Whole-genome analysis of vancomycin-resistant Enterococcus faecium causing nosocomial outbreaks suggests the occurrence of few endemic clonal lineages in Bavaria, Germany.

OBJECTIVES: Infections caused by vancomycin-resistant Enterococcus faecium (VREfm) represent a major public health concern due to limited treatment options. Among invasive isolates of VREfm, ST117, ST80 and ST78 represent the most frequently detected STs by MLST in Germany. In this study, we investigated the genetic diversity of isolates of VREfm recovered from different nosocomial outbreaks in Bavaria, Germany, by WGS. METHODS: Between January 2018 and April 2019, 99 non-replicate isolates of VREfm originating from nosocomial outbreaks at eight different hospitals in Bavaria were investigated for genetic diversity by WGS. In detail, complex types (CTs) were identified by core-genome MLST. Furthermore, an SNP analysis was performed for all VREfm strains. RESULTS: Most of the isolates of this study (76%) belonged to three major clonal groups, which occurred in at least three hospitals: ST80/CT1065 vanB (n = 45; six hospitals), ST117/CT71 vanB (n = 11; four hospitals) and ST78/CT894like vanA (n = 19; three hospitals). Moreover, isolates of the predominant lineage ST80/CT1065 vanB showed a maximum difference of 36 SNPs as revealed by SNP analysis. CONCLUSIONS: Whole-genome analysis of VREfm causing nosocomial outbreaks suggests the occurrence of few endemic clonal lineages in Bavarian hospital settings, namely ST80/CT1065 vanB, ST117/CT71 vanB and ST78/CT894like vanA. Further studies are needed for a better understanding of the factors affecting the successful spread of the above-mentioned lineages.

First report of the new emerging global clone ST1193 among clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli from Germany.

OBJECTIVES: Sequence type 1193 (ST1193) is a new emerging global clone of Escherichia coli. The main goal of this study was to determine the prevalence and molecular characteristics of ST1193 among clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing E. coli from University Hospital of Erlangen, Germany. METHODS: Between November 2015 and February 2016, all consecutive non-duplicate clinical E. coli isolates showing resistance to cefotaxime or ceftazidime were further analysed for ESBL production by the combined disk method. ESBL genes were identified by PCR and sequencing. Bacterial strain typing was performed by PCR-based phylogrouping, MLST and whole-genome sequencing. RESULTS: ESBL production was confirmed in 51 isolates. The globally dominant ST131 occurred at a frequency of 37.3% (n=19). Major non-ST131 sequence types were ST38 (n=4; 7.8%), ST10 (n=3; 5.9%) and ST1193 (n=3; 5.9%). Among the ESBL-producing E. coli ST1193, two expressed CTX-M-14 and one expressed CTX-M-15 ESBL type. All three ST1193 isolates belonged to serogroup O75:H5, phylogroup B2, and harboured IncFIA and IncFIB plasmids and the virulence factors genes iha, sat, gad, vat and senB. Moreover, they showed ciprofloxacin resistance and exhibited a set of four conserved mutations defining quinolone resistance (gyrA S83L, gyrA D87N, parC S80I and parC L416F). CONCLUSIONS: This study revealed for the first time in Germany the occurrence of ST1193 among clinical isolates of ESBL-producing E. coli. Further national or regional multicentre studies are needed to assess the effective relevance of ESBL-producing E. coli ST1193 as a nosocomial pathogen in Germany.

Occurrence of Antibiotic-Resistant Bacteria in Therapy Pools and Surrounding Surfaces.

The number of patients colonized with antibiotic-resistant bacteria is increasing in health care facilities. Because transmission of antibiotic-resistant bacteria is feared, there exist reports that the affected patients are frequently excluded from hydrotherapy, which is a non-invasive and beneficial treatment used for patients with different diseases. Data from the literature suggest that deficient water disinfection measures exist, which are not always sufficient to kill all released bacteria. If the pool water is not disinfected properly, it may also infect the bathers. Immunocompromised patients are particularly susceptible to be infected with (antibiotic-resistant) bacteria. In order to determine the distribution of antibiotic-resistant bacteria in the pool water treatment system and the pool environment and to estimate the associated transmission risk we analyzed samples from eleven health care facilities. Antibiotic-resistant bacteria were found in the water and surface samples collected. One hundred and two antibiotic-resistant isolates from water samples and 307 isolates from surrounding surfaces were obtained, respectively. The majority of the isolates belonged to non-fermenting Gram-negative rods, like Pseudomonas spp. Some isolates were resistant to a wide range of the tested antibiotics. The results indicate a relation between the number of isolates in water samples and the number of patients using the pools in combination with deficiencies in water treatment. In the pool environment the highest number of isolates was obtained from barefoot areas and floor cleaning equipment.

