Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

Antimicrobial resistance and its relationship with antimicrobial use on Austrian dairy farms.

The aim of this study was to investigate the prevalence of ESBL/AmpC-producing E. coli and the resistance pattern of commensal E. coli, as well as the link between the use of antibiotics (AMU) and the occurrence of resistance in E. coli on Austrian dairy farms. AMU data from 51 farms were collected over a one-year period in 2020. Fecal samples were collected from cows, pre-weaned and weaned calves in 2020 and 2022. Samples were then analyzed using non-selective and selective agar plates, E. coli isolates were confirmed by MALDI-TOF analysis. Broth microdilution was used for antimicrobial susceptibility testing. The AMU of each farm was quantified as the number of Defined Daily Doses (nDDDvet) and Defined Course Doses (nDCDvet) per cow and year. Cephalosporins (mean 1.049; median 0.732 DDDvet/cow/year) and penicillins (mean 0.667; median 0.383 DDDvet/cow/year) were the most frequently used antibiotics on these farms, followed by tetracyclines (mean 0.275; median 0.084 DDDvet/cow/year). In 2020, 26.8% of the E. coli isolated were resistant to at least one antibiotic class and 17.7% of the isolates were classified as multidrug resistant (≥3 antibiotic classes). Out of 198 E. coli isolates, 7.6% were identified as extended-spectrum/AmpC beta-lactamase (ESBL/AmpC) producing E. coli. In 2022, 33.7% of E. coli isolates showed resistance to at least one antibiotic and 20.0% of isolates displayed multidrug resistance. Furthermore, 29.5% of the samples carried ESBL/AmpC-producing E. coli. In 2020 and 2022, the most frequently determined antibiotic resistances among commensal E. coli isolates were to tetracyclines, sulfonamides and penicillins. In addition, pre-weaned calves had the highest resistance rates in both years. Statistical analyses showed a significant association between low and high use AMU classifications for penicillins (in nDDDvet/cow/year) and their respective resistance among commensal E. coli isolates in 2020 (p = 0.044), as well as for sulfonamide/trimethoprim (p = 0.010) and tetracyclines (p = 0.042). A trend was also noted between the total amount of antibiotics used on farm in 2020 (by nDDDvet/cow/year) and multidrug resistances in commensal E. coli isolated on farm that year (p = 0.067). In conclusion, the relationship between AMU and antimicrobial resistance (AMR) on dairy farms continues to be complex and difficult to quantify.

Analysis of Antimicrobial Use and the Presence of Antimicrobial-Resistant Bacteria on Austrian Dairy Farms-A Pilot Study.

The assumed link between high levels of antimicrobial use on farms and selection for antimicrobial-resistant (AMR) bacteria on that farm remains difficult to prove. In the pilot study presented here, we analysed total antimicrobial use on 50 dairy farms in Austria and also collected environmental samples to ascertain whether specific AMR bacteria were present. Antimicrobial use (AMU) analysis was based on electronic veterinary treatment records over a one-year period. Faecal samples for the assessment of extended-spectrum beta-lactamase (ESBL)-producing E. coli were collected from cowsheds, calf pens, and youngstock housing areas, as well as dust samples from barns, to isolate methicillin-resistant Staphylococcus aureus (MRSA). Bacteriological cultures were carried out on selective agar. Farms were split into groups of 25 of the highest antimicrobial users and 25 of the lowest users. Overall, samples from 13/50 (26.0%) farms were found to be positive for the presence of ESBL-producing E. coli. Of these, eight farms were in the low user group and five were in the high user group. Only one farm was confirmed to harbour MRSA. Statistical analyses demonstrated that there was no significant difference in this study population between high or low antimicrobial use with respect to the presence of ESBL-producing E. coli on farms (p = 0.33). In conclusion, the presence of specific AMR bacteria on farms in this study population was not found to have a statistically proven relationship with their level of antimicrobial use.

Therapeutic vaccination of chronic hepatitis C nonresponder patients with the peptide vaccine IC41.

