Genomic evolution of ST228 SCCmec-I MRSA 10 years after a major nosocomial outbreak.

In this study, we investigated the genomic changes in a major methicillin-resistant Staphylococcus aureus (MRSA) clone following a significant outbreak at a hospital. Whole-genome sequencing of MRSA isolates was utilized to explore the genomic evolution of post-outbreak MRSA strains. The epidemicity of the clone declined over time, coinciding with the introduction of multimodal infection control measures. A genome-wide association study (GWAS) identified multiple genes significantly associated with either high or low epidemic success, indicating alterations in mobilome, virulence, and defense mechanisms. Random Forest models pinpointed a gene related to fibrinogen binding as the most influential predictor of epidemicity. The decline of the MRSA clone may be attributed to various factors, including the implementation of new infection control measures, single nucleotide polymorphisms accumulation, and the genetic drift of a given clone. This research underscores the complex dynamics of MRSA clones, emphasizing the multifactorial nature of their evolution. The decline in epidemicity seems linked to alterations in the clone's genetic profile, with a probable shift towards decreased virulence and adaptation to long-term carriage. Understanding the genomic basis for the decline of epidemic clones is crucial to develop effective strategies for their surveillance and management, as well as to gain insights into the evolutionary dynamics of pathogen genomes.

Core genome multilocus sequence typing of Clostridioides difficile to investigate transmission in the hospital setting.

PURPOSE: Traditional epidemiological investigations of healthcare-associated Clostridioides difficile infection (HA-CDI) are often insufficient. This study aimed to evaluate a procedure that includes secondary isolation and genomic typing of single toxigenic colonies using core genome multilocus sequence typing (cgMLST) for the investigation of C. difficile transmission. METHODS: We analyzed retrospectively all toxigenic C. difficile-positive stool samples stored at the Lausanne University Hospital over 6 consecutive months. All isolates were initially typed and classified using a modified double-locus sequence typing (DLST) method. Genome comparison of isolates with the same DLST and clustering were subsequently performed using cgMLST. The electronic administrative records of patients with CDI were investigated for spatiotemporal epidemiological links supporting hospital transmission. A comparative descriptive analysis between genomic and epidemiological data was then performed. RESULTS: From January to June 2021, 86 C. difficile isolates were recovered from thawed samples of 71 patients. Thirteen different DLST types were shared by > 1 patient, and 13 were observed in single patients. A genomic cluster was defined as a set of isolates from different patients with ≤ 3 locus differences, determined by cgMLST. Seven genomic clusters were identified, among which plausible epidemiological links were identified in only 4/7 clusters. CONCLUSION: Among clusters determined by cgMLST analysis, roughly 40% included unexplained HA-CDI acquisitions, which may be explained by unidentified epidemiological links, asymptomatic colonization, and/or shared common community reservoirs. The use of DLST, followed by whole genome sequencing analysis, is a promising and cost-effective stepwise approach for the investigation of CDI transmission in the hospital setting.

[Methicillin-resistant Staphylococcus aureus: 15 years of molecular epidemiology in Western Switzerland]. / Staphylococcus aureus résistant à la méticilline : 15 ans de surveillance moléculaire en Suisse romande.

Since the introduction of antibiotics, successive waves of Staphylococcus aureus clones occurred, each one having characteristic susceptibility pattern to antibiotics and virulence factors. We report here the results of a molecular epidemiological surveillance of methicillin-resistant S. aureus (MRSA) in French-speaking Switzerland between 2006 and 2020 showing the emergence and disappearance of clones known for their international dissemination, and the sporadic appearance of other international clones. Since 2012, a marked decrease in the incidence of cases attributable to the biology of the clones and to the control measures taken in the hospitals has been observed. These results highlight the importance of continuous surveillance in order to better assess the burden of this multi-resistant pathogen in our region.

Depuis l'introduction des antibiotiques, des vagues successives de clones de Staphylococcus aureus sont apparues, chacun avec un profil de susceptibilité aux antibiotiques et de virulence caractéristique. Nous rapportons ici les résultats d'une surveillance épidémiologique moléculaire de S. aureus résistant à la méticilline (MRSA) en Suisse romande entre 2006 et 2020 montrant l'émergence et la disparition de clones connus pour leur dissémination internationale, ainsi que l'apparition sporadique d'autres clones internationaux. Depuis 2012, une diminution marquée de l'incidence des cas attribuable à la biologie des clones et aux mesures de contrôle prises dans les hôpitaux est observée. Ces résultats nous montrent l'importance d'une surveillance continue afin de mieux évaluer le fardeau que représente ce germe multirésistant dans notre région.