Global outbreak of severe Mycobacterium chimaera disease after cardiac surgery: a molecular epidemiological study.

BACKGROUND: Since 2013, over 100 cases of Mycobacterium chimaera prosthetic valve endocarditis and disseminated disease were notified in Europe and the USA, linked to contaminated heater-cooler units (HCUs) used during cardiac surgery. We did a molecular epidemiological investigation to establish the source of these patients' disease. METHODS: We included 24 M chimaera isolates from 21 cardiac surgery-related patients in Switzerland, Germany, the Netherlands, and the UK, 218 M chimaera isolates from various types of HCUs in hospitals, from LivaNova (formerly Sorin; London, UK) and Maquet (Rastatt, Germany) brand HCU production sites, and unrelated environmental sources and patients, as well as eight Mycobacterium intracellulare isolates. Isolates were analysed by next-generation whole-genome sequencing using Illumina and Pacific Biosciences technologies, and compared with published M chimaera genomes. FINDINGS: Phylogenetic analysis based on whole-genome sequencing of 250 isolates revealed two major M chimaera groups. Cardiac surgery-related patient isolates were all classified into group 1, in which all, except one, formed a distinct subgroup. This subgroup also comprised isolates from 11 cardiac surgery-related patients reported from the USA, most isolates from LivaNova HCUs, and one from their production site. Isolates from other HCUs and unrelated patients were more widely distributed in the phylogenetic tree. INTERPRETATION: HCU contamination with M chimaera at the LivaNova factory seems a likely source for cardiothoracic surgery-related severe M chimaera infections diagnosed in Switzerland, Germany, the Netherlands, the UK, the USA, and Australia. Protective measures and heightened clinician awareness are essential to guarantee patient safety. FUNDING: Partly funded by the EU Horizon 2020 programme, its FP7 programme, the German Center for Infection Research (DZIF), the Swiss National Science Foundation, the Swiss Federal Office of Public Health, and National Institute of Health Research Oxford Health Protection Research Units on Healthcare Associated Infection and Antimicrobial Resistance.

Screening of ESBL-producing Enterobacteriacae concomitant with low degree of transmission in intensive care and bone marrow transplant units.

BACKGROUND: Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) are spreading worldwide in both hospital and community settings. In this study, the molecular epidemiology and the transmission modalities of ESBL-E in intensive care- and bone marrow transplant were investigated. METHODS: All patients included in this study were screened for presence of ESBL-E on admission and weekly. Relevant ß-lactamase genes were identified by PCR and sequencing. RESULTS: A total of 669 patients were included in this study. On admission, ESBL-producing Escherichia coli were detected in 49 (7.3%) patients and ESBL-producing Klebsiella pneumoniae in one patient. The most common ESBL types among E. coli isolates were CTX-M-15 (38.8%) and CTX-M-1 (38.8%). Furthermore, 12 of 49 (24.5%) ESBL-producing E. coli could be assigned to the epidemic clone ST131. A single patient acquired ESBL-producing E. coli during the hospital stay but cross-transmission could not be demonstrated. Among 1095 environmental samples none revealed ESBL. CONCLUSIONS: Our results suggest that early detection of ESBL-producing Enterobacteriaceae and consequent implementation of basic hygiene measures and contact isolation may reduce the transmission rate during the hospital stay.

Prevalence and genetic diversity of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in nursing homes in Bavaria, Germany.

