Application of three different methods to determine the prevalence, the abundance and the environmental drivers of culturable Vibrio cholerae in fresh and brackish bathing waters.

AIMS: Three cultivation methods were used to study the prevalence and abundance of Vibrio cholerae in Eastern Austrian bathing waters and to elucidate the main factors controlling their distribution. METHODS AND RESULTS: Vibrio cholerae abundance was monitored at 36 inland bathing sites with membrane filtration (MF), a standard most probable number (MPN) approach and direct plating (DP). Membrane filtration yielded the most reliable and sensitive results and allowed V. cholerae detection at 22 sites with concentrations up to 39 000 CFU per 100 ml, all belonging to serogroups other than O1 and O139 and not coding for cholera toxin and toxin coregulated pilus. Direct plating turned out as an easy method for environments with high V. cholerae abundances, conductivity was the only significant predictor of V. cholerae abundance in the bathing waters at warm water temperatures. CONCLUSIONS: Vibrio cholerae nonO1/nonO139 are widely prevalent in Eastern Austrian bathing waters. Instead of the standard MPN approach, MF and DP are recommended for V. cholerae monitoring. Conductivity can be used as a first easy-to-measure parameter to identify potential bathing waters at risk. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio cholerae nonO1/nonO139 infections associated with bathing activities are an increasing public health issue in many countries of the northern hemisphere. However, there are only limited data available on the prevalence and abundance of V. cholerae in coastal and inland bathing waters. For monitoring V. cholerae prevalence and abundance, reliable and simple quantification methods are needed. Moreover, prediction of V. cholerae abundance from environmental parameters would be a helpful tool for risk assessment. This study identified the best culture-based quantification methods and a first quick surrogate parameter to attain these aims.

Whole genome sequencing as a tool to investigate a cluster of seven cases of listeriosis in Austria and Germany, 2011-2013.

A cluster of seven human cases of listeriosis occurred in Austria and in Germany between April 2011 and July 2013. The Listeria monocytogenes serovar (SV) 1/2b isolates shared pulsed-field gel electrophoresis (PFGE) and fluorescent amplified fragment length polymorphism (fAFLP) patterns indistinguishable from those from five food producers. The seven human isolates, a control strain with a different PFGE/fAFLP profile and ten food isolates were subjected to whole genome sequencing (WGS) in a blinded fashion. A gene-by-gene comparison (multilocus sequence typing (MLST)+) was performed, and the resulting whole genome allelic profiles were compared using SeqSphere(+) software version 1.0. On analysis of 2298 genes, the four human outbreak isolates from 2012 to 2013 had different alleles at ≤6 genes, i.e. differed by ≤6 genes from each other; the dendrogram placed these isolates in between five Austrian unaged soft cheese isolates from producer A (≤19-gene difference from the human cluster) and two Austrian ready-to-eat meat isolates from producer B (≤8-gene difference from the human cluster). Both food products appeared on grocery bills prospectively collected by these outbreak cases after hospital discharge. Epidemiological results on food consumption and MLST+ clearly separated the three cases in 2011 from the four 2012-2013 outbreak cases (≥48 different genes). We showed that WGS is capable of discriminating L. monocytogenes SV1/2b clones not distinguishable by PFGE and fAFLP. The listeriosis outbreak described clearly underlines the potential of sequence-based typing methods to offer enhanced resolution and comparability of typing systems for public health applications.

Listeria monocytogenes - characterization of strains isolated from clinical severe cases.

Listeria monocytogenes became an increasing pathogen involved more frequently in sporadic severe illnesses and outbreaks of foodborne infections. This study investigates in vitro susceptibility of 26 strains of Listeria monocytogenes isolated from the clinical specimens collected between March 2009 and November 2013, from 24 patients hospitalized in three medical institutions in Bucharest. All isolates were tested by disk diffusion method to 15 antimicrobial agents, by using disk diffusion tests. Among the 26 clinical L. monocytogenes isolates tested, no multidrug resistant strains were detected, but 18 (72%) were found to be resistant to at least one clinically relevant antibiotic. Among them, 18 clinical isolates were resistant against ciprofloxacin also. Resistance to Ciprofloxacin was particularly noticed to the strains in Romania. Serological and molecular typing by Multiplex PCR method detected two molecular types 1/2 a, 3a and 1/2 b, 3b, as to the more frequent isolated among studied cases. These types of L. monocytogenes could be associated to the higher pathogenic activity of immunodeficient patients.

