Interlaboratory proficiency tests for the detection of staphylococcal enterotoxin type A in food.

Staphylococcal enterotoxins (SEs) are among the leading causes of food intoxications, affecting consumer health even in nanogram (ng) amounts. In the European Union, certain food safety criteria are specified, including the absence of SEs in cheeses, milk powder and whey powder. Until 2019, the analytical reference method used was the European Screening Method, which was replaced by EN ISO 19020. For the official laboratories involved in food control, the German Reference Laboratory for coagulase-positive staphylococci including Staphylococcus aureus organized three interlaboratory proficiency tests (ILPTs) to detect SE type A in food during the years 2013-2018. The selected food products (cream cheese and vanilla pudding) were successfully tested beforehand with regard to easy handling, homogeneity and stability of the added toxin. In 2013, ILPT participants overall were not competent in detecting SE type A in food. The following factors were identified to improve the performance: (i) concentration of sample extract using dialysis; (ii) selection of a sensitive detection kit; and (iii) proper sample handling. By taking these factors into account and instructing and training the laboratories, their competence greatly improved. In 2018, all performance criteria (specificity, sensitivity and accuracy) were >90%, even at very low concentrations of SE type A of approximately 0·01 ng g-1 food.

Detection and quantification of methicillin-resistant Staphylococcus aureus in fresh broiler meat at retail in Germany.

The aim of this study was to detect, to quantify and to characterize MRSA in broiler meat samples with skin. Furthermore, we compared an isolation method using a second selective enrichment step (method A) with a simpler method omitting this step (method B). For quantification we used a direct plating method on selective agar plates and a "Most probable number" (MPN) technique for estimation of low numbers of MRSA. Presumptive MRSA colonies were confirmed by MALDI-TOF and by PCR. After confirmation the isolated MRSA were characterized by spa-typing and, if necessary, by multi-locus sequence typing. Method B detected more MRSA-positive samples (16.7%, n = 215) than method A (12.1%). However, method B also produced more false positive results (28.4%).The highest estimated number of MRSA in fresh broiler meat with skin was 1100 MPN/g, but in most positive samples (80.1%) the estimated numbers of MRSA were lower than 10 MPN/g. Thus, the numbers of MRSA in the samples were too low to detect using the spread plate technique. Ten different spa-types were identified. Six of these with 69% of the isolates were assigned to the clonal complex CC398 (t034; t011; t2576; t571; t5452; t1457). Spa-types t1430, t13177 and t899 can be assigned to CC9. Spa-type t304 was identified as MLST-type ST6. In conclusion, we provide quantitative data on low level contamination of fresh broiler meat with MRSA. Most isolated MRSA were from livestock associated spa-types. Omitting the second enrichment step was associated with an increase in sensitivity but lower specificity of the cultural method.

Staphylococcus aureus food-poisoning outbreak associated with the consumption of ice-cream.

In April 2013, a food poisoning outbreak caused by staphylococcal enterotoxins (SEs) in ice-cream occurred in Freiburg, Germany, among the 31 participants of a christening party. Of the 13 cases, seven were hospitalized or obtained ambulatory treatment. Different types of ice-cream, which was freshly produced at the hotel where the party took place, were found to contain SE and high amounts of coagulase positive staphylococci. Enterotoxigenic Staphylococcus aureus strains isolated from ice-cream and human cases were of the same spa-type (t127), harboured the sea gene and displayed identical phenotypic resistance-, Fourier transform infrared spectroscopy- (FT-IR) and microarray-profiles. Despite the strong microbiological and epidemiological evidence of ice-cream being the incriminated food vehicle of the outbreak, a common source of S. aureus from the ice-cream could not be deduced. As none of the employees carried the outbreak strain, either the equipment used for the production of the ice-cream or a contaminated ingredient is the most likely introduction source.

Methicillin-resistant Staphylococcus aureus in cattle food chains - prevalence, diversity, and antimicrobial resistance in Germany.

Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) have been found in various farm animal species throughout the world. It was the objective of this study to estimate the prevalence of MRSA in different cattle food chains (milk, beef, and veal) in Germany, to analyze the MRSA diversity along each food chain and to compare the characteristics of the different subtypes. Samples were collected between 2009 and 2012 from dairy herds (bulk tank milk), veal herds (dust from the stables), veal calves, and beef cattle at slaughter (nasal swabs) and carcasses of veal calves (surface cuts) and beef as well as veal at retail. Sampling was proportionally distributed over the country according to the cattle population (on-farm sampling), slaughterhouse capacity (abattoir samples), and the human population (meat at retail). Methicillin-resistant S. aureus were isolated using harmonized methods from all sample types and populations investigated. The highest proportion of positive samples was found in nasal swabs from veal calves at slaughter in 2012 (144/320; 45.0%) and the lowest rate in bulk tank milk in 2009 (14/388; 4.1%). Most isolates, irrespective of the origin, were from spa types t011 and t034. Both have been assigned to the clonal complex (CC) 398. Few isolates (15/632; 2.4%) were from spa types not associated with the CC398. Spa-type patterns were similar along individual food chains but differed between food chains. Antimicrobial resistance patterns differed between isolates from the different food chains and spa types. Isolates from the veal chain displayed the highest resistance rates. We conclude that there is substantial diversity in the MRSA prevalence across different cattle production sectors.

Occurrence of livestock-associated methicillin-resistant Staphylococcus aureus in Turkey and broiler barns and contamination of air and soil surfaces in their vicinity.

The emission of microorganisms, especially resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), from poultry farms is of public interest, and its occurrence and relevance are controversially discussed. So far, there are limited data on this issue. In this study, we investigated the occurrence of livestock-associated (LA)-MRSA inside and outside previously tested MRSA-positive poultry barns in Germany. In total, five turkey and two broiler fattening farms were investigated four and three times, respectively. In a longitudinal study during one fattening period, samples were collected from animals, the animals' environment inside the barn, including the air, and the barns' surroundings, such as ambient air and boot swabs of ground surfaces at different distances from the barn. Moreover, a cross-sectional study was carried out once inside the barns on five turkey and four broiler farms during the last third of the fatting period. In the cross-sectional study, LA-MRSA was detected in the air of most barns (7 of 9, 77.8%), as well as in many samples originating from animals, with detections levels of 50 to 54% in broiler and 62 to 77% in turkey farms. In the longitudinal study, LA-MRSA was found in the ambient air outside two turkey barns and on the ground surface on the downwind side of many (44.4%) turkey and broiler farms. The same spa types of isolates were observed inside and outside the barns. Transmission of MRSA within poultry farms, as well as emission via the airborne route, seems to be possible.

Community incidence of pathogen-specific gastroenteritis: reconstructing the surveillance pyramid for seven pathogens in seven European Union member states.

By building reconstruction models for a case of gastroenteritis in the general population moving through different steps of the surveillance pyramid we estimated that millions of illnesses occur annually in the European population, leading to thousands of hospitalizations. We used data on the healthcare system in seven European Union member states in relation to pathogen characteristics that influence healthcare seeking. Data on healthcare usage were obtained by harmonized cross-sectional surveys. The degree of under-diagnosis and underreporting varied by pathogen and country. Overall, underreporting and under-diagnosis were estimated to be lowest for Germany and Sweden, followed by Denmark, The Netherlands, UK, Italy and Poland. Across all countries, the incidence rate was highest for Campylobacter spp. and Salmonella spp. Incidence estimates resulting from the pyramid reconstruction approach are adjusted for biases due to different surveillance systems and are therefore a better basis for international comparisons than reported data.

Prevalence, antimicrobial resistance, and molecular characterization of methicillin-resistant Staphylococcus aureus from bulk tank milk of dairy herds.

It was the objective of the study to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in bulk tank milk from German dairy herds and to characterize isolates from bulk tank milk with respect to their Staph. aureus protein A (spa) and staphylococcal cassette chromosome mec (SCCmec) type, their phenotypic antimicrobial resistance and resistance- resp. virulence-associated genes using broth microdilution and a microarray for Staph. aureus. Bulk tank milk samples (25 mL) were tested for MRSA using a 2-step selective enrichment protocol. Presumptive MRSA were confirmed by PCR. Thirty-six isolates collected from bulk tank milk of dairy herds in 2009 and 2010 were included in the characterization. All isolates displayed spa-types assigned to the clonal complex CC398. Based on the epidemiological cut-off values for the interpretation of minimum inhibitory concentrations isolates were resistant to tetracycline (100%), clindamycin (58%), erythromycin (52%), quinupristin/dalfopristin (36%), and kanamycin (27%). Isolates did not carry genes associated with typical virulence factors for Staph. aureus such as the Panton-Valentine leukocidin. However, they did carry hemolysin genes. Livestock-associated MRSA of CC398 does occur in German dairy herds and the strains have similar properties as described for strains from pigs.

