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.

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.

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.

[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.

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.

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.

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.

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.

In the centre of an epidemic: Fifteen years of LA-MRSA CC398 at the University Hospital Münster.

Ten years after initial publications on livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in 2005, we report on the course of the LA-MRSA CC398 epidemic among patients of the University Hospital Münster. This tertiary care facility is located in the Dutch-German border region (EUREGIO), which is characterized by a high density of livestock production and is hence a hotspot for the occurrence of LA-MRSA CC398. Taking advantage of the unique opportunity to track the emergence and spread of MRSA CC398 among humans from the very beginning of the epidemic until today, a total of 6555 non-duplicate MRSA isolates from all screenings and clinical specimens cultivated within the period from 2000 to 2014 were included in the analysis. Retrospectively, the first MRSA CC398 isolate (spa type t034) was obtained from a screening specimen of a patient in 2000, which represents one of the first human-associated LA-MRSA CC398 isolates reported in Europe. After sporadic detections between 2000 and 2004, this clonal lineage accounted for 9.6% of all local MRSA in 2005; a proportion which increased to 35% in 2013 and became stable since then. Considering the period from 2000 to 2014, the group of MRSA CC398 isolates comprised a total of 45 different spa types among which t011 (48.3%), t034 (39.3%) and t108 (3.5%) were predominant and so far unreported types were found. Overall, LA-MRSA CC398 emerged rapidly during the past decade, developed enormous sublineage diversity and contributed substantially to the total burden of MRSA colonization and infection at the hospital.

Unveiling the molecular basis of antimicrobial resistance in Staphylococcus aureus from the Democratic Republic of the Congo using whole genome sequencing.

Staphylococcus aureus from sub-Saharan Africa is frequently resistant to antimicrobial agents that are commonly used to treat invasive infections in resource-limited settings. The underlying mechanisms of resistance are largely unknown. We therefore performed whole genome sequencing (WGS) on S. aureus from the Democratic Republic of the Congo (DRC) to analyse the genetic determinants of antimicrobial resistance. One hundred S. aureus samples were collected from community-associated asymptomatic nasal carriers in the metropolitan area of Kinshasa, DRC, between 2013 and 2014. Phenotypic resistance against 15 antimicrobial agents was compared to the genotypic results that were extracted from WGS data using Mykrobe predictor and the SeqSphere(+) software that screened for 106 target genes associated with resistance. Isolates were phenotypically resistant against penicillin (97%, n=97), trimethoprim (72%, n=72) and tetracycline (54%, n=45). Thirty-three isolates (33%) were methicillin-resistant S. aureus (MRSA). Of these, nine isolates (27.3%) were oxacillin-susceptible MRSA (OS-MRSA) and belonged to ST8 (t1476). The Y195F mutation of FemA was associated with OS-MRSA (p 0.015). The majority of trimethoprim resistant isolates carried dfrG. Tetracycline resistance was associated with tet(K). The concordance between phenotypic susceptibility testing and both WGS analysis tools was similar and ranged between 96% and 100%. In conclusion, a high proportion of OS-MRSA in the DRC was linked to mutations of FemA. Genotypic and phenotypical antimicrobial susceptibility testing showed high concordance. This encourages the future use of WGS in routine antimicrobial susceptibility testing.

Norovirus outbreak management: how much cohorting is necessary?

For the control of norovirus outbreaks, it is widely recommended that exposed but asymptomatic patients should be cohorted separately from unexposed patients and from symptomatic patients. The frequency of subsequent symptomatic norovirus infection in contact patients has not been investigated systematically. We retrospectively investigated the development of typical norovirus symptoms in contact patients during seven norovirus outbreaks affecting 57 patients between November 2014 and May 2015. Only one of 14 contact patients developed typical norovirus symptoms, calling into question current recommendations to isolate contact patients.

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.

Ensuring backwards compatibility: traditional genotyping efforts in the era of whole genome sequencing.