BACKGROUND & AIMS: IC41 is a synthetic peptide vaccine containing 7 relevant hepatitis C virus (HCV) T-cell epitopes and the T helper cell (Th)1/Tc1 adjuvant poly-L-arginine. IC41 has been shown to be safe and to induce HCV-specific interferon (IFN)-gamma-secreting CD4+ and CD8+ T cells in healthy volunteers. We aimed to investigate whether IC41 is able to induce HCV-specific T-cell responses also in chronic hepatitis C patients. METHODS: Sixty HLA-A2-positive chronic HCV patients not responding to or relapsing from standard therapy were randomized in a double-blind phase II study into 5 groups to receive 6 vaccinations of IC41 (3 different dose groups), HCV peptides alone, or poly-L-arginine alone. RESULTS: IC41 was well tolerated, and no drug-related serious adverse events or induction of hepatitis were observed. T-cell proliferation was recorded in up to 67% of patients in the 3 IC41 vaccine groups but only in 17% of patients treated with peptides alone. IFN-gamma enzyme-linked immunospot assay responses were observed exclusively in the IC41 groups with response rates up to 42%. There were 3 RNA responders with transient >1-log declines of HCV serum RNA associated with the strongest IFN-gamma enzyme-linked immunospot assay values within all 60 patients. CONCLUSIONS: This study showed that the HCV peptide vaccine IC41 can induce HCV-specific Th1/Tc1 responses in a subset of difficult to treat HCV nonresponder patients despite persisting viremia. However, changes in HCV RNA occurred only in single patients. Because strongest T-cell responses were associated with HCV RNA decline, further studies with optimized vaccine regimens and combination therapies have been initiated.

A foodborne outbreak due to norovirus in Austria, 2007.

A foodborne norovirus outbreak occurred after a pre-Christmas celebration among a group of local foresters in Austria in December 2007. A total of 66 persons, 60 participants of the Christmas party and 6 kitchen staff members of the restaurant where the party took place, were identified as the at-risk cohort. Questioning of this cohort was performed by self-report questionnaires or telephone interviews (response rate of 95%). The outbreak attack rate was 33.3% (21 of 63 persons), including two of the kitchen staff. Three stool specimens yielded norovirus genogroup II. Univariate analysis revealed that exposure to a ham roll and pastry was possibly associated with risk of gastroenteritis (risk ratio [RR] of 4.45, 95% CI of 1.91 to 10. RR of 2.44, 95% CI of 0.93 to 6.39). After controlling for the effects of sex, age, and other food items with a log-linear model, ham roll remained significantly associated with disease risk (RR of 3.91, 95% CI of 1.57 to 9.76). Ham roll was most likely contaminated with norovirus during preparation by a disease-free kitchen assistant, whose infant became sick with laboratory-confirmed norovirus gastroenteritis 2 days before the party. Informing food handlers about the possible risk of food contamination with norovirus and training them in the appropriate measures of hand hygiene and environmental disinfection at the working place and at home are essential for preventing food-related norovirus outbreaks. Norovirus-infected household members of healthy food handlers must be considered a possible reservoir for foodborne norovirus outbreaks.

Campylobacteriosis in Austria: situation and trends.

Campylobacter species are among the most commonly identified causative agents of acute bacterial gastroenteritis in humans worldwide. In Austria, campylobacteriosis has been a notifiable disease since 1996 and its incidence has increased continuously over the past years. Improvement of notification has led to an important increase in knowledge of the epidemiology of this disease in Austria. In addition to existing programs monitoring food safety, implementation of a national surveillance program to monitor antimicrobial resistance and the prevalence of campylobacter in food animals allows continuous surveillance of trends in antimicrobial susceptibility patterns in humans, animals and food, as well as a more comprehensive evaluation of the occurrence of campylobacter in the relevant reservoirs of human infection. Such data make it possible to identify the most important areas for intervention and launch of control strategies. As in other countries around the world, antimicrobial resistance of campylobacter from human, animal and food origin, particularly resistance to fluoroquinolones, is of public health concern in Austria. The magnitude of global trade and foreign travel requires EU-wide and worldwide implementation of control strategies to fight campylobacteriosis.

An outbreak of norovirus gastroenteritis in an Austrian hospital, winter 2006-2007.

BACKGROUND: Norovirus is easily spread from person to person by the fecal-oral route and through aerosols or by vehicles such as contaminated food or water. The virus is able to survive in the environment for many days, which enables outbreaks to be prolonged. We describe a norovirus outbreak and its control measures in an Austrian secondary-level hospital during December 2006 - February 2007. METHODS: A descriptive-epidemiological investigation of the outbreak was undertaken. We also determined outbreak costs, including the estimated lost revenue associated with department closures and the cost of sick leave and cleaning expenses. Selected stool specimens were tested for norovirus RNA. RESULTS: In the hospital, 90 persons with symptoms and signs consistent with norovirus gastroenteritis with clinical onset between December 1, 2006 and February 13, 2007 were identified. Out of these, 56 patients and 14 persons among the hospital staff fulfilled the definition of an outbreak case (77.8%), and 20 cases (22.2%) were identified as non-outbreak cases including 13 community-acquired cases of norovirus gastroenteritis and 7 clinical-suspected cases of norovirus gastroenteritis associated with health care facilities other than the affected hospital. The Department of Internal Medicine was the mainly affected department (46 patient-cases and 6 staff-cases). Considering hospital patients, who have been hospitalised between December 1, 2006 and February 13, 2007 as cohort at risk of nosocomial norovirus infection, the nosocomial hospital outbreak attack-rate was 5.9% (56/947). A total of 120 hospital staff members worked in the period from December 1 to February 13, which makes an attack-rate among the hospital staff of 11.7% (14/120). Norovirus strain GII.4 variant 2006b was detected, which has been circulating widely in Europe since 2006. The total cost of the outbreak for the Department of Internal Medicine was 80,138. CONCLUSIONS: The significant disruption of patient care and the cost of this single nosocomial outbreak support strict implementations of adequate and timely control measures based on evidence-based recommendations.

