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The objective of this study was to determine the prevalence, molecular epidemiology, and risk factors for gut colonization with extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), and vancomycin-resistant enterococci (VRE) in healthcare workers (HCWs). In September/October 2022, we performed a cross-sectional study among HCW from 14 institutions in Northeastern Switzerland. HCWs reported risk factors for antimicrobial resistance (covering the last 12-24 months) and provided rectal swabs. Swabs were screened for ESBL-E, CPE, and VRE; whole-genome sequencing (WGS) was performed to assess the genetic relatedness. Logistic regression was used to identify occupational and non-occupational risk factors. Among approximately 22,500 employees, 1,209 participated (median age 46 years, 82% female). Prevalences of ESBL-E ( n = 65) and CPE ( n = 1) were 5.4% [95% confidence interval (CI) 4.2-6.8] and 0.1% (95% CI 0.0-0.5), respectively; no VREs were detected. In the multivariable analysis, non-European ethnicity [adjusted odds ratio (aOR) 7.0, 95% CI 1.4-27.3], travel to high-risk countries (aOR 4.9, 95% CI 2.5-9.3), systemic antibiotics (aOR 2.1, 95% CI 1.1-3.7), antibiotic eye drops (aOR 4.7, 95% CI 1.7-11.9), and monthly sushi consumption (aOR 2.4, 95% CI 1.4-4.3) were positively associated with ESBL-E colonization, whereas alcohol consumption (aOR 0.5 per glass/week, 95% CI 0.3-0.9) was negatively associated with ESBL-E colonization. Occupational factors showed no association. Among ESBL- Escherichia coli , ST131 (15 of 61, 25%) and bla CTX-M-15 (37/61; 61%) were most common; one isolate co-harbored bla OXA-244 . WGS data did not show relevant clustering. Occupational exposure is not associated with ESBL-E colonization in HCW. Given the potential public health and antibiotic stewardship implications, the role of sushi consumption and antibiotic eye drops as risk factors should be further elucidated., Competing Interests: The authors declare no conflict of interest.

An outbreak of OXA-23-producing carbapenem-resistant Acinetobacter baumannii amongst ICU-patients with COVID-19 likely occurred by transmission through inanimate surfaces, potentially facilitated by a contaminated positioning pillow shared between patients. Subsequent rapid spread may have been caused by exposure to respiratory secretions contaminating healthcare worker's gloves and gowns during prone positioning., (© 2024. The Author(s).)

This report describes the characterization of Burkholderia cenocepacia isolates belonging to sequence type (ST)-250, detected in eight patients with cystic fibrosis (CF) in Switzerland. We retrospectively analyzed 18 isolates of B. cenocepacia ST-250 isolated between 2003 and 2015 by whole-genome sequencing and evaluated clinical and epidemiological data. Single nucleotide polymorphism analysis of the B.°cenocepacia ST-250 lineage showed that the isolates from all patients cluster tightly, suggesting that this cluster has a recent common ancestor. Epidemiological investigations showed that six out of eight patients acquired B.°cenocepacia ST-250 in the years 2001-2006, where participation in CF summer camps was common. Two patients were siblings. Genomic relatedness of the B. cenocepacia ST-250 isolates supported transmission by close contact, however, a common source or nosocomial routes cannot be excluded. With respect to the fatal outcome in six patients, our study shows the importance of infection control measurements in CF patients with B.°cenocepacia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Whole genome sequencing (WGS) has become a vital tool in clinical microbiology, playing an important role in outbreak investigations, molecular surveillance, and identification of bacterial species, resistance mechanisms and virulence factors. However, the complexity of WGS data presents challenges in interpretation and reporting, requiring tailored strategies to enhance efficiency and impact. This study explores the diverse needs of key stakeholders in healthcare, including clinical management, laboratory work, public surveillance and epidemiology, infection prevention and control, and academic research, regarding WGS-based reporting of clinically relevant bacterial species. In order to determine preferences regarding WGS reports, human-centered design approach was employed, involving an online survey and a subsequent workshop with stakeholders. The survey gathered responses from 64 participants representing the above mentioned healthcare sectors across geographical regions. Key findings include the identification of barriers related to data accessibility, integration with patient records, and the complexity of interpreting WGS results. As the participants designed their ideal report using nine pre-defined sections of a typical WGS report, differences in needs regarding report structure and content across stakeholders became evident. The workshop discussions further highlighted the need to feature critical findings and quality metrics prominently in reports, as well as the demand for flexible report designs. Commonalities were observed across stakeholder-specific reporting templates, such as the uniform ranking of certain report sections, but preferences regarding the depth of content within these sections varied. Using these findings, we suggest stakeholder-specific structures which should be considered when designing customized reporting templates. In conclusion, this study underscores the importance of tailoring WGS-based reports of clinically relevant bacteria to meet the distinct needs of diverse healthcare stakeholders. The evolving landscape of digital reporting increases the opportunities with respect to WGS reporting and its utility in managing infectious diseases and public health surveillance., Competing Interests: The authors declare that they have no competing interests., (© 2024 Mutschler et al.)

