Comparison of Whole-Genome Sequence-Based Methods and PCR Ribotyping for Subtyping of Clostridioides difficile.

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks the discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better and provide standardized and interlaboratory exchangeable data. Using a well-characterized collection of diverse strains (N = 630; 100 unique ribotypes [RTs]), we compared the discriminatory power of core genome multilocus sequence typing (cgMLST) (SeqSphere and EnteroBase), whole-genome MLST (wgMLST) (EnteroBase), and single-nucleotide polymorphism (SNP) analysis. A unique cgMLST profile (more than six allele differences) was observed in 82 of 100 RTs, indicating that cgMLST could distinguish most, but not all, RTs. Application of cgMLST in two outbreak settings with RT078 and RT181 (known to have low intra-RT allele differences) showed no distinction between outbreak and nonoutbreak strains in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize, and offers higher discrimination. However, adjusted cutoff thresholds and epidemiological data are necessary to recognize outbreaks of some specific RTs. We propose to use an allelic threshold of three alleles to identify outbreaks.

Assessing the spreading potential of an undetected case of COVID-19 in orthopaedic surgery.

BACKGROUND: With the novel coronavirus-induced disease (COVID-19), there is the fear of nosocomial infections and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmissions to healthcare workers (HCW). We report the case of a 64-year-old male patient who underwent explantation of a shoulder prosthesis due to a periprosthetic infection. He was tested SARS-CoV-2 positive 7 days after admission to the orthopaedic department following strict infection control measures, routinely including screening all patients for multi-drug-resistant organism (MDRO) colonization upon admission. Aim of our study is to report on the spreading potential of SARS-CoV-2 in a healthcare setting if standard contact precautions and infection control measures have been established. METHODS: All HCW with exposure to the patient from day of admission until confirmed diagnosis of COVID-19 were identified and underwent oropharyngeal swab testing for SARS-CoV-2 by real-time RT-PCR. RESULTS: Sixty-six HCW were identified: nine orthopaedic surgeons, four anaesthesiologists, 25 orthopaedic nurses, five nurse anesthetists, eight scrub nurses, five nursing students, two medical assistants and seven service employees. Fourteen HCW (21%) showed clinical symptoms compatible with a SARS-CoV-2 infection: cough (n = 4), sore throat (n = 3), nasal congestion (n = 3), dyspnea (n = 2), fever (n = 1), headache and myalgia (n = 1). SARS-CoV-2 was not detected in any of the 66 HCW. CONCLUSION: Hygienic measures and contact precautions, aimed at preventing the spread of MRDO, may have helped to prevent a SARS-CoV-2 transmission to HCW-despite high-risk exposure during intubation, surgical treatment and general care. LEVEL OF EVIDENCE: IV, case series.

Global Epidemiology and Evolutionary History of Staphylococcus aureus ST45.

Staphylococcus aureus ST45 is a major global MRSA lineage with huge strain diversity and a high clinical impact. It is one of the most prevalent carrier lineages but also frequently causes severe invasive disease, such as bacteremia. Little is known about its evolutionary history. In this study, we used whole-genome sequencing to analyze a large collection of 451 diverse ST45 isolates from 6 continents and 26 countries. De novo-assembled genomes were used to understand genomic plasticity and to perform coalescent analyses. The ST45 population contained two distinct sublineages, which correlated with the isolates' geographical origins. One sublineage primarily consisted of European/North American isolates, while the second sublineage primarily consisted of African and Australian isolates. Bayesian analysis predicted ST45 originated in northwestern Europe about 500 years ago. Isolation time, host, and clinical symptoms did not correlate with phylogenetic groups. Our phylogenetic analyses suggest multiple acquisitions of the SCCmec element and key virulence factors throughout the evolution of the ST45 lineage.

Genotypic Characterization of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates of Clonal Complex 398 in Pigsty Visitors: Transient Carriage or Persistence?