A food-borne outbreak of Shigella sonnei gastroenteritis, Austria, 2008.

We report on the first foodborne outbreak of Shigella sonnei described in Austria. On July 14 2008, AGES was informed of a cluster of 22 laboratory-confirmed cases of infection with S. sonnei restricted to public health district X in the province of Salzburg. All cases had attended a youth-group trip to a small village in the province of Tyrol from July 7 to July 9. An outbreak case among the trip participants was a person who (1) attended the trip and (2) fell ill with diarrhea in the period between July 8 and July 12. Among the 61 trip participants, 42 fitted the outbreak case definition, including 31 culture-confirmed cases. A household outbreak case was a person who (1) did not participate in the trip, (2) fell ill with diarrhea not before July 10 and (3) had household contact with an outbreak case between one and three days before onset of illness. Of the 11 household outbreak cases, 10 were tested by stool culture and four of these had a laboratory-confirmed S. sonnei infection. In addition, one of the two hostel staff tested positive for S. sonnei. All 36 isolates were biotype gl, lysotype 12, and were indistinguishable from each other by PFGE. A cohort study among the trip participants revealed a risk ratio of 2.71 for consumption of salad (on at least 1 of the 3 days of the trip) (95% CI: 1.38-5.32, P = 0.004). Among the 42cases, 34 could be explained by consumption of salad. The landlady of the hostel, who prepared the salad with bare hands, was a carrier and was assumed to be the source of contamination of the salad. Without proper epidemiological analytical investigation of this shigellosis outbreak, its association with food consumption would not have been identified.

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.

Estimating sensitivity and specificity of a PCR for boot socks to detect Campylobacter in broiler primary production using Bayesian latent class analysis.

The present study compares three different assays for sample collection and detection of Campylobacter spp. in broiler flocks, based on (i) the collection of faecal samples from intestinal organs (caecum), (ii) individual faecal droppings collected from the bedding and (iii) faecal material collected by socks placed on the outside of a pair of boots (boot socks) and used for walking around in the flock. The two first methods are examined for Campylobacter using a culture method (ISO-10272-2:2006), while the boot socks are tested using PCR. The PCR-assay is a genus specific multiplex PCR with primers targeting 16S rDNA in Campylobacter and primers targeting Yersinia ruckerii. Sixty-seven broiler flocks from Austria and 83 broiler flocks from Denmark were included in this prospective study and 89 of these were found to be positive in at least one method (AT: 49 samples, DK: 40 samples) whereas 61 of these were negative in all assays. In Austria samples for the three assays were collected simultaneously, which facilitates a direct comparison of the diagnostic test performance. In Denmark, however, boot socks and faecal droppings were collected three days before slaughter while caecum samples were collected at slaughter. The results were evaluated in the absence of a gold standard using a Bayesian latent class model. Austrian results showed higher sensitivity for PCR detection in sock samples (0.98; Bayesian credible interval (BCI) [0.93-1]) than for culture of faecal droppings (0.86; BCI [0.76-0.91]) or caecal samples (0.92; BCI [0.85-0.97]). The potential impact of Campylobacter introduction within the final three days before slaughter was observed in Denmark, where four flocks were tested negative three days before slaughter, but were detected positive at the slaughterhouse. Therefore the model results for the PCR sensitivity (0.88; BCI [0.83-0.97]) and cultural ISO-method in faecal samples (0.84; BCI [0.76-0.92]) are lower than for caecal samples (0.93; BCI [0.85-0.98]). In our study, PCR detection on boot sock samples is more sensitive than conventional culture. In view of the advantage of rapid results before slaughter and low costs for sampling, especially in combination with existing Salmonella surveillance systems (just another pair of boot socks needed), this method-matrix combination could be a valuable surveillance tool in the broiler primary production.

Effects of low concentrations of cadmium on immunoglobulin E production by human B lymphocytes in vitro.