Current methods for plasmid-mediated AmpC β-lactamase (pAmpC) detection in routine microbiological laboratories are based on various phenotypic tests. Eazyplex®SuperBug AmpC assay is a molecular assay based on isothermal amplification for rapid detection of the most common pAmpC types from bacterial culture: CMY-2 group, DHA, ACC and MOX. Our aim was to evaluate the diagnostic performance of this assay. The assay was evaluated on 64 clinical isolates of Enterobacterales without chromosomal inducible AmpC, and with phenotypically confirmed AmpC production. The results were confirmed, and isolates further characterized by whole-genome sequencing (WGS). eazyplex®SuperBug AmpC assay correctly detected the two most common pAmpC types CMY-2 group (16/16) and DHA (19/19). Detection of ACC and MOX could not be evaluated on our set of isolates since there was only one isolate harbouring ACC and none with MOX. pAmpC encoding genes could be detected in only eight of 36 investigated Escherichia coli isolates. The remaining 28 E. coli isolates harboured previously described mutations in the bla EC promoter, leading to the overexpression of chromosomally encoded E. coli specific AmpC β-lactamase. All results were 100% concordant with the results of WGS. eazyplex®SuperBug AmpC assay enabled rapid and reliable detection of pAmpC-encoding genes in Enterobacterales like Klebsiella spp. and Proteus spp. and the distinction between plasmid-mediated and chromosomally encoded AmpC in E. coli., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Few methicillin-resistant Staphylococcus aureus (MRSA) from the early years of its global emergence have been sequenced. Knowledge about evolutionary factors promoting the success of specific MRSA multi-locus sequence types (MLSTs) remains scarce. We aimed to characterize a legacy MRSA collection isolated from 1965 to 1987 and compare it against publicly available international and local genomes., Methods: We accessed 451 historic (1965-1987) MRSA isolates stored in the Culture Collection of Switzerland, mostly collected from the Zurich region. We determined phenotypic antimicrobial resistance (AMR) and performed whole genome sequencing (WGS) using Illumina short-read sequencing on all isolates and long-read sequencing on a selection with Oxford Nanopore Technology. For context, we included 103 publicly available international assemblies from 1960 to 1992 and sequenced 1207 modern Swiss MRSA isolates from 2007 to 2022. We analyzed the core genome (cg)MLST and predicted SCCmec cassette types, AMR, and virulence genes., Results: Among the 451 historic Swiss MRSA isolates, we found 17 sequence types (STs) of which 11 have been previously described. Two STs were novel combinations of known loci and six isolates carried previously unsubmitted MLST alleles, representing five new STs (ST7843, ST7844, ST7837, ST7839, and ST7842). Most isolates (83% 376/451) represented ST247-MRSA-I isolated in the 1960s, followed by ST7844 (6% 25/451), a novel single locus variant (SLV) of ST239. Analysis by cgMLST indicated that isolates belonging to ST7844-MRSA-III cluster within the diversity of ST239-MRSA-III. Early MRSA were predominantly from clonal complex (CC)8. From 1980 to the end of the twentieth century, we observed that CC22 and CC5 as well as CC8 were present, both locally and internationally., Conclusions: The combined analysis of 1761 historic and contemporary MRSA isolates across more than 50 years uncovered novel STs and allowed us a glimpse into the lineage flux between Swiss-German and international MRSA across time., (© 2024. The Author(s).)

During the SARS-CoV-2 pandemic, the Dr. Risch medical group employed the multiplex TaqPath TM COVID-19 CE-IVD RT-PCR Kit for large-scale routine diagnostic testing in Switzerland and the principality of Liechtenstein. The TaqPath Kit is a widely used multiplex assay targeting three genes (i.e., ORF1AB, N, S). With emergence of the B.1.1.7 (Alpha) variant, a diagnostic flaw became apparent as the amplification of the S-gene target was absent in these samples due to a deletion (ΔH69/V70) in the Alpha variant genome. This S-gene target failure (SGTF) was the earliest indication of a new variant emerging and was also observed in subsequent variants such as Omicron BA.1 and BA4/BA.5. The Delta variant and Omicron BA.2 did not present with SGTF. From September 2020 to November 2022, we investigated the applicability of the SGTF as a surrogate marker for emerging variants such as B.1.1.7, B.1.617.2 (Delta), and Omicron BA.1, BA.2, and BA.4/BA.5 in samples with cycle threshold (Ct) values < 30. Next to true SGTF-positive and SGTF-negative samples, there were also samples presenting with delayed-type S-gene amplification (higher Ct value for S-gene than ORF1ab gene). Among these, a difference of 3.8 Ct values between the S- and ORF1ab genes was found to best distinguish between "true" SGTF and the cycle threshold variability of the assay. Samples above the cutoff were subsequently termed partial SGTF (pSGTF). Variant confirmation was performed by whole-genome sequencing (Oxford Nanopore Technology, Oxford, UK) or mutation-specific PCR (TIB MOLBIOL). In total, 17,724 (7.4%) samples among 240,896 positives were variant-confirmed, resulting in an overall sensitivity and specificity of 93.2% [92.7%, 93.7%] and 99.3% [99.2%, 99.5%], respectively. Sensitivity was increased to 98.2% [97.9% to 98.4%] and specificity lowered to 98.9% [98.6% to 99.1%] when samples with pSGTF were included. Furthermore, weekly logistic growth rates (α) and sigmoid's midpoint (t 0 ) were calculated based on SGTF data and did not significantly differ from calculations based on comprehensive data from GISAID. The SGTF therefore allowed for a valid real-time estimate for the introduction of all dominant variants in Switzerland and Liechtenstein.

Background: Reliable species identification of cultured isolates is essential in clinical bacteriology. We established a new study algorithm named NOVA - Novel Organism Verification and Analysis to systematically analyze bacterial isolates that cannot be characterized by conventional identification procedures MALDI-TOF MS and partial 16 S rRNA gene sequencing using Whole Genome Sequencing (WGS)., Results: We identified a total of 35 bacterial strains that represent potentially novel species. Corynebacterium sp. (n = 6) and Schaalia sp. (n = 5) were the predominant genera. Two strains each were identified within the genera Anaerococcus, Clostridium, Desulfovibrio, and Peptoniphilus, and one new species was detected within Citrobacter, Dermabacter, Helcococcus, Lancefieldella, Neisseria, Ochrobactrum (Brucella), Paenibacillus, Pantoea, Porphyromonas, Pseudoclavibacter, Pseudomonas, Psychrobacter, Pusillimonas, Rothia, Sneathia, and Tessaracoccus. Twenty-seven of 35 strains were isolated from deep tissue specimens or blood cultures. Seven out of 35 isolated strains identified were clinically relevant. In addition, 26 bacterial strains that could only be identified at the species level using WGS analysis, were mainly organisms that have been identified/classified very recently., Conclusion: Our new algorithm proved to be a powerful tool for detection and identification of novel bacterial organisms. Publicly available clinical and genomic data may help to better understand their clinical and ecological role. Our identification of 35 novel strains, 7 of which appear to be clinically relevant, shows the wide range of undescribed pathogens yet to define., (© 2023. The Author(s).)