Clostridium difficile infection: monoclonal or polyclonal genesis?

Clostridium difficile is considered to be a leading cause of hospital-acquired diarrhea. C. difficile (CDI) infection shows a high rate of recurrence. There would have to be a predominantly monoclonal mechanism of CDI within individual patients in order for molecular epidemiologic tools such as polymerase chain reaction (PCR) ribotyping to be useful in outbreak investigation or differentiation between infection relapse versus re-infection. It was the aim of our study to determine whether CDI is of monoclonal or of polyclonal genesis. Between December 2009 and June 2010, 11 patients with nosocomial CDI were chosen arbitrarily. Five individual colonies of C. difficile were picked from each of the primary culture plates. Of 55 isolates gained, 47 were available for PCR ribotyping (eight isolates failed attempts to re-culture). Among these 47 isolates, eight different PCR ribotypes were identified. Only one of the 11 patients had a stool sample that yielded more than one ribotype (PCR ribotypes 438 and 232); this 67-year-old female cancer patient was already suffering from recurring diarrhea prior to the fatal episode of colitis which was subsequently investigated. We conclude that polyclonal infections may occasionally occur in patients with CDI. Our findings of predominantly monoclonal origin of CDI within patients suggest that molecular epidemiologic investigations can be used reliably for outbreak investigations or discrimination between relapse and re-infection.

Fatal Pseudomonas aeruginosa pneumonia in a previously healthy woman was most likely associated with a contaminated hot tub.

Community-acquired pneumonia due to Pseudomonas aeruginosa in previously healthy individuals is a rare disease that is associated with high fatality. On 14 February 2010 a previously healthy 49-year-old woman presented to an emergency room with signs and symptoms of pneumonia, 2 days after returning from a spa holiday in a wellness hotel. Blood cultures and respiratory specimens grew P. aeruginosa. Despite adequate antimicrobial therapy, the patient died of septic multiorgan failure on day nine of hospitalization. On February 26, nine water samples were taken from the hotel facilities used by the patient: In the hot tub sample 37,000 colony-forming units of P. aeruginosa/100 ml were detected. Two of five individual colonies from the primary plate used for this hot tub water sample were found to be genetically closely related to the patient's isolates. Results from PFGE, AFLP and MLST analysis allowed the two lung isolates gained at autopsy and the whirlpool bathtub isolates to be allocated into one cluster. The patient most likely acquired P. aeruginosa from the contaminated water in the hotel's hot tub. The detection of P. aeruginosa in high numbers in a hot tub indicates massive biofilm formation in the bath circulation and severe deficiencies in hygienic maintenance. The increasing popularity of hot tubs in hotels and private homes demands increased awareness about potential health risks associated with deficient hygienic maintenance.

Update: Multinational listeriosis outbreak due to 'Quargel', a sour milk curd cheese, caused by two different L. monocytogenes serotype 1/2a strains, 2009-2010.

We previously reported an outbreak of listeriosis in Austria and Germany due to consumption of Quargel cheese. It comprised 14 cases (including five fatalities) infected by a serotype 1/2a Listeria monocytogenes (clone 1), with onset of illness from June 2009 to January 2010. A second strain of L. monocytogenes serotype 1/2a (clone 2) spread by this product could be linked to further 13 cases in Austria (two fatal), six in Germany (one fatal) and one case in the Czech Republic, with onset of disease from December 2009 to end of February 2010.

Listeriosis outbreak caused by acid curd cheese Quargel , Austria and Germany 2009.

We report an outbreak of listeriosis in Austria and Germany due to the consumption of Quargel cheese produced by an Austrian manufacturer. At the time of writing this report, the outbreak was known to account for 14 outbreak cases in 2009, including four cases with lethal outcome. On 23 January 2010, the cheese product was voluntarily withdrawn from the market.

