Differences in antimicrobial resistance of commensal Escherichia coli isolated from caecal contents of organically and conventionally raised broilers in Austria, 2010-2014 and 2016.

The prevalence of antimicrobial resistance in commensal Escherichia coli isolated from organically raised broiler flocks was compared to the prevalence in isolates from conventional flocks. From 2010 to 2014, and in 2016, resistance trends and multidrug resistance in isolates from the caecal contents of flocks from both broiler production forms were analyzed. Samples were taken in four abattoirs accounting for at least 90% of the national slaughtered broiler population. In total, 962 commensal E. coli were obtained from organically raised broiler flocks (n = 142) and from conventionally raised broiler flocks (n = 820). The mean prevalence of commensal E. coli isolates, which were fully susceptible to the antimicrobials tested, was 43.3% in organically raised broiler flocks and thus significantly higher (P < 0.001) compared to 16.7% in conventionally operated flocks. During the study period, the proportion of fully susceptible isolates increased significantly in both broiler populations. Antimicrobial resistance rates were significantly lower in commensal E. coli isolated from organic compared to conventional production regarding ciprofloxacin (33.3% versus 69.1%), nalidixic acid (33.7% versus 67.4%), sulfamethoxazole (26.7% versus 39.9%), ampicillin (19.0% versus 33.8%) and trimethoprim (12.8% versus 24.9%). Regarding tetracycline, tigecycline and ceftazidime resistance rates were slightly but not significantly higher in isolates from organic flocks (27.6% versus 25.9%; 4.0% versus 1.4%; 2.0% versus 1.9%). This fact is surprising for tetracycline, as none of the investigated organic flocks had been treated with this antimicrobial during their lifetime. No resistances were found in isolates from both production forms against colistin and meropenem, and from organic flocks against azithromycin. The annual prevalence of resistance against ciprofloxacin and nalidixic acid decreased significantly in isolates from both broiler production forms. In isolates from organic flocks, it also decreased regarding ampicillin and sulfamethoxazole. Significant increasing trends were observed in the resistance prevalence against trimethoprim and borderline significantly for ampicillin in commensal E. coli from conventional flocks. Multidrug resistance was detected at a significantly higher prevalence in isolates from conventionally raised flocks (35.1%) compared to organic flocks (22.7%). Findings from this study clearly indicate the influences of organic compared to conventional broiler production practices on the prevalence of antimicrobial resistance in commensal E. coli from broiler flocks.

[Implementation and results of the EU-wide baseline studies on the prevalence of Salmonella spp. in slaughter and breeding pigs in Austria]. / Umsetzung und Ergebnisse der EU-weiten Grundlagenerhebung zur Prävalenz von Salmonella spp. in Schlacht- und Zucht- schweinen in Osterreich.

The Member States of the European Union are following a common strategy on the control of Salmonella and other foodborne pathogens (Anonym, 2003). Within that framework baseline studies on the most relevant animal populations have been carried out. This paper describes the implementation and the results of the baseline studies on Salmonella spp. in slaughter and breeding pigs in Austria. A total of 647 slaughter pigs were sampled in 28 slaughterhouses between October 2006 and September 2007. Samples were taken from the ileocaecal lymphnodes to detect infection in pigs and from the surface of the carcasses to detect contamination. Out of the 617 datasets included in the final analysis, Salmonella prevalences of 2% in lymphnodes and 1.1% on the carcass surface were observed. S. Derby, S. Enteritidis and S. Typhimurium were the three most frequently identified serovars. In an additional study, a total of 252 holdings with breeding pigs has been sampled between January and December combined multiplier herds. Respectively prevalences of 5, 8, 5, 3 and 9.1% were obtained, with S. Typhimurium being the most frequently isolated serovar. Overall, compared to neighbouring Member States a rather low prevalence of Salmonella spp. in pigs was documented for Austria, in particular in slaughter pigs.The serovar distribution seemed to be similar throughout the pig populations, some also being represented in Austrian human isolates. Contamination of feed seems to play a minor role considering the overall low prevalence, but nevertheless has to be taken into account in any future control or monitoring strategy for Salmonella spp. in pigs.

[Implementation and results of the EU-wide baseline study on the prevalence of Salmonella spp. in flocks of turkeys in Austria]. / Umsetzung und Ergebnisse der EU-weiten Grundlagenstudie zur Prävalenz von Salmonella spp. in Putenherden in Osterreich.