Background: Urinary tract infections (UTIs) are among the most common bacterial infections worldwide, often caused by uropathogenic Escherichia coli. Multiple bacterial virulence factors or patient characteristics have been linked separately to progressive, more invasive infections. In this study, we aim to identify pathogen- and patient-specific factors that drive the progression to urosepsis by jointly analysing bacterial and host characteristics., Methods: We analysed 1076 E. coli strains isolated from 825 clinical cases with UTI and/or bacteraemia by whole-genome sequencing (Illumina). Sequence types (STs) were determined via srst2 and capsule loci via fastKaptive. We compared the isolates from urine and blood to confirm clonality. Furthermore, we performed a bacterial genome-wide association study (bGWAS) (pyseer) using bacteraemia as the primary clinical outcome. Clinical data were collected by an electronic patient chart review. We concurrently analysed the association of the most significant bGWAS hit and important patient characteristics with the clinical endpoint bacteraemia using a generalised linear model (GLM). Finally, we designed qPCR primers and probes to detect papGII-positive E. coli strains and prospectively screened E. coli from urine samples (n = 1657) at two healthcare centres., Results: Our patient cohort had a median age of 75.3 years (range: 18.00-103.1) and was predominantly female (574/825, 69.6%). The bacterial phylogroups B2 (60.6%; 500/825) and D (16.6%; 137/825), which are associated with extraintestinal infections, represent the majority of the strains in our collection, many of which encode a polysaccharide capsule (63.4%; 525/825). The most frequently observed STs were ST131 (12.7%; 105/825), ST69 (11.0%; 91/825), and ST73 (10.2%; 84/825). Of interest, in 12.3% (13/106) of cases, the E. coli pairs in urine and blood were only distantly related. In line with previous bGWAS studies, we identified the gene papGII (p-value < 0.001), which encodes the adhesin subunit of the E. coli P-pilus, to be associated with 'bacteraemia' in our bGWAS. In our GLM, correcting for patient characteristics, papGII remained highly significant (odds ratio = 5.27, 95% confidence interval = [3.48, 7.97], p-value < 0.001). An independent cohort of cases which we screened for papGII-carrying E. coli at two healthcare centres further confirmed the increased relative frequency of papGII-positive strains causing invasive infection, compared to papGII-negative strains (p-value = 0.033, chi-squared test)., Conclusions: This study builds on previous work linking papGII with invasive infection by showing that it is a major risk factor for progression from UTI to bacteraemia that has diagnostic potential., (© 2023. The Author(s).)

The reference method for cefiderocol antimicrobial susceptibility testing is broth microdilution (BMD) with iron-depleted-Mueller-Hinton (ID-MH) medium, whereas breakpoints recommended for disk diffusion (DD) are based on MH-agar plates. We aimed to compare the performance of the commercial BMD tests ComASP (Liofilchem) and UMIC (Bruker), and DD and E-test using MH- and ID-MH-agar plates with the reference BMD method using 100 carbapenem-resistant- A. baumannii isolates. Standard BMD was performed according to the EUCAST guidelines; DD and E-test were carried out using two commercial MH-agar plates (BioMérieux and Liofilchem) and an in-house ID-MH-agar plate, while ComASP and UMIC were performed according to the manufacturer's guidelines. DD performed with the ID-MH-agar plates led to a higher categorical agreement (CA, 95.1%) with standard BMD and fewer categorization errors compared to the commercial MH-agar plates (CA BioMérieux 91.1%, Liofilchem 89.2%). E-test on ID-MH-agar plates exhibited a significantly higher essential agreement (EA, 75%) with standard BMD compared to the two MH-agar plates (EA BioMérieux 57%, Liofilchem 44%), and showed a higher performance in detecting high-level resistance than ComASP and UMIC (mean log2 difference with standard BMD for resistant isolates of 0.5, 2.83, and 2.08, respectively). In conclusion, DD and E-test on ID-MH-agar plates exhibit a higher diagnostic performance than on MH-agar plates and the commercial BMD methods. Therefore, we recommend using ID-MH-agar plates for cefiderocol susceptibility testing of A. baumannii .

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.

Background: We sought to decipher transmission pathways in healthcare-associated infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within our hospital by epidemiological work-up and complementary whole genome sequencing (WGS). We report the findings of the four largest epidemiologic clusters of SARS-CoV-2 transmission occurring during the second wave of the pandemic from 11/2020 to 12/2020., Methods: At the University Hospital Basel, Switzerland, systematic outbreak investigation is initiated at detection of any nosocomial case of SARS-CoV-2 infection, as confirmed by polymerase chain reaction, occurring more than five days after admission. Clusters of nosocomial infections, defined as the detection of at least two positive patients and/or healthcare workers (HCWs) within one week with an epidemiological link, were further investigated by WGS on respective strains., Results: The four epidemiologic clusters included 40 patients and 60 HCWs. Sequencing data was available for 70% of all involved cases (28 patients and 42 HCWs), confirmed epidemiologically suspected in house transmission in 33 cases (47.1% of sequenced cases) and excluded transmission in the remaining 37 cases (52.9%). Among cases with identical strains, epidemiologic work-up suggested transmission mainly through a ward-based exposure (24/33, 72.7%), more commonly affecting HCWs (16/24, 66.7%) than patients (8/24, 33.3%), followed by transmission between patients (6/33, 18.2%), and among HCWs and patients (3/33, 9.1%, respectively two HCWs and one patient)., Conclusions: Phylogenetic analyses revealed important insights into transmission pathways supporting less than 50% of epidemiologically suspected SARS-CoV-2 transmissions. The remainder of cases most likely reflect community-acquired infection randomly detected by outbreak investigation. Notably, most transmissions occurred between HCWs, possibly indicating lower perception of the risk of infection during contacts among HCWs., (© 2023. The Author(s).)