Outbreak of Clostridium difficile 027 infection in Vienna, Austria 2008-2009.

From November 2008 to 15 April 2009, 36 isolates of CD027 identified in Austria, all originating from four hospitals in Vienna. All isolates were positive for toxin A, toxin B and the binary toxin, and showed a characteristic 18 bp deletion in the tcdC gene. Clostridium difficile is an anaerobic spore-forming bacterium. Some strains may cause diarrhoea due to formation of toxins. Symptomatic C. difficile infection (CDI) is primarily linked with hospital admission and antibiotic treatment, although antibiotic exposure is neither necessary nor sufficient for CDI [1,2]. In Belgium, for instance, one third of CDI cases reported in the hospital surveillance system are not hospital-associated [3]. Symptoms range from mild diarrhoea to serious manifestations such as pseudomembranous colitis, toxic megacolon or perforation of the colon. C. difficile challenges hygiene standards as it is forms spores. The risk of infection rises with increasing age, underlying disease and immunodeficiency [4]. In recent years, a particularly virulent strain, ribotype 027 (CD027), has emerged in a number of countries, particularly in connection with hospital outbreaks, but also in community-acquired diarrhoea cases [5]. The risk of serious disease and death associated with CD027 exceeds that of other C. difficile strains. The classical CD027 is characterised - among other things - by an increased production of toxins A and B, production of a binary toxin and resistance to newer fluoroquinolones such as moxifloxacin. The first three Austrian cases of CD027 occurred in 2006 and in March 2008 [6,7]. Since August 2006, the Austrian National Reference Centre for C. difficile has ribotyped approximately 2,700 human C. difficile isolates received from all nine Austrian provinces. In recent months, a drastic increase in CD027 cases has been noted, all originating from four hospitals in Vienna. From November 2008 to 15 April 2009, 36 isolates of CD027 were received at the National Reference Centre. The Figure summarises these C. difficile 027 cases by month of reception of the sample at the reference centre.

Etiology of acute gastroenteritis in three sentinel general practices, Austria 2007.

BACKGROUND: We studied the etiology of acute gastroenteritis in a village with a total population of approximately 6,000. This is the first study in Austria that has investigated a broad range of pathogens recovered from an unselected population of patients who had consulted general practitioners because of gastroenteritis. MATERIALS AND METHODS: In 2007, all patients who visited one of three local general practitioners for acute gastroenteritis were invited to provide stool specimens to be tested for Salmonella, Shigella, Campylobacter, enterohemorrhagic Escherichia coli (EHEC) (mTSB enrichment [R-Biopharm] followed by toxin ELISA plus culture), enteropathogenic E. coli (EPEC), Yersinia, Vibrio cholerae, Clostridium difficile (toxin plus culture), rotavirus plus adenovirus (RIDA) Quick Rotavirus/Adenovirus Combi test), Giardia duodenalis plus Cryptosporidium parvum (RIDA) Quick Cryptosporidium/Giardia Combi test), astrovirus (ELISA), and norovirus (reverse-transcriptase PCR). RESULTS: Stool specimens were provided by 306 patients (161 female) with acute diarrhea. The ages of the patients ranged from 1 to 89 years (mean 37, median 36). Pathogens were detected in 71 (23.2%) patients, with incidence peaks in February and June. Norovirus accounted for 36.0% of positive results, C. difficile for 18.7%, rotavirus for 17.3%, Campylobacter for 9.3%, Salmonella for 6.6%, adenovirus for 5.4%, G. duodenalis and C. parvum for 2.7% each, and Yersinia enterocolitica for 1.3%. No cases of shigellosis or infection with EHEC, EPEC, or astrovirus were diagnosed. Viruses accounted for 58.7% of the 75 positive results, bacteria for 36.0%, and parasites for 5.3%. CONCLUSION: Our study underlines a dominant role of norovirus and toxigenic C. difficile as etiologic agents of acute gastroenteritis among the patients of general practitioners.

Characterization of Clostridium difficile isolates using capillary gel electrophoresis-based PCR ribotyping.