Prevalence of types of methicillin-resistant Staphylococcus aureus in turkey flocks and personnel attending the animals.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have been isolated from a number of livestock species and persons involved in animal production. We investigated the prevalence of LA-MRSA in fattening turkeys and people living on farms that house fattening turkeys. Eighteen (90%) of 20 investigated flocks were positive for MRSA, and on 12 of the farms 22 (37·3%) of 59 persons sampled were positive for MRSA. People with frequent access to the stables were more likely to be positive for MRSA. In most flocks MRSA that could be assigned to clonal complex (CC) 398 were detected. In five flocks MRSA of spa-type t002 that is not related to CC398 were identified. Moreover, other methicillin-resistant Staphylococcus spp. were detected on 11 farms and in eight people working on the farms.

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources.

A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6')-Ie-aph(2″)-Ia and/or ant(4')-Ia but also to aph(3')-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.

Methicillin-resistant Staphylococcus aureus (MRSA) in three dairy herds in southwest Germany.

The objective of this study was to analyse the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in three dairy herds in the southwest of Germany that had experienced individual cases of clinical and subclinical mastitis associated with MRSA. The herds were identified by the detection of MRSA during routine resistance testing of mastitis pathogens. All quarters of all cows in the herds that were positive on California Mastitis Test were sampled for bacteriological analysis on two occasions. Bulk tank milk samples were also tested. Furthermore, nasal swabs were collected from people working on the farms and from cattle. Environmental samples were collected from associated pig holdings. Isolates were characterized using spa-typing and testing for antimicrobial resistance. Our results revealed a substantial spread of MRSA in the three dairy herds. In the first of the two investigations carried out on all cows in the three herds, milk samples of 5.1-16.7% of dairy cows were found positive for MRSA. The respective proportions in the second herd level investigation were 1.4-10.0%. Quarters harbouring MRSA had higher somatic cell counts than quarters that were negative on culture. Methicillin-resistant Staphylococcus aureus were also detected in nasal swabs of staff (7/9), cows (7/15) and calves (4/7), bulk tank milk samples (3/3) and environmental samples from pig premises (4/5) on the farm. Herds B and C had no contact to herd A. However, in all three herds MRSA of spa-type t011 were detected in milk samples. Results show that MRSA of spa-type t011 is a problem in dairy farms that needs urgent attention.

Livestock associated methicillin-resistant Staphylococcus aureus (LaMRSA) isolated from lesions of pigs at necropsy in northwest Germany between 2004 and 2007.

An increasing number of reported detections of methicillin-resistant Staphylococcus aureus (MRSA) in food animals since 2007 has led to the assumption that there is an emerging zoonotic problem with livestock associated (la)MRSA potentially aggravating the MRSA problem in humans. It was the objective of the study to investigate, whether MRSA was present in clinical specimens of pigs collected at post-mortem since 2004 and to further characterize these isolates. We studied 138 isolates of S. aureus collected between 2004 and 2007 from various pathological lesions of pigs at necropsy. Potential MRSA were identified by growth on selective chromogenic media. Isolates were confirmed as MRSA using multiplex PCR. Confirmed isolates were spa- and SCCmec-typed and were tested for antimicrobial resistance. Overall, 60 (43%) S. aureus isolates were identified as MRSA. The majority (57/60) of the MRSA isolates found in the altered porcine tissues were spa-types associated with MRSA ST398. Three MRSA were ST97 isolates, a type that has not been described as an MRSA in pigs before. Other clonal complexes (ST9, ST30) dominated among the methicillin-sensitive S. aureus. MRSA were found in similar frequency in all 4 years. We assume that MRSA in pigs may have occurred earlier than 2004 and might be not really 'emerging', but rather have been overlooked until recently. The potentially causative role of the MRSA in the lesions warrants further investigation.

High heterogeneity within methicillin-resistant Staphylococcus aureus ST398 isolates, defined by Cfr9I macrorestriction-pulsed-field gel electrophoresis profiles and spa and SCCmec types.