Campylobacteriosis is the most common cause of acute gastrointestinal bacterial infection in Europe, with most infections linked to the consumption of contaminated food. While previous studies found an increasing rate of antimicrobial resistance (AMR) in Campylobacter spp. over the past decades, the investigation of additional clinical isolates is likely to provide novel insights into the population structure and mechanisms of virulence and drug resistance of this important human pathogen. Therefore, we combined whole-genome sequencing and antimicrobial-susceptibility testing of 340 randomly selected Campylobacter jejuni isolates from humans with gastroenteritis, collected in Switzerland over an 18 year period. In our collection, the most common multilocus sequence types (STs) were ST-257 ( n =44), ST-21 ( n =36) and ST-50 ( n =35); the most common clonal complexes (CCs) were CC-21 ( n =102), CC-257 ( n =49) and CC-48 ( n =33). High heterogeneity was observed among STs, with the most abundant STs recurring over the entire study period, while others were observed only sporadically. Source attribution based on ST assigned more than half of the strains to the 'generalist' category ( n =188), 25  % as 'poultry specialist' ( n =83), and only a few to 'ruminant specialist' ( n =11) or 'wild bird' origin ( n =9). The isolates displayed an increased frequency of AMR from 2003 to 2020, with the highest rates of resistance observed for ciprofloxacin and nalidixic acid (49.8 %), followed by tetracycline (36.9 %). Quinolone-resistant isolates carried chromosomal gyrA mutations T86I (99.4 %) and T86A (0.6 %), whereas tetracycline-resistant isolates carried tet(O ) (79.8 %) or mosaic tetO/32/O (20.2 %) genes. A novel chromosomal cassette carrying several resistance genes, including aph(3')-III , satA and aad (6), and flanked by insertion sequence elements was detected in one isolate. Collectively, our data revealed an increasing prevalence of resistance to quinolones and tetracycline in C. jejuni isolates from Swiss patients over time, linked to clonal expansion of gyrA mutants and acquisition of the tet(O ) gene. Investigation of source attribution suggests that infections are most likely related to isolates from poultry or generalist backgrounds. These findings are relevant to guide future infection prevention and control strategies.

The increasing integration of genomics into routine clinical diagnostics requires reliable computational tools to identify determinants of antimicrobial resistance (AMR) from whole-genome sequencing data. Here, we developed PorinPredict, a bioinformatic tool that predicts defects of the Pseudomonas aeruginosa outer membrane porin OprD, which are strongly associated with reduced carbapenem susceptibility. PorinPredict relies on a database of intact OprD variants and reports inactivating mutations in the coding or promoter region. PorinPredict was validated against 987 carbapenemase-negative P. aeruginosa genomes, of which OprD loss was predicted for 454 out of 522 (87.0%) meropenem-nonsusceptible and 46 out of 465 (9.9%) meropenem-susceptible isolates. OprD loss was also found to be common among carbapenemase-producing isolates, resulting in even further increased MICs. Chromosomal mutations in quinolone resistance-determining regions and OprD loss commonly co-occurred, likely reflecting the restricted use of carbapenems for multidrug-resistant infections as recommended in antimicrobial stewardship programs. In combination with available AMR gene detection tools, PorinPredict provides a robust and standardized approach to link P. aeruginosa phenotypes to genotypes. IMPORTANCE Pseudomonas aeruginosa is a major cause of multidrug-resistant nosocomial infections. The emergence and spread of clones exhibiting resistance to carbapenems, a class of critical last-line antibiotics, is therefore closely monitored. Carbapenem resistance is frequently mediated by chromosomal mutations that lead to a defective outer membrane porin OprD. Here, we determined the genetic diversity of OprD variants across the P. aeruginosa population and developed PorinPredict, a bioinformatic tool that enables the prediction of OprD loss from whole-genome sequencing data. We show a high correlation between predicted OprD loss and meropenem nonsusceptibility irrespective of the presence of carbapenemases, which are a second widespread determinant of carbapenem resistance. Isolates with resistance determinants to other antibiotics were disproportionally affected by OprD loss, possibly due to an increased exposure to carbapenems. Integration of PorinPredict into genomic surveillance platforms will facilitate a better understanding of the clinical impact of OprD modifications and transmission dynamics of resistant clones.

A novel multidrug resistance conjugative 177,859-bp IncC plasmid pJEF1-OXA-181 coharboring the carbapenemase-coding bla OXA181 and the aminoglycoside resistance 16S rRNA methyltransferase-coding armA genes was detected in two unrelated Escherichia coli gut isolates of ST196 and ST648, as well as two ST35 Klebsiella pneumoniae gut and sputum isolates of a cystic fibrosis patient. The armA gene was located within the antimicrobial resistance island ARI-A and the bla OXA181 gene, which was preceded by IS 903 and IS Ecp1Δ was inserted within the transfer genes region without affecting conjugation ability. Comparative plasmid analysis with other related IncC plasmids showed the presence of bla OXA181 , as well as its integration site, are thus far unique for these types of plasmids. This study illustrates the potential of a promiscuous multidrug resistance plasmid to acquire antibiotic resistance genes and to disseminate in the gut of the same host. IMPORTANCE Colocalization of carbapenemases and aminoglycoside resistance 16S rRNA methylases on a multidrug resistance conjugative plasmid poses a serious threat to public health. Here, we describe the novel IncC plasmid pJEF1-OXA-181 cocarrying bla OXA-181 and armA as well as several other antimicrobial resistance genes (ARGs) in different Enterobacterales isolates of the sputum and gut microbiota of a cystic fibrosis patient. IncC plasmids are conjugative, promiscuous elements which can incorporate accessory antimicrobial resistance islands making them key players in ARGs spread. This plasmid was thus far unique among IncC plasmids to contain a bla OXA-181 which was integrated in the transfer gene region without affecting its conjugation ability. This study highlights that new plasmids may be introduced into a hospital through different species hosted in one single patient. It further emphasizes the need of continuous surveillance of multidrug-resistant bacteria in patients at risk to avoid spread of such plasmids in the health care system.