Main goal of this study was to determine the prevalence and molecular epidemiology of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae among 156 nursing home residents in Bavaria and to compare the results with healthy individuals from the Bavarian community. Intestinal colonisation by ESBL-producing Escherichia coli was detected in 23 nursing home residents (14.7%) using MacConkey agar supplemented with cefotaxime (1mg/L) for screening and the combined disc method for ESBL confirmation. Antimicrobial susceptibility testing revealed co-resistance to ciprofloxacin in 86.9% of the ESBL-producers. All isolates harboured CTX-M-ESBL with CTX-M-15 (65.2%) and CTX-M-27 (21.7%) as the most common types. Moreover, 16 isolates (69.6%) could be assigned by PCR-typing to the epidemic clonal lineage E. coli O25b-ST131. Further typing by rep-PCR and XbaI-macrorestriction with subsequent pulsed-field gel electrophoresis, respectively, revealed that two or more residents shared the same ESBL-producing E. coli clone in four nursing homes. In conclusion, we could show a high prevalence of ESBL-producing E. coli in Bavarian nursing homes (14.7%) compared to the healthy population (6.3%). Although the prevalence of ESBL-type CTX-M-15 in E. coli was similar in nursing home residents (65.2%) and healthy individuals (46%) the presence of E. coli O25b-ST131 clones differed substantially (69.6% and 14.2%, respectively). Furthermore, this study demonstrates that a person-to-person transmission or a common source of infection for ESBL-producing microorganisms may occur in these facilities. Therefore, basic hygiene measures should be assiduously implemented to prevent the further spread of these multidrug-resistant bacteria.

Detection of Legionella-contaminated aerosols in the vicinity of a bio-trickling filter of a breeding sow facility - A pilot study.

The urbanization of agricultural areas results in a reduction of distances between residential buildings and livestock farms. In the public debate, livestock farming is increasingly criticized due to environmental disturbance and odor nuisance originating from such facilities. One method to reduce odor and ammonia is by exhaust air treatment, for example, by biological exhaust air purification processes with bio-trickling filters filled with tap water. Higher temperatures in the summer time and the generation of biofilms are ideal growth conditions for Legionella. However, there are no studies on the presence of Legionella in the water of bio-trickling filters and the release of Legionella-containing aerosols. Therefore, the aim of this study was to investigate Legionella in wash water and emitted bioaerosols of a bio-trickling filter system of a breeding sow facility. For this purpose, measurements were carried out using a cyclone sampler. In addition, samples of wash water were taken. Legionella were not found by culture methods. However, using molecular biological methods, Legionella spp. could be detected in wash water as well as in bioaerosol samples. With antibody-based methods, Legionella pneumophila were identified. Further studies are needed to investigate the environmental health relevance of Legionella-containing aerosols emitted by such exhaust air purification systems.

Association between the ambient temperature and the occurrence of human Salmonella and Campylobacter infections.

Salmonella spp. and thermotolerant Campylobacter spp. are the most important causes of human bacterial diarrheal infections worldwide. These bacterial species are influenced by several factors like behaviour of the host, shedding, environment incl. directly or indirectly through ambient temperature, and the infections show seasonality. Therefore, the aim of our study was to investigate the association between the occurrence of human campylobacteriosis and salmonellosis and the ambient temperature. The number of campylobacteriosis and salmonellosis cases in two German metropolises, Munich and Berlin, and three rural regions was analysed with simultaneous consideration of the ambient temperature over a period of four years (2001 to 2004) using regression, time series, and cross-correlation analysis. The statistical analysis showed that an increase in the ambient temperature correlated positively with an increase in human Salmonella and Campylobacter cases. The correlation occurred with a delay of approximately five weeks. The seasonal rise in ambient temperature correlated with increased incidence of bacterial diarrheal infections.

Contamination during production of heater-cooler units by Mycobacterium chimaera potential cause for invasive cardiovascular infections: results of an outbreak investigation in Germany, April 2015 to February 2016.

Invasive infections with Mycobacterium chimaera were reported in patients with previous open chest surgery and exposure to contaminated heater-cooler units (HCUs). We present results of the surveillance of clinical cases and of contaminated HCUs as well as environmental investigations in Germany up until February 2016. Clinical infections occurred in five male German cases over 50 years of age (range 53-80). Cases had been exposed to HCUs from one single manufacturer during open chest surgery up to five years prior to onset of symptoms. During environmental investigations, M. chimaera was detected in samples from used HCUs from three different countries and samples from new HCUs as well as in the environment at the manufacturing site of one manufacturer in Germany. Our investigation suggests that at least some of the M. chimaera infections may have been caused by contamination of HCUs at manufacturing site. We recommend that until sustainable measures for safe use of HCUs in operation theatres are implemented, users continue to adhere to instructions for use of HCUs and Field Safety Notices issued by the manufacturer, implement local monitoring for bacterial contamination and continuously check the websites of national and European authorities for current recommendations for the safe operation of HCUs.