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex (CC) 398 has become a rising issue for public health. While it is known that >80% of pig farmers are colonized with LA-MRSA, only a few studies have assessed the situation for humans with occasional livestock contact. Recently it was shown that over 75% of scientific fieldworkers visiting pigsties were temporarily carrying LA-MRSA. To find out whether they were transiently or permanently colonized, we used whole-genome sequencing (WGS) data to analyze the relatedness of isolates from these recurrently LA-MRSA-positive fieldworkers and from corresponding pigsties. Sequences were analyzed using in silico typing (spa and core genomic multilocus sequence typing [cgMLST]), and the BEAST software package was used to examine phylogeny. In total, 81 samples from three fieldworkers on eight different pigsties over a period of 2.5 years were sequenced. All isolates belonged to spa type t011, t034, or t2011, with different types found in the same fieldworker at different time points. Analysis of cgMLST revealed nine genotypic clusters, mostly correlating with the pigsty on which they were sampled. Fieldworker isolates clustered with the samples from farms that were visited on the same day. BEAST analysis corroborated the cgMLST-based clustering and suggests an origin of the lineage about 22 years ago. We conclude that nasal LA-MRSA colonization among humans with occasional livestock contact is common but most likely only temporary. Furthermore, we showed that the Western German LA-MRSA CC398 originated in the late 1990s and diversified into farm-specific genotypes, which stay relatively consistent over time.

[Hygiene aspects of multidrug-resistant pathogens in the operating room and intensive care unit]. / Hygieneaspekte bei multiresistenten Erregern im OP und auf der Intensivstation.

The major multidrug-resistant pathogens (MRE) in human medicine are methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) and multidrug-resistant Gram-negative rod bacteria (MRGN). MRE are a very heterogeneous group with respect to epidemiology and therapeutic or hospital hygiene consequences. After MRSA played an important role among MREs at the beginning of the twenty-first century, VRE and MRGN have come to the fore in recent years. During work in the operating room and on the intensive care unit, there are many possibilities for transmission of MRE between the patient environment and the patient, especially via the hands, e. g. during intubation or catheterization in vessels, tissues or the urinary tract. For this reason, hand and surface hygiene is of particular relevance in the prevention of nosocomial colonization or infection, in particular with MRE.

Low-level antimicrobial resistance of Enterobacteriaceae isolated from the nares of pig-exposed persons.

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) have recently emerged in livestock and humans. Therefore, this study assessed the carriage of Enterobacteriaceae in the anterior nares and associated antimicrobial resistance in pig-exposed persons. Nasal swabs were enriched in non-selective broth and then plated on MacConkey and ESBL-selective agars. Species was confirmed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-ToF MS). Antimicrobial susceptibility testing was performed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Of 114 pig-exposed persons tested, Enterobacteriaceae were detected in the nares of 76 (66·7%) participants. The predominant species were Proteus mirabilis (n = 17, 14·9%), Pantoea agglomerans (n = 13, 11·4%), Morganella morganii (n = 9, 7·9%), Citrobacter koseri (n = 9, 7·9%), Klebsiella pneumoniae, Escherichia coli and Proteus vulgaris (each n = 8, 7·0%). ESBL-E were not detected. Of all isolates tested, 3·4% were resistant against ciprofloxacin, 2·3% against gentamicin, 23·9% against trimethoprim-sulfamethoxazole and 44·3% against tigecycline. Despite the high prevalence of ESBL-E in livestock, pig-exposed persons did not carry ESBL-E in their nares. This finding is important, because colonization of the nasal reservoir might cause endogenous infections or facilitate transmission of ESBL-E in the general population.

Clonal structure of Staphylococcus aureus colonizing children with sickle cell anaemia and healthy controls.