The Bacillus cereus -group ( B. cereus sensu lato ) includes common, usually avirulent species, often considered contaminants of patient samples in routine microbiological diagnostics, as well as the highly virulent B. anthracis . Here we describe 16 isolates from 15 patients, identified as B. cereus -group using a MALDI-TOF MS standard database. Whole genome sequencing (WGS) analysis identified five of the isolates as B. anthracis species not carrying the typical virulence plasmids pXO1 and pXO2, four isolates as B. paranthracis, three as B. cereus sensu stricto , two as B. thuringiensis , one as B. mobilis , and one isolate represents a previously undefined species of Bacillus ( B. basilensis sp. nov.). More detailed analysis using alternative MALDI-TOF MS databases, biochemical phenotyping, and diagnostic PCRs, gave further conflicting species results. These cases highlight the difficulties in identifying avirulent B. anthracis within the B. cereus -group using standard methods. WGS and alternative MALDI-TOF MS databases offer more accurate species identification, but so far are not routinely applied. We discuss the diagnostic resolution and discrepancies of various identification methods., (© 2022 The Authors.)

Background: The source of transmission of Clostridioides difficile in healthcare institutions is frequently unknown. The aim of this prospective cohort study was to assess the association between strains cultured from patients and shoe soles of healthcare workers (HCWs), as already shown in the operating theatre, but not on general hospital wards in an acute-care institution., Methods: We conducted a study at a university tertiary care centre in Switzerland. From October 2019 to July 2020, shoe soles of HCWs were cultured for C. difficile twice per shift while taking care of a patient infected with toxigenic C. difficile. Additional risk factors were assessed by interviewing involved HCWs. Patients' faecal samples were processed by routine microbiological methods. Similarity of the HCWs' and patients' strains was determined by whole-genome sequencing (WGS)., Results: A total of 103 HCWs exposed to 42 hospitalized patients participated in the study, providing 206 samples. Contamination of shoe soles with C. difficile was detected in 37 samples (17.8%) of HCWs taking care of patients infected with C. difficile. Overall, transmission was suspected by epidemiological link and matching strains demonstrated by WGS in 74%., Conclusions: HCWs' shoe soles were positive in 17.8% with C. difficile strains linked epidemiologically and confirmed by WGS to infected patients suggesting potential transmission by HCWs' shoe soles. This pilot study provides sufficient evidence to further evaluate this potential mode of healthcare-associated transmission of C. difficile by a larger clinical trial., (Copyright © 2022 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

(1) Background: Some COVID-19 vaccine recipients show breakthrough infection. It remains unknown, which factors contribute to risks and severe outcomes. Our aim was to identify risk factors for SCoV2 breakthrough infections in fully vaccinated individuals. (2) Methods: We conducted a retrospective case-control study from 28 December 2020 to 25 October 2021. Data of all patients with breakthrough infection was compared to data of all vaccine recipients in the Canton of Basel-City, Switzerland. Further, breakthrough infections by Alpha- and Delta-variants were compared. (3) Results: Only 0.39% (488/126,586) of all vaccine recipients suffered from a breakthrough infection during the observational period, whereof most cases were asymptomatic or mild (97.2%). Breakthrough infections after full vaccination occurred in the median after 78 days (IQR 47-123.5). Factors with lower odds for breakthrough infection were age (OR 0.987) and previous COVID-19 infection prior to vaccination (OR 0.296). Factors with higher odds for breakthrough infection included vaccination with Pfizer/BioNTech instead of Moderna (OR 1.459), chronic disease (OR 2.109), and healthcare workers (OR 1.404). (4) Conclusions: Breakthrough infections are rare and mild but can occur early after vaccination. This implies that booster vaccination might be initiated earlier, especially for risk groups. Due to new variants emerging repeatedly, continuous monitoring of breakthrough infections is crucial.

Background: The optimal extent of screening of contact patients (CoPat) after exposure to patients infected or colonized with vancomycin-resistant enterococci (VRE) remains controversial., Methods: We retrospectively developed a new risk stratification for screening patients exposed to VRE, based on data from three outbreaks-two with Enterococcus faecium vanB and one with Enterococcus faecium vanA involving 1096 CoPat-in a low endemic setting. We classified them into four risk groups: three on environmental exposure, one by healthcare exposure: high (sharing the same room/bathroom with a VRE-colonized patient), medium (hospitalization in the same room after a VRE-colonized patient's discharge until terminal disinfection including ultraviolet C (UVc)-disinfection), low (hospitalized in the same room within three weeks before the VRE-colonized patient), and "staff" (screening of patients having the same medical care team)., Results: VRE-transmission occurred in 7.9% in the high-risk group compared to 0.6% and 0% in the medium and low risk groups. There was a significant trend to higher rates of transmission by risk level of exposure (p < 0.001). In the "staff" group, VRE transmission rate was 2.3%., Conclusion: Based on this stratification, we recommend to focus screening of exposed CoPat on the high-risk and "staff" group, saving resources and costs, but larger studies will allow to further improve the yield of VRE screening in the outbreak setting., (© 2022. The Author(s).)