The member states of the European Union (EU) are following a common strategy on the control of Salmonella and other foodborne zoonotic pathogens (Anonym, 2003). In order to establish the prevalence of Salmonella (S.) spp. in turkeys, a baseline survey was organised in between October 2006 and September 2007. In Austria a total of 202 turkey flocks in 104 holdings was included. By doing so, all holdings with fattening turkeys were covered by the sampling scheme, whereas breeding turkey flocks did not exist in the survey period. A prevalence of Salmonella spp. of 17.3% in flocks or 25% of holdings was obtained. A total of nine different serovars were identified, S. Hadar being the most frequently isolated with eleven flocks infected representing 31.4% of all. S. Montevideo and S. Saintpaul were both found in eight flocks, and S. Senftenberg in six flocks. Two flocks were colonized with S. Newport, while one flock each had S. Typhimurium, S. Derby, S. Blockley and a monophasic strain of Salmonella group B. Of the two serovars of highest relevance for human health, S. Typhimurium was detected in only 0.5% of flocks or 1% of holdings tested and S. Enteritidis was not detected at all. Taking into account the high prevalence of the various Salmonella serovars however control measures to prohibit horizontal transmission and entry of Salmonella spp. from the turkey reservoir into the food chain in Austria seem justified.

Role of the GapA and CrmA cytadhesins of Mycoplasma gallisepticum in promoting virulence and host colonization.

Mycoplasma gallisepticum is an important avian pathogen that commonly induces chronic respiratory disease in chicken. To better understand the mycoplasma factors involved in host colonization, chickens were infected via aerosol with two hemadsorption-negative (HA(-)) mutants, mHAD3 and RCL2, that were derived from a low passage of the pathogenic strain R (Rlow) and are both deficient in the two major cytadhesins GapA and CrmA. After 9 days of infection, chickens were monitored for air sac lesions and for the presence of mycoplasmas in various organs. The data showed that mHAD3, in which the crmA gene has been disrupted, did not promote efficient colonization or significant air sac lesions. In contrast, the spontaneous HA(-) RCL2 mutant, which contains a point mutation in the gapA structural gene, successfully colonized the respiratory tract and displayed an attenuated virulence compared to that of Rlow. It has previously been shown in vitro that the point mutation of RCL2 spontaneously reverts with a high frequency, resulting in on-and-off switching of the HA phenotype. Detailed analyses further revealed that such an event is not responsible for the observed in vivo outcome, since 98.4% of the mycoplasma populations recovered from RCL2-infected chickens still display the mutation and the associated phenotype. Unlike Rlow, however, RCL2 was unable to colonize inner organs. These findings demonstrate the major role played by the GapA and CrmA proteins in M. gallisepticum host colonization and virulence.

Prevalence of Salmonella spp. in Austrian broiler flocks in the context of the EU-wide baseline survey 2005-2006.

In Austria an EU-wide baseline survey on the prevalence of Salmonella spp. in broilers organized by the EU commission was conducted from October 2005 to September 2006. The aim of this study was to produce comparable data on the prevalence of Salmonella in broiler flocks and holdings for all member states and for the EU-Commission to set EU-wide targets for the control of Salmonella in the broiler populations. A randomised sampling plan was designed according to EU-commission parameters (p = 50%; CI = 95%, a = 5%). Sampling was carried out regularly throughout the whole year. On every farm one flock was sampled with five pairs of boot swabs and analysed in the lab according to appendix D of ISO 6579 (2002). In Austria, 363 flocks on farms consisting of at least 5000 broilers each were tested. 28 flocks (7.7%) showed infections with Salmonella spp., eight flocks (2.2%) had either S. Enteritidis (six flocks) or S. Typhimurium (two flocks). In detail, S. Enteritidis (1.7%), S. Typhimurium (0.6%), S. Montevideo (4.1%), S. Infantis 0.6%, S. Senftenberg, S. Tennessee and S. Virchow (0.3% each) have been found. Data indicated that the risk of vertical transmission of Salmonella spp. to broiler flocks has almost been kept at bay; however, the risk of horizontal transmission still needs attention. Contamination of feeding stuff, possible persistence, spreading between barns of a farm as well as introduction of Salmonella spp. through individuals or materials are important factors for future control strategies.

Foodborne outbreaks, Austria 2007.