Phenotypic and Genomic Analyses of Burkholderia stabilis Clinical Contamination, Switzerland.

A recent hospital outbreak related to premoistened gloves used to wash patients exposed the difficulties of defining Burkholderia species in clinical settings. The outbreak strain displayed key B. stabilis phenotypes, including the inability to grow at 42°C; we used whole-genome sequencing to confirm the pathogen was B. stabilis. The outbreak strain genome comprises 3 chromosomes and a plasmid, sharing an average nucleotide identity of 98.4% with B. stabilis ATCC27515 BAA-67, but with 13% novel coding sequences. The genome lacks identifiable virulence factors and has no apparent increase in encoded antimicrobial drug resistance, few insertion sequences, and few pseudogenes, suggesting this outbreak was an opportunistic infection by an environmental strain not adapted to human pathogenicity. The diversity among outbreak isolates (22 from patients and 16 from washing gloves) is only 6 single-nucleotide polymorphisms, although the genome remains plastic, with large elements stochastically lost from outbreak isolates.

Whole-genome sequencing revealed independent emergence of vancomycin-resistant Enterococcus faecium causing sequential outbreaks over 3 years in a tertiary care hospital.

Vancomycin-resistant Enterococcus faecium (VREfm) emerged as an important cause of nosocomial infections worldwide. Previous studies based on molecular typing revealed that VREfm outbreaks are mainly associated with a particular genetic lineage, namely clonal complex 17 (CC17), which harbours either vanA or vanB gene cluster. The University Hospital of Lausanne faced several VREfm episodes of transmissions between 2014 and 2017. In this study, we used whole-genome sequencing (WGS) to investigate the relatedness of 183 VREfm isolates collected from 156 patients. Sequence types (ST) 17, ST80 and ST117 were the most predominant clones. Based on epidemiological data, 10 outbreaks were identified, which were caused by at least 13 distinct genotypes. The majority of isolates involved in outbreaks (91%) differed by only 0 to 3 SNPs. Four outbreaks involved more than one genotype and half of the cases considered as sporadic were possibly linked to an outbreak. By sequencing all isolates, we were able to better understand our local epidemiology of VREfm. The polyclonal structure observed between the different outbreaks strains, the high level of recombination detected in isolates, the time elapsed between admission and the first VREfm detection and the negative screening at admission support the hypothesis of the emergence of new VREfm clones within the hospitalised population.

Molecular epidemiology of Clostridioides (previously Clostridium) difficile isolates from a university hospital in Minas Gerais, Brazil.

The molecular epidemiology of 38 non-duplicate toxigenic Clostridioides (previously Clostridium) difficile isolates from inpatients from a hospital in Brazil during a 6-year period (2012-2017) were investigated by multilocus sequence typing (MLST) and ribotyping. These isolates were classified into 20 sequence types (ST), six (30%) of which were novel, revealing a high diversity in a single hospital. Classic hypervirulent strains ST1/RT027 and ST11/RT078 were not identified, while ST42 (almost all RT106) was the most common type, being detected in 11 (28.9%) strains. Noteworthy, six (15.8%) isolates were classified into five STs from clade 2, four of which were new ST and RT. Our study suggests that possible hypervirulent strains other than ST1/RT027 might be inadvertently circulating in Brazilian hospitals and highlights the importance of permanent surveillance on circulating strains in a national scale.

Rodents are carriers of Clostridioides difficile strains similar to those isolated from piglets.

Features of Clostridioides difficile transmission in swine and the role of rodents as C. difficile reservoir are not clear. To investigate if rodents can carry strains of C. difficile that are genetically similar to those isolated from swine, 97 fecal samples from neonatal piglets and 41 intestinal contents from rodents were collected in two farms. All samples were subjected to C. difficile culture and the presence of A/B toxins in piglet feces were accessed by commercial enzyme imunoassay (EIA). C. difficile isolates were typed by double- (DLST) and multi-locus sequence typing (MLST). C. difficile was isolated from 15.5% of piglets and 31.7% of rodents. Most isolates were identified as DLST type 4-4 and 17-5 (both are ST11), which were found in both rodents and piglets. Results of this study suggested that rodents may have a role on the transmission and spread of C. difficile strains to swine.

Clostridioides difficile infection in dogs with chronic-recurring diarrhea responsive to dietary changes.