We present polyphasic taxonomic data to demonstrate that strain 125703-2019 T , a human blood isolate, represents a novel species within the genus Pseudoclavibacter, and to reclassify the illegitimate Zimmermannella alba Lin et al., 2004 as Pseudoclavibacter albus comb. nov. Upon primary isolation, strain 125703-2019 T could not be identified reliably using MALDI-TOF mass spectrometry during routine diagnostic work, but partial 16S rRNA gene sequence analysis revealed that it belonged to the genus Pseudoclavibacter. Average nucleotide identity and digital DNA-DNA hybridisation analyses confirmed that it represented a novel species within this genus. A detailed physiological characterisation yielded differential tests between the novel species and its nearest neighbor taxa, which could also be differentiated using MALDI-TOF mass spectrometry. We propose to formally classify this strain into the novel species Pseudoclavibacter triregionum sp. nov., with strain 125703-2019 T (= R-76471 T , LMG 31777 T , CCUG 74796 T ) as the type strain. The whole-genome assembly of strain 125703-2019 T has a size of 2.4 Mb and a G + C content of 72.74%. A Pseudoclavibacter pangenome analysis revealed that 667 gene clusters were exclusively present in strain 125703-2019 T . While these gene clusters were enriched in several COG functional categories, this analysis did not reveal functions that explained the occurrence of this species in human infection. Finally, several phylogenetic and phylogenomic analyses demonstrated that the genus Pseudoclavibacter is polyphyletic with Pseudoclavibacter soli and Pseudoclavibacter caeni representing a unique and deeply branching line of descent within the family Microbacteriaceae. We therefore also propose to reclassify both species into the novel genus Caespitibacter gen. nov. as Caespitibacter soli comb. nov. and Caespitibacter caeni comb. nov., respectively, and with C. soli comb. nov. as the type species., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Transmission chains within small urban areas (accommodating ∼30 per cent of the European population) greatly contribute to case burden and economic impact during the ongoing coronavirus pandemic and should be a focus for preventive measures to achieve containment. Here, at very high spatio-temporal resolution, we analysed determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in a European urban area, Basel-City (Switzerland). We combined detailed epidemiological, intra-city mobility and socio-economic data sets with whole-genome sequencing during the first SARS-CoV-2 wave. For this, we succeeded in sequencing 44 per cent of all reported cases from Basel-City and performed phylogenetic clustering and compartmental modelling based on the dominating viral variant (B.1-C15324T; 60 per cent of cases) to identify drivers and patterns of transmission. Based on these results we simulated vaccination scenarios and corresponding healthcare system burden (intensive care unit (ICU) occupancy). Transmissions were driven by socio-economically weaker and highly mobile population groups with mostly cryptic transmissions which lacked genetic and identifiable epidemiological links. Amongst more senior population transmission was clustered. Simulated vaccination scenarios assuming 60-90 per cent transmission reduction and 70-90 per cent reduction of severe cases showed that prioritising mobile, socio-economically weaker populations for vaccination would effectively reduce case numbers. However, long-term ICU occupation would also be effectively reduced if senior population groups were prioritised, provided there were no changes in testing and prevention strategies. Reducing SARS-CoV-2 transmission through vaccination strongly depends on the efficacy of the deployed vaccine. A combined strategy of protecting risk groups by extensive testing coupled with vaccination of the drivers of transmission (i.e. highly mobile groups) would be most effective at reducing the spread of SARS-CoV-2 within an urban area., (© The Author(s) 2022. Published by Oxford University Press.)

Background: Agrobacterium spp. are infrequent agents of bloodstream infections linked to healthcare-associated outbreaks. However, it is unclear if outbreaks also occur across larger geographic areas. Triggered by two local clusters from putative point sources, our aim was to detect potential additional clusters in Switzerland., Methods: We performed a nationwide descriptive study of cases in Switzerland based on a prospective surveillance system (Swiss Centre for Antibiotic Resistance, anresis.ch), from 2008 to 2019. We identified patients with Agrobacterium spp. isolated from blood cultures and used a survey to collect clinical-epidemiological information and susceptibility testing results. We performed whole genome sequencing (WGS) of available clinical isolates and determined their relatedness by single nucleotide polymorphism (SNP) variant calling analysis., Results: We identified a total of 36 cases of Agrobacterium spp. from blood samples over 10 years. Beyond previously known local clusters, no new ones were identified. WGS-based typing was performed on 22 available isolates and showed no clonal relationships between newly identified isolates or to those from the known clusters, with all isolates outside these clusters being at least 50 SNPs apart., Conclusion and Relevance: Agrobacterium spp. bacteraemia is infrequently detected and, given that it may be healthcare-associated and stem from a point source, occurrence of multiple episodes should entail an outbreak investigation. With the help of the national antimicrobial resistance surveillance system we identified multiple clinical cases of this rare pathogen but found no evidence by WGS that suggested a nation-wide outbreak., (© 2022. The Author(s).)

Objectives: We aimed to assess the burden of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales in Swiss long-term care facilities (LTCFs) to describe the molecular epidemiology, describe the intrainstitutional and regional clusters of resistant pathogens, and identify independent institution- and resident-level factors associated with colonization., Design: Cross-sectional study., Setting and Participants: From August to October 2019, we performed a point prevalence study among residents from 16 LTCFs in Western and Eastern Switzerland (8 per region)., Methods: Residents underwent screening for ESBL-producing Enterobacterales (ESBL-E); whole-genome sequencing (WGS) was performed. We gathered institution-level (eg, number of beds, staff-resident ratio, alcoholic hand rub consumption) and resident-level [eg, anthropometric data, time in facility, dependency, health care exposure, antibiotic treatment, proton-pump inhibitor (PPI) use] characteristics. Factors associated with colonization were identified using a generalized linear model., Results: Among 1185 eligible residents, 606 (51%) consented to the study. ESBL-E prevalence was 11.6% (70/606), ranging from 1.9% to 33.3% between institutions, with a median of 12.5% in the West and 6.9% in the East (P = .03). Among 59 Escherichia coli (from 58 residents), multilocus sequence type (ST) 131 was most common (n = 43/59, 73%), predominantly its subclone H30R1 (n = 37/43, 86%). WGS data identified multiple intrainstitutional and regional clusters. Independent risk factors for ESBL carriage were previous ESBL colonization [adjusted odds ratio (aOR) 23.5, 95% confidence interval (CI) 6.6-83.8, P < .001), male gender (aOR 2.6, 95% CI 1.5-4.6, P = .002), and use of PPIs (aOR 2.2, 95% CI 1.2-3.8, P = .01)., Conclusions and Implications: Overall ESBL-E prevalence in Swiss LTCF residents is low. Yet, we identified several clusters of residents with identical pathogens within the same institution. This implies that particularly affected institutions might benefit from targeted infection control interventions. PPI use was the only modifiable factor associated with carriage of ESBL producers. This study adds to the growing list of adverse outcomes associated with PPIs, calling for action to restrict their use in the long-term care setting., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