We have developed a Clostridium difficile PCR ribotyping method based on capillary gel electrophoresis and have compared it with conventional PCR ribotyping. A total of 146 C. difficile isolates were studied: five isolates were reference strains (PCR ribotypes 001, 014, 017, 027 and 053); 141 were clinical isolates comprising 39 Austrian PCR ribotypes collected in the period 2006-2007 at 25 Austrian healthcare facilities. Capillary gel electrophoresis yielded up to 11 fragments per isolate and 47 ribotype patterns. All but one of the five PCR ribotypes of reference strains were clearly reflected in the chromatograms of capillary-based typing. Capillary gel electrophoresis divided 24 isolates belonging to PCR ribotype type 014 into seven subgroups, whereas subtyping the same isolates using multiple-locus variable-number tandem-repeat analysis yielded three unrelated subgroups, without obvious correlation to sr subgroups. Using a web-based software program (http://webribo.ages.at), we were able to correctly identify these 014 isolates by simply allocating the seven subgroup patterns to one ribotype, i.e. to PCR ribotype 014. We consider capillary gel electrophoresis-based PCR ribotyping to be a way of overcoming the problems associated with inter-laboratory comparisons of typing results, while at the same time substantially diminishing the hands-on time for PCR ribotyping.

Characterization of clinical Clostridium difficile isolates by PCR ribotyping and detection of toxin genes in Austria, 2006-2007.

In order to assess the lethality of Clostridium difficile-associated disease (CDAD) and the PCR ribotypes prevalent in Austria, the Austrian Agency for Health and Food Safety requested isolates of C. difficile from patients in a structured but arbitrary sampling scheme. In the allocated period from February 2006 to January 2007, local hospital laboratories within each of the nine provinces were asked to submit C. difficile isolates from at least ten cases of CDAD. Confirmation of species identification, toxin detection, susceptibility testing against four antimicrobial agents and typing using a PCR ribotyping method were performed at the reference laboratory. In total, 149 isolates of putative C. difficile were submitted, from which 142 were included for study. Antimicrobial susceptibility patterns revealed resistance to clindamycin in 57% and high-level resistance to moxifloxacin in 38% of isolates tested. CDAD manifested as diarrhoea (including eight cases of bloody diarrhoea) in 126 cases (88.7%), as pseudomembranous colitis in 15 cases (10.6%) and as toxic megacolon in one case. Twelve of the 142 patients died within 30 days of specimen collection (8.45% lethality). A lethal outcome occurred in 2/15 cases (13.3%) when pseudomembranous colitis was present and in 10/126 cases (7.9%) in the absence of pseudomembranous colitis or toxic megacolon. Among the 142 isolates from 25 health-care facilities, 41 PCR ribotype patterns were found. The most frequent ribotypes were AI-5 (including six lethal cases out of 26 patients), 014 (two out of 24) and 053 (one out of 24). The typing patterns demonstrated the occurrence of clusters in hospitals.

Outbreak of acute gastroenteritis in an Austrian boarding school, September 2006.

An outbreak of acute gastroenteritis occurred in September 2006 in a boarding school in eastern Austria. Of 113 cases, 101 were hospitalised. In order to identify the outbreak source, a retrospective cohort study on the group at risk was performed, including 222 pupils and 30 staff members. Food exposure in the canteen of the school was identified as the most relevant common link among the cases in the case series investigation. Although the preliminary microbiological investigation made Norovirus infections possible, an in-depth descriptive epidemiological investigation later pointed to food intoxication rather than a viral infection as the cause of the outbreak. The analytical epidemiological investigation implicated boiled rice and chicken wings served in the canteen as the most likely source of the outbreak. Staphylococcus aureus was identified as the causative agent. Further molecular characterisation revealed that the predominant S. aureus type in this outbreak was a new spa type, t2046. The same spa type was isolated from stool specimens of the majority of the cases investigated, from samples of the incriminated boiled rice, and also from a swab of a palmar skin lesion of one of the healthy kitchen workers, who is therefore the most likely source of contamination. This outbreak underlines again the importance of compliance with the basic guidelines for kitchen hygiene.