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.

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.

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.

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.

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.

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.

[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.

MRSA surveillance programmes worldwide: moving towards a harmonised international approach.

Multinational surveillance programmes for methicillin-resistant Staphylococcus aureus (MRSA) are dependent on national structures for data collection. This study aimed to capture the diversity of national MRSA surveillance programmes and to propose a framework for harmonisation of MRSA surveillance. The International Society of Antimicrobial Chemotherapy (ISAC) MRSA Working Group conducted a structured survey on MRSA surveillance programmes and organised a webinar to discuss the programmes' strengths and challenges as well as guidelines for harmonisation. Completed surveys represented 24 MRSA surveillance programmes in 16 countries. Several countries reported separate epidemiological and microbiological surveillance. Informing clinicians and national policy-makers were the most common purposes of surveillance. Surveillance of bloodstream infections (BSIs) was present in all programmes. Other invasive infections were often included. Three countries reported active surveillance of MRSA carriage. Methodology and reporting of antimicrobial susceptibility, virulence factors, molecular genotyping and epidemiological metadata varied greatly. Current MRSA surveillance programmes rely upon heterogeneous data collection systems, which hampers international epidemiological monitoring and research. To harmonise MRSA surveillance, we suggest improving the integration of microbiological and epidemiological data, implementation of central biobanks for MRSA isolate collection, and inclusion of a representative sample of skin and soft-tissue infection cases in addition to all BSI cases.

Prevalence of multidrug-resistant bacteria colonisation among asylum seekers in western Switzerland.

The recent increase of migration to Europe represents a risk of increased the prevalence of multidrug-resistant (MDR) bacteria. We conducted a cross-sectional study among asylum seekers admitted at two hospitals in Switzerland. Of the 59 patients included, 9 (14%) were colonised by a MDR bacteria, including 5 (8.5%) methicilin-resistant Staphylococcus aureus (MRSA) and 4 (6.8%) extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. No patient carried both ESBL-producing bacteria and MRSA. None of the patients carried a vancomycin-resistant Enterococcus (VRE) or a carbapenem-resistant Enterobacteriaceae (CRE). Colonisation with MDR bacteria was not associated with hospitalisation abroad or recent arrival in Switzerland. Whole genome sequencing analysis allowed us to exclude transmission between patients. The prevalence of MDR bacteria carriage is moderate among asylum seekers in western Switzerland. Further surveillance studies are necessary to determine if there is a risk of dissemination of pathogens into the local population.

Phylogeographical Analysis Reveals the Historic Origin, Emergence, and Evolutionary Dynamics of Methicillin-Resistant Staphylococcus aureus ST228.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a common healthcare-associated pathogen that remains a major public health concern. Sequence type 228 (ST228) was first described in Germany and spread to become a successful MRSA clone in several European countries. In 2000, ST228 emerged in Lausanne and has subsequently caused several large outbreaks. Here, we describe the evolutionary history of this clone and identify the genetic changes underlying its expansion in Switzerland. MATERIALS AND METHODS: We aimed to understand the phylogeographic and demographic dynamics of MRSA ST228/ST111 by sequencing 530 representative isolates of this clone that were collected from 14 European countries between 1997 and 2012. RESULTS: The phylogenetic analysis revealed distinct lineages of ST228 isolates associated with specific geographic origins. In contrast, isolates of ST111, which is a single locus variant of ST228 sharing the same spa type t041, formed a monophyletic cluster associated with multiple countries. The evidence points to a German origin of the sampled population, with the basal German lineage being characterized by spa type t001. The highly successful Swiss ST228 lineage diverged from this progenitor clone through the loss of the aminoglycoside-streptothricin resistance gene cluster and the gain of mupirocin resistance. This lineage was introduced first in Geneva and was subsequently introduced into Lausanne. CONCLUSION: Our results reveal the radiation of distinct lineages of MRSA ST228 from a German progenitor, as the clone spread into different European countries. In Switzerland, ST228 was introduced first in Geneva and was subsequently introduced into Lausanne.

Comparison of Whole Genome (wg-) and Core Genome (cg-) MLST (BioNumericsTM) Versus SNP Variant Calling for Epidemiological Investigation of Pseudomonas aeruginosa.