The new SARS-CoV-2 Omicron variant (B.1.1.529) has been recently declared a Variant of Concern due to a series of important mutations in the viral spike protein and especially in the receptor-binding domain. While investigations into the spread of this new variant are ongoing, the first cases have been detected in Switzerland. Important questions have been raised: (1) Will the PCR assays commonly used to detect SARS-CoV-2 still work for the Omicron variant? (2) Can specific PCR features, e.g. S-gene dropout, be used to identify potential Omicron samples? In this minireview we provide current knowledge on the Omicron variant and guidance on its PCR validation.

Background: Colistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e. mgrB, phoPQ, pmrCAB). Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97 Enterobacterales and non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation., Results: Of 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these, mcr-1 was detected in four isolates and mcr-2 in one isolate. Non-synonymous mutations in mgrB, phoQ, pmrA, pmrB, and pmrC genes were encountered in Klebsiella pneumoniae, Escherichia coli, and Acinetobacter bereziniae resistant isolates. Mutations found in mgrB and pmrB were only identified in isolates exhibiting MICs of ≥16 mg/L., Conclusions: The Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance., (© 2021. The Author(s).)

We present genomic, phylogenomic, and phenotypic taxonomic data to demonstrate that three human ear isolates represent a novel species within the genus Gulosibacter. These isolates could not be identified reliably using MALDI-TOF mass spectrometry during routine diagnostic work, but partial 16S rRNA gene sequence analysis revealed that they belonged to the genus Gulosibacter. Overall genomic relatedness indices between the draft genome sequences of the three isolates and of the type strains of established Gulosibacter species confirmed that the three isolates represented a single novel Gulosibacter species. A biochemical characterisation yielded differential tests between the novel and established Gulosibacter species, which could also be differentiated using MALDI-TOF mass spectrometry. We propose to formally classify these three isolates into Gulosibacter hominis sp. nov., with 401352-2018  T (= LMG 31778  T , CCUG 74795  T ) as the type strain. The whole-genome sequence of strain 401352-2018  T has a size of 2,340,181 bp and a G+C content of 62.04 mol%. A Gulosibacter pangenome analysis revealed 467 gene clusters that were exclusively present in G. hominis genomes. While these G. hominis specific gene clusters were enriched in several COG functional categories, this analysis did not reveal functions that suggested a role in the human microbiome, nor did it explain the occurrence of G. hominis in ear infections. The absence of acquired antimicrobial resistance determinants and virulence factors in the G. hominis genomes, and an analysis of publicly available 16S rRNA gene sequences and 16S rRNA amplicon sequencing data sets suggested that G. hominis is a member of the human skin microbiota that may occasionally be involved in opportunistic infections., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Conflicting results have been published on the impact of contact precautions (CPs) on reduction of transmission of multi-drug-resistant micro-organisms (MDROs) in the endemic setting. Ambiguous definitions coupled with low adherence partly explain these differences., Aim: We prospectively monitored the level of adherence to CPs and aimed to relate it to in-hospital transmission of MDROs., Methods: Between January 2016 and March 2018, all patients under CPs underwent continuous monitoring of adherence to CPs by routine on-site visits on days 0, 3 and 7 after initiating CPs using a standardized checklist. The protocol included 10 interventions that were routinely checked such as CP sign at the door as well as wearing of gowns and gloves upon entry to the patient room. Patients requiring CPs were defined as colonized or infected with MDROs (meticillin-resistant Staphylococcus aureus (MRSA), non-Escherichia coli extended-spectrum beta lactamase (ESBL) Enterobacterales, vancomycin-resistant enterococci (VRE) and carbapenem-resistant Gram-negative micro-organisms (CRGN)) as well as patients infected with respiratory viruses, norovirus, scabies and hypervirulent strains of Clostridioides difficile., Findings: Overall, data from 13,756 CP records from 1378 visits of 812 patients were analysed. Adherence varied between 93% and 100% for each intervention, except for "separate space for contaminated material" with an adherence of 5.3-6.1%. The incidence of in-hospital transmission during the study period was extremely low for MRSA, VRE, non-E.coli ESBL Enterobacterales and CRGN with 0.00-0.064 cases/1000 patient days., Conclusion: High adherence coupled with continuous monitoring of CPs correlated with a very low in-hospital transmission rate. These results indicate that CPs are highly effective if routine monitoring of adherence is implemented., (Copyright © 2021 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Background: Klebsiella spp. are opportunistic pathogens which can cause severe infections, are often multi-drug resistant and are a common cause of hospital-acquired infections. Multiple new Klebsiella species have recently been described, yet their clinical impact and antibiotic resistance profiles are largely unknown. We aimed to explore Klebsiella group- and species-specific clinical impact, antimicrobial resistance (AMR) and virulence., Methods: We analysed whole-genome sequence data of a diverse selection of Klebsiella spp. isolates and identified resistance and virulence factors. Using the genomes of 3594 Klebsiella isolates, we predicted the masses of 56 ribosomal subunit proteins and identified species-specific marker masses. We then re-analysed over 22,000 Matrix-Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectra routinely acquired at eight healthcare institutions in four countries looking for these species-specific markers. Analyses of clinical and microbiological endpoints from a subset of 957 patients with infections from Klebsiella species were performed using generalized linear mixed-effects models., Results: Our comparative genomic analysis shows group- and species-specific trends in accessory genome composition. With the identified species-specific marker masses, eight Klebsiella species can be distinguished using MALDI-TOF MS. We identified K. pneumoniae (71.2%; n = 12,523), K. quasipneumoniae (3.3%; n = 575), K. variicola (9.8%; n = 1717), "K. quasivariicola" (0.3%; n = 52), K. oxytoca (8.2%; n = 1445), K. michiganensis (4.8%; n = 836), K. grimontii (2.4%; n = 425) and K. huaxensis (0.1%; n = 12). Isolates belonging to the K. oxytoca group, which includes the species K. oxytoca, K. michiganensis and K. grimontii, were less often resistant to 4th-generation cephalosporins than isolates of the K. pneumoniae group, which includes the species K. pneumoniae, K. quasipneumoniae, K. variicola and "K. quasivariicola" (odds ratio = 0.17, p < 0.001, 95% confidence interval [0.09,0.28]). Within the K. pneumoniae group, isolates identified as K. pneumoniae were more often resistant to 4th-generation cephalosporins than K. variicola isolates (odds ratio = 2.61, p = 0.003, 95% confidence interval [1.38,5.06]). K. oxytoca group isolates were found to be more likely associated with invasive infection to primary sterile sites than K. pneumoniae group isolates (odds ratio = 2.39, p = 0.0044, 95% confidence interval [1.05,5.53])., Conclusions: Currently misdiagnosed Klebsiella spp. can be distinguished using a ribosomal marker-based approach for MALDI-TOF MS. Klebsiella groups and species differed in AMR profiles, and in their association with invasive infection, highlighting the importance for species identification to enable effective treatment options., (© 2021. The Author(s).)