Children with sickle cell anaemia (SCA) might carry hospital-associated bacterial lineages due to frequent hospital stays and antibiotic treatments. In this study we compared Staphylococcus aureus from SCA patients (n=73) and healthy children (n=143) in a cross-sectional study in Gabon. S. aureus carriage did not differ between children with SCA (n=34, 46∙6%) and controls matched for age, residence and sex (n=67, 46∙9%). Both groups shared similar S. aureus genotypes. This finding points towards a transmission of S. aureus between both groups in the community. We conclude that resistance rates from population-based studies with healthy participants could therefore also be used to guide treatment and prophylaxis of endogenous infections in children with SCA despite a different selection pressure.

[Evolution and infection biology of hemolytic-uremic syndrome (HUS) associated E. coli (HUSEC)]. / Evolution und Infektionsbiologie der mit dem hämolytisch-urämischen Syndrom (HUS) assoziierten E. coli (HUSEC).

Shiga toxin (Stx)-producing Escherichia coli (STEC), which cause hemolytic-uremic syndrome (HUS), are designated as HUSEC. Their exceptional genome variability driven by evolutionary diversification permits fast adaptation to changed environmental conditions. The HUSEC collection (http://www.ehec.org), which has been established at the Institute for Hygiene in Münster, contains 42 EHEC reference strains (HUSEC001-HUSEC042). It represents a unique repository collection of pathogens and is extremely helpful for the analysis of evolutionary changes and fixed properties in the STEC that cause the most severe host injury. Such genomic attributes include slowly evolving loci, mobile genetic elements that often encode virulence factors and are assimilated via horizontal gene transfer. Current evolutionary models indicate that numerous outbreak strains evolved recently and that highly pathogenic HUSEC descend from less pathogenic progenitors. However, additional data suggest that HUSEC have small effective population sizes. The HUSEC collection is also a valuable resource with which to study important non-Shiga toxin virulence factors.

Do complement factor H 402Y and C7 M allotypes predispose to (typical) haemolytic uraemic syndrome?

Typical haemolytic uraemic syndrome (HUS) is mainly caused by infections with enterohaemorrhagic Escherichia coli, whereas in atypical, nonbacteria-associated HUS, complement plays a dominant role. Recently, complement has also been shown to be involved in typical HUS. In this study, mostly weakly significant associations with homozygosities of complement allotype C7 M and inversely with factor H 402H were found, suggesting that 402Y and C7 M allotypes predispose to (typical) haemolytic uraemic syndrome.

Semi-selective broth improves screening for methicillin-resistant Staphylococcus aureus.

OBJECTIVES: To evaluate two enrichment broths for methicillin-resistant Staphylococcus aureus (MRSA) detection and compare results with direct plating. METHODS: Swabs from 1224 patients were re-analysed for MRSA in a central laboratory (Münster) using six methods. Swabs were suspended in 0.5 mL of non-selective enrichment broth (NB) and vortexed. Aliquots of 100 microL were inoculated on/into: (I) ChromID MRSA agar; (II) Columbia sheep blood (5%) agar (BA) and ChromID MRSA; (III, IV) NB incubated overnight followed by plating on BA and ChromID MRSA; and (V, VI) a semi-selective broth containing cefoxitin and aztreonam (TSB-SSI) incubated overnight followed by plating on BA and ChromID MRSA. In III-VI, 100 microL of the enriched broth was plated on each agar. RESULTS: The combined MRSA-positive rate was 21.5%. MRSA isolates detected by each method were: TSB-SSI, n = 223; NB, n = 205; BA and ChromID MRSA, n = 203; ChromID MRSA alone, n = 183. TSB-SSI detected more positive throat samples than the comparators and significantly reduced methicillin-susceptible S. aureus (MSSA) growth. The maximum sensitivity obtained was only 85%, possibly due to the study design using pre-used swabs and dilution of swab material. For 997 samples, results from Münster were compared with initial results. Peripheral laboratories identified 172 MRSA compared with Münster where 186, 186 and 204 MRSA were found for direct plating, NB and TSB-SSI broth, respectively. CONCLUSIONS: TSB-SSI was superior to both NB and direct plating on ChromID MRSA and BA. Despite re-using swabs for the study, we showed that routine diagnostic screening could be significantly improved, using a semi-selective enrichment broth.