Whole genome sequencing (WGS) is increasingly used for epidemiological investigations of pathogens. While SNP variant calling is currently considered as the most suitable method, the choice of a representative reference genome and the isolate dependency of results limit standardization and affect resolution in an unknown manner. Whole or core genome Multi Locus Sequence Typing (wg-, cg-MLST) represents an attractive alternative. Here, we assess the accuracy of wg- and cg-MLST by comparing results of four Pseudomonas aeruginosa datasets for which epidemiological and genomic data were previously described. Three datasets included 155 isolates from three different sequence types (ST) of P. aeruginosa collected in our ICUs over a 5-year period. The fourth dataset consisted of 10 isolates from an investigation of P. aeruginosa contaminated hand soap. All isolates were previously analyzed by a core SNP approach. In this study, wg- and cg-MLST were performed in BioNumericsTM using a scheme developed by Applied-Maths. Correlation between SNP calling and wg- or cg-MLST results were evaluated by calculating linear regressions and their coefficient of correlations (R 2) between the number of SNPs and the number of allele differences in pairwise comparison of isolates. The number of SNPs and allele difference between isolates with close epidemiological linkage varies between 0-26 and 0-13, respectively. When compared to core-SNP calling, a higher coefficient of correlation was obtained with cgMLST (R 2 of 0.92-0.99) than with wgMLST (0.78-0.99). In one dataset, a putative homologous recombination of a large DNA fragment (202 loci) was identified among these isolates, affecting its phylogeny, but with no impact on the epidemiological analysis of outbreak isolates. In conclusion, we showed that the P. aeruginosa wgMLST scheme in BioNumericsTM is as discriminatory as the core-SNP calling approach and apparently useful for outbreak investigations. We also showed that epidemiological linked isolates showed less than 26 SNPs or 13 allele differences. These are important figures for the distinction between outbreak and non-outbreak isolates when interpreting WGS results. However, as P. aeruginosa is highly recombinant, a cgMLST approach is preferable and caution should be addressed to possible recombination of large DNA fragments.

Evolution and Population Dynamics of Clonal Complex 152 Community-Associated Methicillin-Resistant Staphylococcus aureus.

Since the late 1990s, changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognized with the emergence of community-associated MRSA (CA-MRSA). CA-MRSA belonging to clonal complex 152 (CC152), carrying the small staphylococcal cassette chromosome mec (SCCmec) type V and encoding the Panton-Valentine leukocidin (PVL), has been observed in Europe. The aim of this study was to investigate its origin, evolution, and dissemination. Whole-genome sequencing was performed on a global collection of 149 CC152 isolates spanning 20 years (93 methicillin-susceptible S. aureus [MSSA] and 56 MRSA isolates). Core genome phylogeny, Bayesian inference, in silico resistance analyses, and genomic characterization were applied. Phylogenetic analysis revealed two major distinct clades, one dominated by MSSA and the other populated only by MRSA. The MSSA isolates were predominately from sub-Saharan Africa, whereas MRSA was almost exclusively from Europe. The European MRSA isolates all harbored an SCCmec type V (5C2&5) element, whereas other SCCmec elements were sporadically detected in MRSA from the otherwise MSSA-dominated clade, including SCCmec types IV (2B), V (5C2), and XIII (9A). In total, 93% of the studied CC152 isolates were PVL positive. Bayesian coalescent inference suggests an emergence of the European CC152-MRSA in the 1990s, while the CC152 lineage dates back to the 1970s. The CA-MRSA CC152 clone mimics the European CC80 CA-MRSA lineage by its emergence from a PVL-positive MSSA ancestor from North Africa or Europe. The CC152 lineage has acquired SCCmec several times, but acquisition of SCCmec type V (5C2&5) seems associated with expansion of MRSA CC152 in Europe.IMPORTANCE Understanding the evolution of CA-MRSA is important in light of the increasing importance of this reservoir in the dissemination of MRSA. Here, we highlight the story of the CA-MRSA CC152 lineage using whole-genome sequencing on an international collection of CC152. We show that the evolution of this lineage is novel and that antibiotic usage may have the potential to select for the phage-encoded Panton-Valentine leukocidin. The diversity of the strains correlated highly to geography, with higher level of resistance observed among the European MRSA isolates. The mobility of the SCCmec element is mandatory for the emergence of novel MRSA lineages, and we show here distinct acquisitions, one of which is linked to the successful clone found throughout Europe today.

Combining Standard Molecular Typing and Whole Genome Sequencing to Investigate Pseudomonas aeruginosa Epidemiology in Intensive Care Units.