Clostridioides difficile causes nosocomial outbreaks which can lead to severe and even life-threatening colitis. Rapid molecular diagnostic tests allow the identification of toxin-producing, potentially hypervirulent strains, which is critical for patient management and infection control. PCR-ribotyping has been used for decades as the reference standard to investigate transmission in suspected outbreaks. However, the introduction of whole genome sequencing (WGS) for molecular epidemiology provides a realistic alternative to PCR-ribotyping. In this transition phase it is crucial to understand the strengths and weaknesses of the two technologies, and to assess their correlation. We aimed to investigate ribotype prediction from WGS data, and options for analysis at different levels of analytical granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons including the ribotype-defining ISR fragments collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. Ribotype extraction from long read Oxford nanopore data also requires optimization. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from Switzerland, and in addition from the Enterobase database (n=1778). Our results show that while multi-locus sequence type (MLST) often correlates with a specific ribotype, the agreement is not complete, and for some ribotypes the resolution is insufficient. Using core genome MLST (cgMLST) analysis, there is an improved resolution and ribotypes can often be predicted within clusters, using cutoffs of 30-50 allele differences. The exceptions are ribotypes within known ribotype complexes such as RT078/RT106, where the genome differences in cgMLST do not reflect the ribotype segregation. We show that different ribotype clusters display different degrees of diversity, which could be important for the definition of ribotype cluster specific cutoffs. WGS-based analysis offers the ultimate resolution to the SNP level, enabling exploration of patient-to-patient transmission. PCR-ribotyping does not sufficiently discriminate to prove nosocomial transmission with certainty. We discuss the associated challenges and opportunities in a switch to WGS from conventional ribotyping for C. difficile ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Seth-Smith, Biggel, Roloff, Hinic, Bodmer, Risch, Casanova, Widmer, Sommerstein, Marschall, Tschudin-Sutter and Egli.)

Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis , is caused by strains from the LGV biovar, most commonly represented by ompA -genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples ( n =321) from eight countries collected between 2011 and 2017 (Spain n =97, Netherlands n =67, Switzerland n =64, Australia n =53, Sweden n =37, Hungary n =31, Czechia n =30, Slovenia n =10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA -genotype L2b. However, analysis of the ompA gene shows ompA -genotype L2b ( n =83), ompA -genotype L2 ( n =180) and several variants of these ( n =52; 12 variant types), as well as other/mixed ompA -genotypes ( n =6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA -genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA -genotypes derive from an ompA -genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA -genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA -genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA -genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.

A variety of antiviral treatments for COVID-19 have been investigated, involving many repurposed drugs. Currently, the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp, encoded by nsp12-nsp7-nsp8 ) has been targeted by numerous inhibitors, e.g., remdesivir, the only provisionally approved treatment to-date, although the clinical impact of these interventions remains inconclusive. However, the potential emergence of antiviral resistance poses a threat to the efficacy of any successful therapies on a wide scale. Here, we propose a framework to monitor the emergence of antiviral resistance, and as a proof of concept, we address the interaction between RdRp and remdesivir. We show that SARS-CoV-2 RdRp is under purifying selection, that potential escape mutations are rare in circulating lineages, and that those mutations, where present, do not destabilise RdRp. In more than 56,000 viral genomes from 105 countries from the first pandemic wave, we found negative selective pressure affecting nsp12 (Tajima's D = -2.62), with potential antiviral escape mutations in only 0.3% of sequenced genomes. Potential escape mutations included known key residues, such as Nsp12:Val473 and Nsp12:Arg555. Of the potential escape mutations involved globally, in silico structural models found that they were unlikely to be associated with loss of stability in RdRp. No potential escape mutation was found in a local cohort of remdesivir treated patients. Collectively, these findings indicate that RdRp is a suitable drug target, and that remdesivir does not seem to exert high selective pressure. We anticipate our framework to be the starting point of a larger effort for a global monitoring of drug resistance throughout the COVID-19 pandemic.

Mycoplasma hominis is a common colonizer of the lower genitourinary tract. Although its clinical relevance for causing urogenital infections in immunocompetent individuals is controversial, this bacterium has been involved in severe invasive infections in allograft recipients. In this report, we describe two cases of M. hominis infection in two young renal transplant recipients within the first month post-transplant. Although at first no epidemiological link between the two cases had been suspected, whole-genome sequencing (WGS) analysis showed that both isolates were identical, highly suggestive of an origin with the common organ donor.