Prevalence, antimicrobial susceptibility, and genetic diversity of Pseudomonas aeruginosa as intestinal colonizer in the community.

In this study we determined the prevalence of intestinal carriage, the antimicrobial susceptibility rates, and the genetic diversity of Pseudomonas aeruginosa in the community. From July 2010 to December 2011, a total of 2110 nonreplicate fecal samples from individuals living in Bavaria were collected. Samples were screened for P. aeruginosa by a selective medium and antimicrobial susceptibility was determined by disc diffusion technique. Genetic diversity was assessed by multilocus sequence typing (MLST). Intestinal colonization was detected in 31 of 2110 (1.47%) individuals. None of the isolates showed resistance to aztreonam, imipenem, meropenem, ciprofloxacin, amikacin or colistin. Twenty-five isolates could be assigned to 20 different sequence types (STs), whereas the remaining 6 could not be assigned. Interestingly, four isolates belonged to ST253. These data show that intestinal colonization by P. aeruginosa occurs in the community with high genetic diversity and low rates of antimicrobial resistance.

[Cultural detection of thermotolerant Campylobacter spp. in food--potentials and limitations of diagnostic tools in the context of official food control]. / Kultureller Nachweis thermophiler Campylobacter spp. in Lebensmitteln--Möglichkeiten und Grenzen der Diagnostik im Rahmen der amtlichen Überwachung.

Thermotolerant Campylobacter spp. rank among the most important foodborne pathogens in Germany. Therefore a necessity for rapid and routinely useable detection methods exists also in the area of food microbiology. A reliable, cultura qualitative, but also quantitative detection of thermotolerant Campylobacter spp. pose a challenge, at least concerning special food matrices, especially because in the context of official food control the cultural detection of thermotolerant Campylobacter spp. is needed. This was the reason, why different cultural detection methods, beside the standard procedure of ISO 10272:2006, in combination with molecular and immunological screening methods were tested at the Bavarian Health and Food Safety Authority (LGL) during the last years for the use in routine diagnostic using different food matrices of animal and plant origin. The results of the comparative studies showed clearly that no enrichment broth tested gave completely satisfactory results for an only culture-based detection the combination with a screening method is therefore recommended for a rapid and reliable detection. But in this case the user should take into account that the sensitivity of such molecular and immunological methods is normally so high that in some cases, depending on the food matrix and processing step, the isolation of the pathogen would not be possible in samples, which were positive in the screening methods.

Assessing the environmental health relevance of cooling towers--a systematic review of legionellosis outbreaks.

Bioaerosols from cooling towers are often suspected to cause community-acquired legionellosis outbreaks. Although Legionella infections can mostly be assigned to the emission sources, uncertainty exists about the release and distribution into the air, the occurrence of the respirable virulent form and the level of the infective concentration. Our study aimed to evaluate studies on legionellosis outbreaks attributed to cooling towers published within the last 11 years by means of a systematic review of the literature. 19 legionellosis outbreaks were identified affecting 12 countries. Recurring events were observed in Spain and Great Britain. In total, 1609 confirmed cases of legionellosis and a case-fatality rate of approximately 6% were reported. Duration of outbreaks was 65 days on average. For diagnosis the urinary antigen test was mainly used. Age, smoking, male sex and underlying diseases (diabetes, immunodeficiency) could be confirmed as risk factors. Smoking and underlying diseases were the most frequent risk factors associated with legionellosis in 11 and 10 of the 19 studies, respectively. The meteorological conditions varied strongly. Several studies reported a temporal association of outbreaks with inadequate maintenance of the cooling systems. A match of clinical and environmental isolates by serotyping and/or molecular subtyping could be confirmed in 84% of outbreaks. Legionella-contaminated cooling towers as environmental trigger, in particular in the neighbourhood of susceptible individuals, can cause severe health problems and even death. To prevent and control Legionella contamination of cooling towers, maintenance actions should focus on low-emission cleaning procedures of cooling towers combined with control measurements of water and air samples. Procedures allowing rapid detection and risk assessment in the case of outbreaks are essential for adequate public health measures. Systematic registration of cooling towers will facilitate the identification of the source of outbreaks and help to shorten their duration.