Five dogs with chronic-recurring diarrhea were positive for Clostridioides difficile infection (CDI), but were unresponsive to treatment with metronidazole. One of these animals was subjected to a colonoscopy, which revealed eosinophilic infiltration of the colon. All five animals completely recovered after dietary changes. The present work suggests that CDI might occur in dogs with other intestinal alterations. In addition, this report suggests that dysbiosis should be considered in animals that have chronic-recurring diarrhea and test positive for C. difficile.

[Vancomycine-resistant enterocci (VRE) : a new reality in our hospitals]. / Entérocoque résistant à la vancomycine (ERV) : une nouvelle réalité dans nos hôpitaux.

Limiting the emergence and spread of multi-resistant bacteria is a global concern and the management of colonized patient represents a real challenge, especially in the hospital setting, where risks of acquisition and transmission are increased. Switzerland is not protected from undesirable trends : for instance, recent outbreaks of vancomycin-resistant enterococci (VRE) have been reported in several hospitals in western Switzerland. Since 2011, more than 250 patients have been tested positive during these outbreak episodes and the molecular analysis of the documented strains shows an unexpected diversity, including both sporadic and epidemic strains. This emerging threat requires strict monitoring, prevention and infection control strategies in our healthcare facilities.

Limiter l'émergence et la diffusion des bactéries multirésistantes (BMR) est une urgence mondiale et la gestion des patients porteurs représente un véritable défi, notamment en milieu hospitalier, où les risques d'acquisition et de transmission de ces germes sont multipliés. La Suisse n'est pas épargnée par ce phénomène. En témoignent les épidémies récentes à entérocoques résistant à la vancomycine (ERV) dans plusieurs hôpitaux de Suisse romande. Depuis 2011, plus de 250 patients ont été dépistés positifs durant ces épisodes et l'analyse moléculaire par séquençage complet de génome montre une diversité inattendue des souches, qu'elles soient sporadiques ou à potentiel épidémique. Cette menace émergente, bien réelle, implique une stratégie de surveillance, prévention et contrôle de l'infection stricte dans nos établissements de soins.

The prevalence of optic neuropathy in alcoholic patients--a pilot study.

BACKGROUND: Alcohol has particularly toxic effects on the central and peripheral nervous systems. Optic neuropathy (ON) is one of these neurological complications. Its diagnosis has not been codified, and its prevalence is poorly known. The aim of this pilot study was to assess the prevalence of ON and identify risk factors in a cohort of patients hospitalized for alcohol withdrawal. METHODS: This was a single-center prospective study. A complete standardized eye examination was performed during the patient's alcohol withdrawal; The data collected included: sociodemographic status; the number of withdrawals; the type and amount of alcohol drunk, tobacco, and illicit drug consumption; and ophthalmological results. RESULTS: One hundred patients were included prospectively from January 2010 to June 2011 (67 men and 33 women) with a mean age of 47 ± 12 and 46 ± 10 years, respectively. The average alcohol consumption was higher for men than women: 207 ± 122 vs. 146 ± 92 g/d, p = 0.013. The most frequent definition of ON in the literature is a decrease in visual acuity associated with impaired color vision. Thirteen percent of men and 3% of women met these criteria. But monocular ON was observed in 22% of men and 18% women, and partial damage was demonstrated in 27% of men and 7% of women. CONCLUSIONS: ON is a relatively rare complication of chronic alcohol consumption, but the high prevalence of incomplete forms should prompt screening and early treatment.

Impact of mutations in the mtrR, rpdlVD and rrl genes on azithromycin resistance in Neisseria gonorrhoeae.