In 2007 Austria reported a total of 438 foodborne outbreaks affecting 1715 people, including 286 hospitalized patients and one death. Salmonella spp. and Campylobacter spp. accounted for 95% of all reported outbreaks. Forty-eight (11%) of the 438 Austrian outbreaks were acquired abroad. Of the 390 domestically acquired foodborne outbreaks, bacterial infection caused 376, viruses (norovirus and 1-time hepatitis A virus) caused 11, and intoxications (Staphylococcus aureus enterotoxins, alkaloid toxins) caused two. In one outbreak the causative agent was unknown. Salmonella spp. caused 264 (70%) of the bacterial outbreaks, Campylobacter spp. caused 104 (28%), enterohemorrhagic Escherichia coli (EHEC O145:H-, O157:H-, O157:H7, O182: H49, O91:H7, ONT:H4) caused six, Shigella flexneri and Shigella sonnei each caused two. The hospitalization rates were 22% for domestically acquired infections with Salmonella spp. and 14% for Campylobacter spp. Among outbreaks where the source was known, eggs were implicated in 49%, meat products (especially poultry) in 44% and fish in 2%. The ratio of household outbreaks to general outbreaks was 82.3% to 17.7%. In 54 of the 62 general domestic outbreaks the following locations of exposure were documented: commercial food suppliers (e.g. restaurants, cafeterias) 24 times, family celebrations, nursery schools, take-aways and barbecues 22 times, nursing homes and hospitals eight times. It is likely that the relatively high number of household outbreaks reflects an insufficient level of epidemiological investigation of outbreaks in Austria. More resources may be needed for identification of individual clusters that belong to larger foodborne outbreaks exceeding district or provincial borders.

Clostridium difficile: a new zoonotic agent?

Clostridium difficile is mainly considered a nosocomial pathogen associated with diarrhea and pseudomembranous colitis in hospitalized patients. Austrian hospitals reported 2761 cases of C. difficile infection (including 277 lethal outcomes) in 2007, compared with 777 cases (including 54 lethal outcomes) in 2003. The occurrence of community-acquired C. difficile infection is also increasingly reported. Recent studies have shown the occurrence of C. difficile in food and animals. The aim of the present study was to determine the occurrence of C. difficile in food and animals in Austria. Between March and July 2008, gut or fecal samples from 67 cows, 61 pigs and 59 broiler chickens were collected at Austrian abattoirs. Between February and April 2008 meat samples (51 beef [25 ground], 27 pork [17 ground] and 6 samples of chicken meat) were purchased at retail outlets. Of the 187 samples tested, eight yielded C. difficile: in cows 3/67 samples (4.5%) were positive, in pigs 2/61 (3.3%), in broiler chickens 3/59 (5%). Six of the eight isolates yielded toxigenic C. difficile (toxins A and B): 2/67 (3%) cow samples, 2/61 (3.3%) pig samples, 2/59 (3.4%) chicken samples. Genes for the binary toxin were detected in one of the two pig isolates, a PCR ribotype 126 strain. None of the 84 meat samples yielded C. difficile. The results of this Austrian study suggest that animal reservoirs are possible sources, via food, of human C. difficile infection.

Methicillin-resistant Staphylococcus aureus: a new zoonotic agent?

Staphylococcus aureus is a major cause of infection in hospitals and the community. One third of the general population is colonized by the bacterium, constituting a risk factor for acquisition of infection with this pathogen. Worldwide, the increasing antibiotic resistance of S. aureus complicates treatment of infection and control measures. Soon after the introduction of methicillin, the first isolates resistant to this antibiotic were reported and named methicillin-resistant S. aureus (MRSA). During the past decade a major change in MRSA epidemiology has been observed: whereas in the past MRSA was almost exclusively regarded a hospital pathogen, the advent of community-acquired MRSA has led to infections in people without hospital-related risk factors. Recent evidence has also identified a link between colonization of livestock and MRSA carriage and infections in people who work with animals. Screening of pigs and pig farmers in the Netherlands revealed high prevalence of MRSA sequence type (ST) 398 and it has become clear that the emergence of ST398 is not just a Dutch problem, as reports on livestock colonization and human infections are appearing worldwide. In Austria, the ST398 lineage has been detected in dust samples from pig breeding facilities and in food samples. Since the first Austrian detection of this emerging lineage in 2006, 21 human isolates, partially associated with infections, have been observed. MRSA has to be regarded as a new emerging zoonotic agent and livestock may constitute a growing reservoir of the ST398 lineage. More information is needed so that control measures to reduce the impact of the emerging MRSA ST398 lineage on public health can be developed and implemented.

A foodborne outbreak of Salmonella Enteritidis phage type 6 in Austria, 2008.