Extended-spectrum-ß-lactamase-producing Escherichia coli as intestinal colonizers in the German community.

We determined the presence of extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli among 3,344 study participants from the German community. Intestinal colonization was detected in 211 persons (6.3%), without significant differences among the different age groups. The majority (95.2%) of isolates harbored CTX-M-type ESBL, with CTX-M-15 (46%) and CTX-M-1 (24.2%) as the most common types. The finding of ESBL producers and one isolate additionally producing carbapenemase OXA-244 indicates a risk of dissemination of resistant bacteria outside the hospitals.

Surveillance of adenoviruses and noroviruses in European recreational waters.

Exposure to human pathogenic viruses in recreational waters has been shown to cause disease outbreaks. In the context of Article 14 of the revised European Bathing Waters Directive 2006/7/EC (rBWD, CEU, 2006) a Europe-wide surveillance study was carried out to determine the frequency of occurrence of two human enteric viruses in recreational waters. Adenoviruses were selected based on their near-universal shedding and environmental survival, and noroviruses (NoV) selected as being the most prevalent gastroenteritis agent worldwide. Concentration of marine and freshwater samples was done by adsorption/elution followed by molecular detection by (RT)-PCR. Out of 1410 samples, 553 (39.2%) were positive for one or more of the target viruses. Adenoviruses, detected in 36.4% of samples, were more prevalent than noroviruses (9.4%), with 3.5% GI and 6.2% GII, some samples being positive for both GI and GII. Of 513 human adenovirus-positive samples, 63 (12.3%) were also norovirus-positive, whereas 69 (7.7%) norovirus-positive samples were adenovirus-negative. More freshwater samples than marine water samples were virus-positive. Out of a small selection of samples tested for adenovirus infectivity, approximately one-quarter were positive. Sixty percent of 132 nested-PCR adenovirus-positive samples analysed by quantitative PCR gave a mean value of over 3000 genome copies per L of water. The simultaneous detection of infectious adenovirus and of adenovirus and NoV by (RT)PCR suggests that the presence of infectious viruses in recreational waters may constitute a public health risk upon exposure. These studies support the case for considering adenoviruses as an indicator of bathing water quality.

Qualitative and quantitative detection of human pathogenic Yersinia enterocolitica in different food matrices at retail level in Bavaria.

Yersinia enterocolitica is a major foodborne pathogen and the third most important bacteriological cause of diarrhea in Germany. However, studies investigating the occurrence of human pathogenic Y. enterocolitica in food at the retail level are very rare. Most of the studies published so far show qualitative but not quantitative data concerning the prevalence of this human pathogen. In this study the qualitative and quantitative assessment of human pathogenic Y. enterocolitica in different food matrices was investigated. For the qualitative analysis we used an enrichment method according to the International Organisation of Standardization (ISO) standard in combination with a real-time polymerase chain reaction (PCR) method detecting the ail gene of Y. enterocolitica. After detecting Y. enterocolitica in a sample, a quantitative investigation on Cefsulodin-Irgasan-Novobiocin (CIN) Agar was done to get information about the contamination level of the different samples. During the years 2008 and 2009, 446 samples of pork and pork products, 51 samples of game meat, and 61 raw milk samples were investigated for the presence of human pathogenic Y. enterocolitica. The samples were collected at the retail level in Bavaria. From the pork samples investigated, 81 samples (18%) were positive for the ail gene by real-time PCR, but human pathogenic Y. enterocolitica O:3 were found only in 46 (10%) pork samples by culture; the concentration in the samples ranged between 0.04 cfu/g and 2.30 × 10(5) cfu/g. Three game meat samples were positive by real-time PCR, but not by the cultural detection. All raw milk samples were negative by real-time PCR and culture.

Prevalence of and risk factors for carriage of Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus among residents and staff of a German nursing home.

The aims of this cross-sectional study were to determine the prevalence of and risk factors for carriage of Panton-Valentine leukocidin-producing methicillin-resistant Staphylococcus aureus (PVL-MRSA) in residents and personnel of a nursing home in Germany. In this study, PVL-MRSA carriage status among nursing home residents was associated with risk factors reflecting their dependence on nursing care. No specific risk factors were detected among staff.