Pseudomonas aeruginosa is one of the main pathogens responsible for nosocomial infections, particularly in Intensive Care Units (ICUs). Due to the complexity of P. aeruginosa ecology, only powerful typing methods can efficiently allow its surveillance and the detection during expanding outbreaks. An increase in P. aeruginosa incidence was observed in the ICUs of the Lausanne University Hospital between 2010 and 2014. All clinical and environmental isolates retrieved during this period were typed with Double locus sequence typing (DLST), which detected the presence of three major genotypes: DLST 1-18, DLST 1-21, and DLST 6-7. DLST 1-18 (ST1076) isolates were previously associated with an epidemiologically well-described outbreak in the burn unit. Nevertheless, DLST 1-21 (ST253) and DLST 6-7 (ST17) showed sporadic occurrence with only few cases of possible transmission between patients. Whole genome sequencing (WGS) was used to further investigate the epidemiology of these three major P. aeruginosa genotypes in the ICUs. WGS was able to differentiate between outbreak and non-outbreak isolates and confirm suspected epidemiological links. Additionally, whole-genome single nucleotide polymorphisms (SNPs) results considered isolates as closely related for which no epidemiological links were suspected, expanding the epidemiological investigation to unsuspected links. The combination of a first-line molecular typing tool (DLST) with a more discriminatory method (WGS) proved to be an accurate and cost-efficient typing strategy for the investigation of P. aeruginosa epidemiology in the ICUs.

NGS-Based S. aureus Typing and Outbreak Analysis in Clinical Microbiology Laboratories: Lessons Learned From a Swiss-Wide Proficiency Test.

Whole genome sequencing (WGS) enables high resolution typing of bacteria up to the single nucleotide polymorphism (SNP) level. WGS is used in clinical microbiology laboratories for infection control, molecular surveillance and outbreak analyses. Given the large palette of WGS reagents and bioinformatics tools, the Swiss clinical bacteriology community decided to conduct a ring trial (RT) to foster harmonization of NGS-based bacterial typing. The RT aimed at assessing methicillin-susceptible Staphylococcus aureus strain relatedness from WGS and epidemiological data. The RT was designed to disentangle the variability arising from differences in sample preparation, SNP calling and phylogenetic methods. Nine laboratories participated. The resulting phylogenetic tree and cluster identification were highly reproducible across the laboratories. Cluster interpretation was, however, more laboratory dependent, suggesting that an increased sharing of expertise across laboratories would contribute to further harmonization of practices. More detailed bioinformatic analyses unveiled that while similar clusters were found across laboratories, these were actually based on different sets of SNPs, differentially retained after sample preparation and SNP calling procedures. Despite this, the observed number of SNP differences between pairs of strains, an important criterion to determine strain relatedness given epidemiological information, was similar across pipelines for closely related strains when restricting SNP calls to a common core genome defined by S. aureus cgMLST schema. The lessons learned from this pilot study will serve the implementation of larger-scale RT, as a mean to have regular external quality assessments for laboratories performing WGS analyses in a clinical setting.

Automated Incubation and Digital Image Analysis of Chromogenic Media Using Copan WASPLab Enables Rapid Detection of Vancomycin-Resistant Enterococcus.

Objective: The aim of the present study was to assess whether the WASPLab automation enables faster detection of vancomycin-resistant Enterococcus (VRE) on chromogenic VRE-specific plates by shortening the incubation time. Methods: Ninety different VRE culture negative rectal ESwab specimens were spiked with various concentrations (ranging from 3 × 102 to 3 × 107 CFU/ml) of 10 Enterococcus faecium strains (vancomycin MICs ranging from 32 to >256 mg/l), 3 E. faecium VanB strains (vancomycin MICs: 4, 8, and 16 mg/l), and 2 E. faecium VanB strains displaying vancomycin heteroresistance (vancomycin MICs: 64 and 96 mg/l). Results: Besides the two strains exhibiting vancomycin heteroresistance, all the other 13 VRE strains included in this study were detected as early as 24 h on the WASPLab even if the inoculum was low (3 × 103 CFU/ml). When the vancomycin MICs were high, all strains were detected as early as at 18 h. However, 30 h was a conservative time point for finalizing the analysis of chromogenic cultures. Conclusion: These results suggested that the WASPLab automated incubation could allow decreasing the initial incubation time to 18 h, followed by an intermediate time at 24 h and a final incubation period of 30 h for VRE culture screening, to deliver rapid results without affecting the analytical sensitivity.