In April 2008, a general foodborne outbreak of Salmonella Enteritidis phage type 6 affected nine persons (two households in Salzburg and one household in Tyrol; eight microbiologically confirmed cases and one possible case). Epidemiological investigation revealed that all cases had eaten lunch together at a farm. Homemade bread dumpling loaf, prepared with eggs from the farm, was the most likely vehicle for the pathogen. Six eggs were bacteriologically tested and yielded the outbreak strain on the egg shells. The farm's egg laying flock consisted of 12 hens, which were identified as the source of infection by isolating the outbreak strain from samples of hen fecal material and dust. Molecular subtyping using multiple loci variable number of tandem repeats analysis (MLVA) revealed a pattern unique to isolates from the outbreak, different from nine epidemiologically unrelated isolates tested for comparison. The laying hens were culled and no further cases of salmonellosis were associated with the farm. This example demonstrates the considerable potential of outbreak investigation to elucidate the transmission of infection along the food chain and to provide information essential for implementation of targeted measures for prevention of foodborne illness.

An outbreak of febrile gastroenteritis associated with jellied pork contaminated with Listeria monocytogenes.

In September 2008, the Austrian Agency for Health and Food Safety (AGES) learned of an outbreak of diarrheal illness that included a 71-year-old patient hospitalized for gastroenteritis with a blood culture positive for Listeria monocytogenes. Three stool specimens provided by seven of 19 persons attending a day trip to a foreign city, including a final break at an Austrian tavern, yielded L. monocytogenes. All isolates were of serovar 4b and had fingerprints indistinguishable from each other. A cohort study revealed that the outbreak of gastroenteritis occurred among 16 persons who had eaten dinner at the wine tavern on September 6. Of the 15 persons who ate from platters of mixed cold-cuts, 12 (80%) developed symptoms of febrile gastroenteritis within 24-48 h. The median age of those who became ill was 62 years. A 72-year-old patient recovered from gastroenteritis but was hospitalized with bacterial meningitis on day 19 after the dinner. The epidemiological investigation identified the consumption of mixed cold-cuts (including jellied pork) at the wine tavern as the most likely vehicle of the foodborne outbreak (P = 0.0015). This hypothesis was confirmed by microbiological investigation of jellied pork produced by the tavern owner on September 3. L. monocytogenes was isolated from leftover food in numbers of 3 x 10(3)-3 x 10(4) colony forming units/g and was indistinguishable from the clinical outbreak isolates. Symptoms reported by the 12 patients included unspecified fever (12x), diarrhea (9x), headache (5x), vomiting (4x), body aches (2x) and sore throat (1x). Active case finding identified one case of rhombencephalitis (female, age 48) among another group of four guests, among whom only the patient and her asymptomatic husband had eaten jellied pork on September 6. This is the first outbreak of L. monocytogenes-associated gastroenteritis reported in Austria. The occurrence of a secondary case of meningitis (diagnosed on day 19 after consumption of jellied pork) indicates a significant risk of systemic listeriosis among elderly patients with febrile gastroenteritis caused by L. monocytogenes; antibiotic therapy should therefore be considered in such cases of documented listerial gastroenteritis.

[Food borne infectious outbreaks, Austria 2005]. / Lebensmittelbedingte infektiöse Krankheitsausbrüche, Osterreich 2005.

In Austria, a total of 606 food borne outbreaks, affecting 1,910 people altogether (including 368 hospitalized patients and 1 fatal outcome) were documented in 2005. Salmonella spp. and Campylobacter spp. accounted for 99% of all reported outbreaks. Fortysix (8%) of the 606 outbreaks were acquired abroad. Bacteria caused all but one of the 560 domestically acquired food borne outbreaks: 427 (76%) were due to Salmonella spp., 128 (23%) due to Campylobacter spp. and two outbreaks each due to enterohaemorrhagic Escherichia coli and Yersinia enterocolitica. Norovirus caused an outbreak affecting 22 persons. The respective hospitalization rate for domestically acquired Salmonella spp. and Campylobacter spp. infections was 20% and 16%. Of those outbreaks where information as to the source was provided, eggs were implicated in 57%, meat products (especially poultry) in 30% and milk or dairy products (especially raw milk) in 4%. The ratio between family outbreaks and general outbreaks was 89% to 11%. For general outbreaks the following locations of exposure were given: commercial food suppliers (incl. restaurants, cafeterias) 34 x, family celebrations 14 x, nursery schools 4 x, festivities (e.g. fairs) 3 x, nursing homes twice and once a mixed outbreak involving commercial food suppliers plus homes (Austria-wide S. Enteritidis PT19 outbreak). In our opinion, the relatively high number of family outbreaks merely reflects the still insufficient quality of epidemiological outbreak investigation in Austria, i.e. lack of consolidating individual clusters into larger food borne outbreaks which exceed district or provincial borders.

An Austrian outbreak of Salmonella enteritidis phage type 36 in 2004.