A child with soft-tissue infection and lymphadenitis.

We report a case of a soft-tissue infection with Francisella philomiragia, a rare opportunistic pathogen in individuals with chronic granulomatous disease.

[Handling of COVID-19 in the emergency department : Field report of the emergency ward of the University Hospital Münster]. / Umgang mit COVID-19 in der Notaufnahme : Erfahrungsbericht der interdisziplinären Notaufnahme des Universitätsklinikums Münster.

With the COVID-19 pandemic, emergency rooms are faced with major challenges because they act as the interface between outpatient and inpatient care. The dynamics of the pandemic forced emergency care at the University Hospital Münster to extensively adjust their processes, which had to be carried out in the shortest time possible. This included the establishment of an outpatient coronavirus test center and a medical student-operated telephone hotline. Inside the hospital, new isolation capacities in the emergency room and a dedicated COVID-19 ward were set up. The patient flow was reorganized using flow diagrams for both the outpatient and inpatient areas. The general and special emergency management was optimized for the efficient treatment of COVID-19-positive patients and the staff were trained in the use of protective equipment. This report of our experience is intended to support other emergency departments in their preparation for the COVID-19 pandemic.

High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a rapid, cost-effective alternative for bacterial species identification. Identifying 60 blind-coded nonfermenting bacteria samples, this international study (using eight laboratories) achieved 98.75% interlaboratory reproducibility. Only 6 of the 480 samples were misidentified due to interchanges (4 samples) or contamination (1 sample) or not identified because of insufficient signal intensity (1 sample).

Whole genome sequencing reveals a prolonged and spatially spread nosocomial outbreak of Panton-Valentine leucocidin-positive meticillin-resistant Staphylococcus aureus (USA300).

BACKGROUND: Whole genome sequencing (WGS) helps to better investigate the transmission and characterization of meticillin-resistant Staphylococcus aureus (MRSA) strains. AIM: We describe the detection and unfolding of a prolonged and spatially distributed nosocomial outbreak of Panton-Valentine leucocidin (PVL)-positive MRSA ST8 (USA300). METHODS: The outbreak was detected by the combination of whole genome sequence (WGS)-based typing, which is implemented for routine surveillance of multidrug-resistant bacteria in our institution, and in-depth epidemiological investigation. To investigate the source, processes were observed and environmental sampling performed. To contain the outbreak, regular and direct personal contact with the healthcare workers (HCWs) was maintained and staff education implemented. FINDINGS: The outbreak took place between October 2016 and November 2017 and included five patients who were treated in two different departments as inpatients and outpatients; three were infected, two were colonized. Additionally, three HCWs carried the outbreak strain. The strain was not found in the hospital environment. Only through non-mediated communication did the source become apparent. Decolonization of HCWs and infection control measures led to a resolution of the outbreak. CONCLUSION: WGS helped to reveal an outbreak that otherwise might have stayed undetected. Nonetheless, epidemiological investigation is needed to trace the nosocomial transmission. The importance of personal communication in infection control cannot be overstated.

Acquisition and colonization dynamics of antimicrobial-resistant bacteria during international travel: a prospective cohort study.