INTRODUCTION: Bacterial sexually transmitted infections (STIs) pose a major public health problem. The emergence of antibiotic-resistant strains of Neisseria gonorrhoeae represents a serious threat to successful treatment and epidemiological control. The first extensively drug-resistant (XDR) strains (ceftriaxone-resistant and high-level azithromycin-resistant [HLR AZY]) have been reported. AIMS: To identify molecular mechanisms implicated in azithromycin resistance in strains isolated from patients over a three-year period in a university hospital in Switzerland. MATERIAL AND METHODS: From January 2020 to December 2022, 34 isolates (one per patient) were recovered from samples analyzed at the University Hospital of Lausanne. Eight genes involved in azithromycin resistance were sequenced: mtrR repressor (mtrCDE operon repressor) and his promotor mtrR-pr, rplD gene (L4 ribosomal protein), rplV gene (L22 ribosomal protein) and the four alleles of the rrl gene (23S rRNA). RESULTS: With a cutoff value of 1 mg/L, 15 isolates were considered as being resistant to azithromycin, whereas the remaining 19 were susceptible. The C2597T mutation in 3 or 4 of the rrl allele confer a medium-level resistance to azithromycin (MIC = 16 mg/L, N = 2). The following mutations were significantly associated with MIC values ≥1 mg/L: the three mutations V125A, A147G, R157Q in the rplD gene (N = 10) and a substitution A->C in the mtrR promotor (N = 9). Specific mutations in the mtrR repressor and its promotor were observed in both susceptible and resistant isolates. CONCLUSIONS: Resistance to azithromycin was explained by the presence of mutations in many different copies of 23S RNA ribosomal genes and their regulatory genes. Other mutations, previously reported to be associated with azithromycin resistance, were documented in both susceptible and resistant isolates, suggesting they play little role, if any, in azithromycin resistance.

Investigations of an increased incidence of non-Aspergillus invasive mould infections in an onco-haematology unit.

AIMS OF THE STUDY: Invasive mould infections are life-threatening complications in patients with haematologic cancer and chemotherapy-induced neutropenia. While invasive aspergillosis represents the main cause of invasive mould infections, non-Aspergillus mould infections, such as mucormycosis, are increasingly reported. Consequently, their local epidemiology should be closely monitored. The aim of this study was to investigate the causes of an increased incidence of non-Aspergillus mould infections in the onco-haematology unit of a Swiss tertiary care hospital. METHODS: All cases of proven and probable invasive mould infections were retrospectively identified via a local registry for the period 2007-2021 and their incidence was calculated per 10,000 patient-days per year. The relative proportion of invasive aspergillosis and non-Aspergillus mould infections was assessed. Factors that may affect invasive mould infections' incidence, such as antifungal drug consumption, environmental contamination and changes in diagnostic approaches, were investigated. RESULTS: A significant increase of the incidence of non-Aspergillus mould infections (mainly mucormycosis) was observed from 2017 onwards (Mann and Kendall test p = 0.0053), peaking in 2020 (8.62 episodes per 10,000 patient-days). The incidence of invasive aspergillosis remained stable across the period of observation. The proportion of non-Aspergillus mould infections increased significantly from 2017 (33% vs 16.8% for the periods 2017-2021 and 2007-2016, respectively, p = 0.02). Building projects on the hospital site were identified as possible contributors of this increase in non-Aspergillus mould infections. However, novel diagnostic procedures may have improved their detection. CONCLUSIONS: We report a significant increase in non-Aspergillus mould infections, and mainly in mucormycosis infections, since 2017. There seems to be a multifactorial origin to this increase. Epidemiological trends of invasive mould infections should be carefully monitored in onco-haematology units in order to implement potential corrective measures.

Diversity and evolution of an abundant ICEclc family of integrative and conjugative elements in Pseudomonas aeruginosa.

IMPORTANCE: Microbial populations swiftly adapt to changing environments through horizontal gene transfer. While the mechanisms of gene transfer are well known, the impact of environmental conditions on the selection of transferred gene functions remains less clear. We investigated ICEs, specifically the ICEclc-type, in Pseudomonas aeruginosa clinical isolates. Our findings revealed co-evolution between ICEs and their hosts, with ICE transfers occurring within strains. Gene functions carried by ICEs are positively selected, including potential virulence factors and heavy metal resistance. Comparison to publicly available P. aeruginosa genomes unveiled widespread antibiotic-resistance determinants within ICEclc clades. Thus, the ubiquitous ICEclc family significantly contributes to P. aeruginosa's adaptation and fitness in diverse environments.

The Swiss Pathogen Surveillance Platform - towards a nation-wide One Health data exchange platform for bacterial, viral and fungal genomics and associated metadata.

The Swiss Pathogen Surveillance Platform (SPSP) is a shared secure surveillance platform between human and veterinary medicine, to also include environmental and foodborne isolates. It enables rapid and detailed transmission monitoring and outbreak surveillance of pathogens using whole genome sequencing data and associated metadata. It features controlled data access, complex dynamic queries, dedicated dashboards and automated data sharing with international repositories, providing actionable results for public health and the vision to improve societal well-being and health.

Cross-reaction of naturally-produced ß-lactamases from Citrobacter farmeri and Citrobacter amalonaticus with immunological detection of CTX-M enzymes.