In June 2004, an egg packing station was epidemiologically incriminated as possibly distributing the vehicle of the Salmonella enterica subsp. enterica ser. Typhimurium definitive type (DT) U291 outbreak which began in September 2003 and--as of June 2004 affected more than 100 persons all over Austria and 8 German tourists. A total of 68 egg producing farms were screened for salmonella. Whereas all samples tested negative for S. Typhimurium DT U291, one batch of laying hens was identified as being heavily contaminated with a rare Salmonella enterica subsp. enterica ser. Enteritidis phage type, namely PT 36. This phage type was cultured from 6 of 7 swabs from pooled fecal samples (drag swabs), from 1 of 4 samples of pooled yolks, and from 7 of 11 hens tested (ovaries, liver and intestinal content pooled per hen). In 2004, a total of 36 culture-confirmed human PT 36 infections (19 manifest cases, 12 asymptomatic carriers, and 5 excreters with unknown clinical status) were documented in 4 of the 9 Austrian provinces. It was possible to demonstrate that eggs from this single farm were the probable source of infection for 30 of 31 culture-confirmed human infections. Only in the case of one patient, who regularly purchased eggs at the local market, could no connection to the farm be proven due to inability to trace the market's egg source. The PT 36 outbreak ceased after elimination of the contaminated flock. This episode underlines the considerable potential of adequate epidemiological and microbiological investigations of food-borne outbreaks to improve prevention and control of communicable diseases. The source of the S. Typhimurium DT U291 outbreak--which initially prompted this investigation--is still not identified and accounts for more than 300 human cases until August 2005.

Identifying and subtyping species of dangerous pathogens by automated ribotyping.

An investigation of dangerous bacterial pathogens was conducted to determine the usefulness of automated rRNA operon ribotyping (RiboPrinter system) to identify species. A total of 26 isolates comprising Bacillus anthracis, Brucella spp., Burkholderia mallei, Francisella tularensis, and Yersinia pestis were tested using restriction endonucleases EcoRI, PstI, PvuII and AseI. The main problem was that the system's database-relying on EcoRI as restriction enzyme-does not contain the essential dangerous pathogens. B. anthracis was misidentified as B. cereus and Y. pestis as Y. pseudotuberculosis. Two isolates of F. tularensis ssp. holarctica were falsely identified as Vibrio cholerae. This study underscores that riboprint patterns generated with a single restriction enzyme are not always unique for each of the species tested. Using more than one enzyme, the RiboPrinter proved to be a valuable primary typing method. Databases of commercially available systems for the identification of bacteria should include the most important dangerous pathogens.

Phenotypic switching in Mycoplasma gallisepticum hemadsorption is governed by a high-frequency, reversible point mutation.

Mycoplasma gallisepticum is a flask-shaped organism that commonly induces chronic respiratory disease in chickens and infectious sinusitis in turkeys. Phenotypic switching in M. gallisepticum hemadsorption (HA) was found to correlate with phase variation of the GapA cytadhesin concurrently with that of the CrmA protein, which exhibits cytadhesin-related features and is encoded by a gene located downstream of the gapA gene as part of the same transcription unit. In clones derived from strain R(low), detailed genetic analyses further revealed that on-off switching in GapA expression is governed by a reversible base substitution occurring at the beginning of the gapA structural gene. In HA(-) variants, this event generates a stop codon that results in the premature termination of GapA translation and consequently affects the expression of CrmA. Sequences flanking the mutation spot do not feature any repeated motifs that could account for error-prone mutation via DNA slippage and the exact mechanism underlying this high-frequency mutational event remains to be elucidated. An HA(-) mutant deficient in producing CrmA, mHAD3, was obtained by disrupting the crmA gene by using transposition mutagenesis. Despite a fully functional gapA gene, the amount of GapA detected in this mutant was considerably lower than in HA(+) clonal variants, suggesting that, in absence of CrmA, GapA might be subjected to a higher turnover.

Mycoplasma gallisepticum: Influence of cell invasiveness on the outcome of experimental infection in chickens.

Recently we have shown that a low (R(low)) and a high laboratory passage (R(high)) of the poultry pathogen Mycoplasma gallisepticum prototype strain R differ markedly in their capability to invade non-phagocytic eukaryotic cells. In the present study the infection traits of these two mycoplasma passages were compared in an in vivo setting. After aerosol inoculation of chickens, M. gallisepticum was re-isolated from the inner organs of birds infected with R(low), whereas no mycoplasma was recovered from the inner organs of birds infected with R(high). These results indicate that the two mycoplasma populations derived from strain R differ in their capacity to cross the mucosal barrier and suggest that cell invasion may play a major role in the observed systemic spreading of M. gallisepticum in its chicken host.