Exposure to cadmium (Cd) can cause a variety of biological effects including alterations of immune responses in animals and humans. Both immunosuppression and immunoenhancement have been reported. The present study was aimed at investigating the consequences of exposure to Cd on the human immunoglobulin (Ig) E synthesis, using purified peripheral blood B lymphocytes and IL-4 and anti-human CD40 monoclonal antibody (a-CD40 mAb) as stimuli. Low concentrations of Cd (0.1-10 microM) markedly inhibited production of IgE in a concentration-dependent manner. IgG production, in contrast to IgE, showed a tendency towards being enhanced by Cd, although with a certain individual variability; IgM production was not affected. Cd failed to alter immediate surface expression of the activation markers CD69 and CD23 indicating that early activation events were not impaired. However, the portion of activated B cells was diminished by Cd after stimulation for more than 24 h, paralleled by a concomitant decrease in viability and a subsequent reduction in proliferation. These data suggest that the mechanism of Cd action on activated B cells involved pathways that interrupted an effectively initiated cell activation and induced a cytotoxic signal. Results from this study thus provide further evidence for and new information on the immunotoxic and immunomodulatory effects of Cd on human immune responses.

Functional and phenotypic characterization of peptide-vaccine-induced HCV-specific CD8+ T cells in healthy individuals and chronic hepatitis C patients.

Only very limited information on phenotype and function of vaccine-induced CD8+ T cells is available for humans. We investigated hepatitis C virus-specific CD8+ T cells after vaccination with the HCV peptide-vaccine IC41 which includes 5 MHC-class I and 3 MHC class-II-restricted epitopes. In healthy subjects, IC41 induced both HCV-specific central memory as well as effector CD8+ T cells which rapidly expanded upon antigen exposure in vitro. IFNgamma production was dependent on formulation of the synthetic peptides with the adjuvant poly-l-arginine. In chronic HCV patients, the frequency of HCV-specific CD8+ T cells increased after vaccination with a decline of CD45RA-positive effector memory cells in some but not all patients. Thus, this study suggests that HCV-specific memory cells can be induced by peptide vaccination and that a reversion of functional impaired phenotypes by therapeutic vaccination is possible in chronic HCV infection.

Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects.

As interferon/ribavirin-based standard therapy is curative in only about half of HCV patients, there remains an important need for alternatives including vaccines. The novel peptide vaccine IC41 consists of five synthetic peptides harboring HCV T cell epitopes and poly-L-arginine as synthetic adjuvant. In this randomized, placebo-controlled trial, 128 HLA-A2 positive healthy volunteers received four s.c. vaccinations of seven different doses IC41, HCV peptides alone, poly-l-arginine alone or saline solution, every 4 weeks. IC41 was safe and well tolerated. Mild to moderate local reactions were transient. Immunogenicity was assessed using T cell epitope specific [3H]-thymidine proliferation, IFN-gamma ELIspot and HLA-tetramer assays. IC41 induced responses in all dose groups. Higher responder rates were recorded in higher dose groups and increasing number of vaccinations were associated with higher responder rates and more robust responses. Poly-L-arginine was required for the aimed-for Th1/Tc1-type immunity (IFN-gamma secreting T cells).

Stimulation of the immune system by different TBE-virus vaccines.

Tick-borne encephalitis (TBE) is one of the most frequent arthropod-transmitted viral diseases in Europe. Different vaccines against TBE-virus have been developed; a thimerosal-free and also albumin-free vaccine [Ticovac (Baxter Hyland Immuno, Vienna)] was approved in 2000. Contrary to previous experience, 779 cases of fever occurred following the first vaccination of children under 15 years of age and in 62 children febrile convulsions were even observed. Consequently, the composition of the vaccine was changed and albumin was again added [FSME-Immun (Baxter Vaccines, Vienna)] in 2001. The new Encepur Kinder (Chiron-Behring, Marburg) from 2002 is a TBE-vaccine for children without any protein as stabilizer but with a relatively high concentration of sucrose, while the former vaccine Encepur K from 1991 contained polygeline as the stabilizer. The induction of the immune system by the different TBE virus vaccines was compared in an in vitro test in order to find an explanation for the unexpected fever attacks. Whole blood was stimulated with complete vaccine suspension, and TNF-alpha, IL-1beta, IL-6, and IL-8 were determined from heparin/EDTA-plasma and culture supernatants. It was shown that Ticovac and the new Encepur Kinder can induce relatively high amounts of TNF-alpha and lower amounts of IL-1beta. An increase of both cytokines was first observed following an incubation of 4 hours, with a maximum after 15 hours. Concentrations returned to base-line values within 26 hours. The behaviour of both cytokines correlates with the febrile phases in children up to two years old. Albumin or other proteins like polygeline and also immunoglobulins prevented a rise of cytokines.