OBJECTIVES: Travellers may be colonized with antimicrobial-resistant bacteria on return, but little is known about colonization during travel. Our objectives were to assess the acquisition and colonization dynamics during the stay abroad for a broad range of antimicrobial-resistant bacteria and resistance phenotypes and to identify risk factors for faecal carriage of antimicrobial-resistant bacteria. METHODS: German and Dutch participants (n = 132) of this prospective cohort study (2016-2018) completed a questionnaire on risk factors and provided daily stool samples before, during, and after travel. Samples were screened for extended-spectrum ß-lactamase producing Enterobacterales (ESBL-E), carbapenem-resistant (CarbR-GN), and non-intrinsically colistin-resistant Gram-negative rods (ColR-GN), vancomycin-resistant Enterococcus faecium/faecalis (VRE), and Clostridioides difficile. RESULTS: Colonization rates reached a plateau within a week after departure fluctuating around 48.5% (63/130) and 58.4% (45/77, ESBL-E), 10.4% (11/106) and 23.4% (18/77, ColR-GN), or 3.0% (4/132) and 6.8% (8/118, CarbR-GN). Colonization rates after the travel were 46.2% (61/132, ESBL-E), 9.0% (12/132, ColR-GN), and 3.4% (5/132, CarbR-GN). Travellers carried mcr-1- (15/132; 11.4%) or blaNDM-positive (4/132; 3.0%) Enterobacterales. A vegetarian diet was associated with a lower risk for the acquisition of ESBL-E (OR = 0.4, p 0.04) and ColR-GN (OR = 0.1, p 0.01) during travel in a multivariable model. Similarly, travellers visiting friends and relatives had a lower risk for the acquisition of ESBL-E (OR = 0.3, p 0.009) and CarbR-GN (OR = 0.3, p 0.01). VRE and C. difficile were not detected. CONCLUSION: The number of travellers with a temporary colonization during the journey exceeded the number of travellers still colonized after return.

Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to 16S rRNA gene sequencing for species identification of nonfermenting bacteria.

Nonfermenting bacteria are ubiquitous environmental opportunists that cause infections in humans, especially compromised patients. Due to their limited biochemical reactivity and different morphotypes, misidentification by classical phenotypic means occurs frequently. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for species identification. By using 248 nonfermenting culture collection strains composed of 37 genera most relevant to human infections, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurement and MALDI BioTyper software (Bruker Daltonik GmbH, Leipzig, Germany), i.e., by using a mass range of 2,000 to 20,000 Da and a new pattern-matching algorithm. To evaluate the database, 80 blind-coded clinical nonfermenting bacterial strains were analyzed. As a reference method for species designation, partial 16S rRNA gene sequencing was applied. By 16S rRNA gene sequencing, 57 of the 80 isolates produced a unique species identification (>or=99% sequence similarity); 11 further isolates gave ambiguous results at this threshold and were rated as identified to the genus level only. Ten isolates were identified to the genus level (>or=97% similarity); and two isolates had similarity values below this threshold, were counted as not identified, and were excluded from further analysis. MALDI-TOF MS identified 67 of the 78 isolates (85.9%) included, in agreement with the results of the reference method; 9 were misidentified and 2 were unidentified. The identities of 10 randomly selected strains were 100% correct when three different mass spectrometers and four different cultivation media were used. Thus, MALDI-TOF MS-based species identification of nonfermenting bacteria provided accurate and reproducible results within 10 min without any substantial costs for consumables.

EUREGIO MRSA-net Twente/Münsterland--a Dutch-German cross-border network for the prevention and control of infections caused by methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with increased mortality and morbidity and a leading cause of hospital-acquired infections. Community-acquired (CA)-MRSA are a growing concern worldwide. In the last 10 years, an increase in the MRSA rate from 2% to approximately 23% has been observed in Germany, while a rate under 5% has been recorded for many years in the Netherlands and Scandinavia. In the Netherlands in particular, MRSA rates have become very low in stationary care due to a consistent 'search and destroy' policy. The main focus in Germany lies on hospital-acquired MRSA, whereas the Netherlands focus on the control of the importation of MRSA cases from abroad and on CA-MRSA. As MRSA in hospitals and in the community can be a problem in cross-border health care, the European Union-funded EUREGIO MRSA-net project was established in the bordering regions Twente/Achterhoek, the Netherlands and Münsterland, Germany. The main aim of the project is the creation of a network of the major health care providers in the EUREGIO and the surveillance and prevention of MRSA infections. A spa-typing network was established in order to understand the regional and cross-border dissemination of epidemic and potentially highly virulent MRSA genotypes. As the reduction of differences in health care quality is an important prerequisite for cross-border health care, a transborder quality group comprising hospitals, general practitioners, public health authorities, laboratories, and insurerance companies has been established since 2005 equalising the quality criteria for the control of MRSA on both sides of the border.

