. There were neither macroscopic symptoms nor mortalities. ELISA and PCR tests showed that infection started to take hold after 3 wk. The challenge trials demonstrated that Arctic charr is susceptible to R. salmoninarum. Cohabitation and inflow of water from tanks with infected fish provide useful models for further studies on R. salmoninarum infection acquired in a natural way in Arctic charr.

Small distances can keep bacteria at bay for days.

Transmission of pathogens between spatially separated hosts, i.e., indirect transmission, is a commonly encountered phenomenon important for epidemic pathogen spread. The routes of indirect transmission often remain untraced, making it difficult to develop control strategies. Here we used a tailor-made design to study indirect transmission experimentally, using two different zoonotic bacteria in broilers. Previous experiments using a single bacterial species yielded a delay in the onset of transmission, which we hypothesized to result from the interplay between diffusive motion of infectious material and decay of infectivity in the environment. Indeed, a mathematical model of diffusive pathogen transfer predicts a delay in transmission that depends both on the distance between hosts and on the magnitude of the pathogen decay rate. Our experiments, carried out with two bacterial species with very different decay rates in the environment, confirm the difference in transmission delay predicted by the model. These results imply that for control of an infectious agent, the time between the distant exposure and the infection event is important. To illustrate how this can work we analyzed data observed on the spread of vancomycin-resistant Enterococcus in an intensive care unit. Indeed, a delayed vancomycin-resistant Enterococcus transmission component was identified in these data, and this component disappeared in a study period in which the environment was thoroughly cleaned. Therefore, we suggest that the impact of control strategies against indirect transmission can be assessed using our model by estimating the control measures' effects on the diffusion coefficient and the pathogen decay rate.

Persistent Bacillus cereus Bacteremia in 3 Persons Who Inject Drugs, San Diego, California, USA.

Bacillus cereus is typically considered a blood culture contaminant; however, its presence in blood cultures can indicate true bacteremia. We report 4 episodes of B. cereus bacteremia in 3 persons who inject drugs. Multilocus sequence typing showed that the temporally associated infections were caused by unrelated clones.

Real-Time Genome Sequencing of Resistant Bacteria Provides Precision Infection Control in an Institutional Setting.

The increasing prevalence of multidrug-resistant (MDR) bacteria is a serious global challenge. Here, we studied prospectively whether bacterial whole-genome sequencing (WGS) for real-time MDR surveillance is technical feasible, returns actionable results, and is cost-beneficial. WGS was applied to all MDR isolates of four species (methicillin-resistant Staphylococcus aureus [MRSA], vancomycin-resistant Enterococcus faecium, MDR Escherichia coli, and MDR Pseudomonas aeruginosa) at the University Hospital Muenster, Muenster, Germany, a tertiary care hospital with 1,450 beds, during two 6-month intervals. Turnaround times (TAT) were measured, and total costs for sequencing per isolate were calculated. After cancelling prior policies of preemptive isolation of patients harboring certain Gram-negative MDR bacteria in risk areas, the second interval was conducted. During interval I, 645 bacterial isolates were sequenced. From culture, TATs ranged from 4.4 to 5.3 days, and costs were €202.49 per isolate. During interval II, 550 bacterial isolates were sequenced. Hospital-wide transmission rates of the two most common species (MRSA and MDR E. coli) were low during interval I (5.8% and 2.3%, respectively) and interval II (4.3% and 5.0%, respectively). Cancellation of isolation of patients infected with non-pan-resistant MDR E. coli in risk wards did not increase transmission. Comparing sequencing costs with avoided costs mostly due to fewer blocked beds during interval II, we saved in excess of €200,000. Real-time microbial WGS in our institution was feasible, produced precise actionable results, helped us to monitor transmission rates that remained low following a modification in isolation procedures, and ultimately saved costs.