The NG-Test CTX-M MULTI immunochromatographic assay has been developed to identify CTX-M-type ß-lactamases in Enterobacterales, being the most widespread extended-spectrum ß-lactamases. We showed here that the chromosomally-encoded ß-lactamases from Citrobacter farmeri and Citrobacter amalonaticus generated false-positive NG-Test CTX-M MULTI results, compromising the specificity of the test.

Molecular typing of Clostridioides difficile from frozen stool samples to investigate cross-transmissions: A proof of concept.

PURPOSE: Toxigenic Clostridioides difficile is responsible for up to one third of post antibiotic diarrhea and for more than 95% of pseudomembranous colitis. Nowadays, diagnosis relies on the documentation of the presence of the toxin in stools by specific antigenic or PCR tests. Stool cultures have been mostly abandoned, leading to the absence of isolates for further epidemiological analyses. METHODS: Aliquots of stool samples, frozen for up to two years, were thawed and inoculated onto commercial C. difficile media. Eighteen stools were recovered from patients hospitalized in the pediatric ward where at that time a chain of transmission was suspected. Eleven stools were recovered from patients hospitalized in a medical ward over a three months period with no suspected transmission event. Up to 16 characteristic colonies were isolates per culture. PCR of toxins genes and molecular typing by Double Locus Sequence Typing (DLST) were performed on these colonies. Whole genome multi locus sequence typing (wgMLST) was performed on selected isolates. RESULTS: Among the 29 stool specimens, no growth was observed for four stools and only one colony grew for one stool. Except the latter, all 16 colonies of the 24 stools showed identical toxin genes profiles than the original stool. However, variant DLST genotypes was observed within 20% of investigated stools. The majority of variants were single locus variant due to an IN/DEL of the repeat in one of the two DLST locus. Despite this variation, results of molecular typing overrule the putative transmission chain in the pediatric ward and revealed undetected chains of transmission in the medical ward. These results were confirmed with wgMLST. CONCLUSIONS: The developed protocol allows prospective and retrospective molecular and genomic epidemiological investigation of C. difficile infections for infection control purpose.

Parallel Evolution of Pseudomonas aeruginosa during a Prolonged ICU-Infection Outbreak.

Most knowledge about Pseudomonas aeruginosa pathoadaptation is derived from studies on airway colonization in cystic fibrosis; little is known about adaptation in acute settings. P. aeruginosa frequently affects burned patients and the burn wound niche has distinct properties that likely influence pathoadaptation. This study aimed to genetically and phenotypically characterize P. aeruginosa isolates collected during an outbreak of infection in a burn intensive care unit (ICU). Sequencing reads from 58 isolates of ST1076 P. aeruginosa taken from 23 patients were independently mapped to a complete reference genome for the lineage (H25338); genetic differences were identified and were used to define the population structure. Comparative genomic analysis at single-nucleotide resolution identified pathoadaptive genes that evolved multiple, independent mutations. Three key phenotypic assays (growth performance, motility, carbapenem resistance) were performed to complement the genetic analysis for 47 unique isolates. Population structure for the ST1076 lineage revealed 11 evolutionary sublineages. Fifteen pathoadaptive genes evolved mutations in at least two sublineages. The most prominent functional classes affected were transcription/two-component regulatory systems, and chemotaxis/motility and attachment. The most frequently mutated gene was oprD, which codes for outer membrane porin involved in uptake of carbapenems. Reduced growth performance and motility were found to be adaptive phenotypic traits, as was high level of carbapenem resistance, which correlated with higher carbapenem consumption during the outbreak. Multiple prominent linages evolved each of the three traits in parallel providing evidence that they afford a fitness advantage for P. aeruginosa in the context of human burn infection. IMPORTANCE Pseudomonas aeruginosa is a Gram-negative pathogen causing infections in acutely burned patients. The precise mechanisms required for the establishment of infection in the burn setting, and adaptive traits underpinning prolonged outbreaks are not known. We have assessed genotypic data from 58 independent P. aeruginosa isolates taken from a single lineage that was responsible for an outbreak of infection in a burn ICU that lasted for almost 2.5 years and affected 23 patients. We identified a core set of 15 genes that we predict to control pathoadaptive traits in the burn infection based on the frequency with which independent mutations evolved. We combined the genotypic data with phenotypic data (growth performance, motility, antibiotic resistance) and clinical data (antibiotic consumption) to identify adaptive phenotypes that emerged in parallel. High-level carbapenem resistance evolved rapidly, and frequently, in response to high clinical demand for this antibiotic class during the outbreak.