Carbapenem-resistant Enterobacteriaceae in wildlife, food-producing, and companion animals: a systematic review.

OBJECTIVES: The spread of carbapenem-resistant Enterobacteriaceae (CRE) in healthcare settings challenges clinicians worldwide. However, little is known about dissemination of CRE in livestock, food, and companion animals and potential transmission to humans. METHODS: We performed a systematic review of all studies published in the PubMed database between 1980 and 2017 and included those reporting the occurrence of CRE in samples from food-producing and companion animals, wildlife, and exposed humans. The primary outcome was the occurrence of CRE in samples from these animals; secondary outcomes included the prevalence of CRE, carbapenemase types, CRE genotypes, and antimicrobial susceptibilities. RESULTS: We identified 68 articles describing CRE among pigs, poultry, cattle, seafood, dogs, cats, horses, pet birds, swallows, wild boars, wild stork, gulls, and black kites in Africa, America, Asia, Australia, and Europe. The following carbapenemases have been detected (predominantly affecting the genera Escherichia and Klebsiella): VIM, KPC, NDM, OXA, and IMP. Two studies found that 33-67% of exposed humans on poultry farms carried carbapenemase-producing CRE closely related to isolates from the farm environment. Twenty-seven studies selectively screened samples for CRE and found a prevalence of <1% among livestock and companion animals in Europe, 2-26% in Africa, and 1-15% in Asia. Wildlife (gulls) in Australia and Europe carried CRE in 16-19%. CONCLUSIONS: The occurrence of CRE in livestock, seafood, wildlife, pets, and directly exposed humans poses a risk for public health. Prospective prevalence studies using molecular and cultural microbiological methods are needed to better define the scope and transmission of CRE.

Infection of exposed patients during norovirus outbreaks: are there predictive parameters?

BACKGROUND: Norovirus outbreak management comprises isolation and cohorting of patients. In this context, exposed patients are preferably cohorted separately from symptomatic and unexposed asymptomatic patients, since they potentially develop symptoms of norovirus gastroenteritis. Whether routinely examined clinical or laboratory parameters can help to predict occurrence of gastroenteritis symptoms in those patients has not yet been examined. AIM: To evaluate routinely examined clinical and laboratory parameters as predictive values for the development of norovirus symptoms in exposed patients during outbreaks. METHODS: Exposed patients during norovirus outbreaks were observed throughout a two-year period in the university hospital of Muenster. The development of laboratory-confirmed norovirus gastroenteritis symptoms was examined in exposed patients, and clinical as well as laboratory parameters prior to onset of the outbreak were compared in exposed symptomatic and asymptomatic patients. FINDINGS: We detected 42 exposed patients within 10 outbreaks. Of these, 33 remained asymptomatic, whereas nine patients developed norovirus gastroenteritis. Exposed symptomatic patients were significantly older (50±10.51 vs 28±4.68 years), had significantly higher blood sodium concentration (142.5±1.48 vs 138.8±0.47mmol/L) and higher systolic blood pressure (119.3±3.84 vs 108.5±2.41mmHg). Development of symptoms among exposed patients was significantly associated with blood type O (75% vs 20%). CONCLUSION: In order to minimize patient-to-patient transmission within norovirus outbreaks in hospital, risk stratification of exposed patients is helpful. To achieve this, routinely detected clinical and laboratory parameters can be useful to predict development of symptoms in these patients.