When using next-generation whole genome sequencing (WGS), extraction of spa types from WGS data is essential for backwards compatibility with Sanger sequencing-based spa typing of methicillin-resistant Staphylococcus aureus (MRSA). We evaluated WGS-based spa typing with a 2×250 bp protocol in a diverse collection of 423 MRSA isolates using two pipelines that executed sequence quality-trimming and de novo assembly before spa typing. The SeqSphere(+) pipeline correctly typed 419 isolates (99.1%) whereas the CLCbio pipeline succeeded in 249 isolates (58.9%). In summary, WGS combined with an optimized de novo assembly enables nearly full compatibility with Sanger sequencing-based spa typing data.

Staphylococcus aureus spa type t437: identification of the most dominant community-associated clone from Asia across Europe.

Methicillin-resistant Staphylococcus aureus (MRSA) belonging to the multilocus sequence type clonal complex 59 (MLST CC59) is the predominant community-associated MRSA clone in Asia. This clone, which is primarily linked with the spa type t437, has so far only been reported in low numbers among large epidemiological studies in Europe. Nevertheless, the overall numbers identified in some Northern European reference laboratories have increased during the past decade. To determine whether the S. aureus t437 clone is present in other European countries, and to assess its genetic diversity across Europe, we analysed 147 S. aureus t437 isolates from 11 European countries collected over a period of 11 years using multiple locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and MLST. Additionally 16 S. aureus t437 isolates from healthy carriers and patients from China were included. Most isolates were shown to be monophyletic with 98% of the isolates belonging to the single MLVA complex 621, to which nearly all included isolates from China also belonged. More importantly, all MLST-typed isolates belonged to CC59. Our study implies that the European S. aureus t437 population represents a genetically tight cluster, irrespective of the year, country and site of isolation. This underpins the view that S. aureus CC59 has been introduced into several European countries, not being restricted to particular geographical regions or specific host environments. The European S. aureus t437 isolates thus bear the general hallmarks of a high-risk clone.

State-wide surveillance of antibiotic resistance patterns and spa types of methicillin-resistant Staphylococcus aureus from blood cultures in North Rhine-Westphalia, 2011-2013.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of bacteraemia. We aimed to obtain a complete picture of severe MRSA infections by characterizing all MRSA isolates from bloodstream infections in the largest German federal state (North Rhine-Westphalia, 18 million inhabitants) using S. aureus protein A (spa) sequence-typing and antimicrobial susceptibility testing. MRSA isolates (n = 1952) were collected prospectively (2011-2013) and spa-typed. Among 181 different spa types, t003 (n = 746 isolates; 38.2%) and t032 (n = 594; 30.4%) were predominant. Analysis of the geographical occurrence of spa clonal complexes (spa-CCs) and spa types revealed divergent distribution between federal state districts for spa-CCs 003 (p < 0.001; including t003, p < 0.001 and t264, p < 0.001), 008 (p 0.021), 011 (p 0.002), 032 (p < 0.001; including t022, p 0.014 and t032, p < 0.001) and spa type t2807 (p < 0.001). MICs of antimicrobial substances were tested using broth microdilution. Of all isolates, 96% were resistant to fluoroquinolones, 78% to erythromycin, 70% to clindamycin, 4% to gentamicin, 2% to rifampicin, 0.4% to daptomycin, 0.1% to linezolid and 0% to vancomycin, respectively. Vancomycin MICs of 2 mg/L involved 0.5% of the isolates. In conclusion, the detection of regional molecular clusters added valuable information for epidemiological case tracing and allowed conclusions to be reached on the importance of newly emerging MRSA reservoirs, such as livestock (spa-CC011), for MRSA bacteraemia in some parts of the federal state. Susceptibility testing revealed broad resistance to substances used for oral treatment, but demonstrated that those antibiotics that are mostly applied for treatment of MRSA bacteraemia and important combination partners were highly susceptible.