During recent years, the animal-associated methicillin-resistant Staphylococcus aureus clone ST398 has extensively been studied. The DNA of these isolates turned out to be refractory to SmaI restriction, and consequently, SmaI is unsuitable for subtyping this clone by standard pulsed-field gel electrophoresis (PFGE). Very recently, ST398 DNA was shown to be digested by Cfr9I, a neoschizomer of SmaI. In the present study, we employed Cfr9I PFGE on 100 German and 5 Dutch ST398 isolates and compared their PFGE profiles, protein A gene variable repeat regions (spa types), and types of the staphylococcal cassette chromosome mec (SCCmec). The isolates (from healthy carrier pigs, clinical samples from pigs, dust from farms, milk, and meat) were assigned to 35 profiles, which were correlated to the SCCmec type. A dendrogram with the Cfr9I patterns assigned all profiles to two clusters. Cluster A grouped nearly all isolates with SCCmec type V, and cluster B comprised all SCCmec type IVa and V* (a type V variant first identified as III) carriers plus one isolate with SCCmec type V. Both clusters also grouped methicillin-susceptible S. aureus isolates. The association of the majority of isolates with SCCmec type V in one large cluster indicated the presence of a successful subclone within the clonal complex CC398 from pigs, which has diversified. In general, the combination of Cfr9I PFGE with spa and SCCmec typing demonstrated the heterogeneity of the series analyzed and can be further used for outbreak investigations and traceability studies of the MRSA ST398 emerging clone.

Prevalence of MRSA types in slaughter pigs in different German abattoirs.

To investigate the prevalence of types of meticillin-resistant Staphylococcus aureus (MRSA) in slaughter pigs in German abattoirs, nasal swabs were collected from a total of 1026 pigs in five abattoirs after stunning in the course of two studies, and examined for MRSA. Study 1 included four abattoirs; study 2 was carried out in one large abattoir. Isolates were tested for antimicrobial susceptibility and characterised using spa-typing, multilocus sequence typing (MLST) and typing of the staphylococcal cassette chromosome, SCCmec. Overall, MRSA was isolated from 70.8 per cent of 520 samples in study 1 and from 49.0 per cent of 506 samples in study 2. The proportion of positive samples varied substantially between the abattoirs in study 1. Most isolates belonged to spa-types t011 and t034 and SCCmec types III and V. MLST of selected isolates revealed that they were all MLST ST398. Besides beta-lactams, 100 per cent of the isolates were resistant to tetracycline, 80.5 per cent were resistant to erythromycin and 80.7 per cent were resistant to clindamycin. Less than 5 per cent of the isolates were resistant to other antimicrobials.

Prevalence of equine herpesvirus type 2 (EHV-2) DNA in ocular swabs and its cell tropism in equine conjunctiva.

Equine herpes virus 2 (EHV-2), a gamma(2)-herpesvirus, is common in horses of all ages. Its role as a primary pathogen is unclear but there is an association between EHV-2, respiratory disease and keratoconjunctivitis. The purpose of this study was to gain more information on the prevalence of EHV-2 DNA in conjunctival swabs from horses with and without ocular disease and to define the anatomical site and cell type harbouring viral genome or antigen. By polymerase chain reaction (PCR) 22 out of 77 (28.6%) ocular swabs of clinically healthy and only 4 out of 48 (8.3%) samples from diseased horses were positive. To define the main virus reservoir ocular tissue from 13 randomly selected horses without pathological evidence of ocular disease were analysed by nested PCR. In two horses optic nerve, lacrimal gland and conjunctiva, in further two cases lacrimal gland and conjunctiva and in four horses the conjunctiva only were EHV-2 PCR positive. For specifying the target cell we focused on conjunctivae and selected 3 out of 15 clinically healthy slaughterhouse horses positive for EHV-2 by PCR. In situ hybridisation on sections of these paraffin embedded conjunctivae localized viral genome in histiocyte-like cells of the submucosa. Immunohistochemical staining with an EHV-2 or S100 specific polyclonal antiserum demonstrated that Langerhans cells were co-localized in the same region of the sample section where virus positive cells were detected. Furthermore, we concluded that detection of viral antigen revealed a productive virus infection.

Effects of hydrogen bonding to a bacteriochlorophyll-bacteriopheophytin dimer in reaction centers from Rhodobacter sphaeroides.

The properties of the primary electron donor in reaction centers from Rhodobacter sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)--bacteriopheophytin (BPhe) dimer with and without hydrogen bonds to the conjugated carbonyl groups. The heterodimer mutation His M202 to Leu was combined with each of the following mutations: His L168 to Phe, which should remove an existing hydrogen bond to the BChl molecule; Leu L131 to His, which should add a hydrogen bond to the BChl molecule; and Leu M160 to His and Phe M197 to His, each of which should add a hydrogen bond to the BPhe molecule [Rautter, J., Lendzian, F., Schulz, C., Fetsch, A., Kuhn M., Lin, X., Williams, J. C., Allen J. P., & Lubitz, W. (1995) Biochemistry 34, 8130-8143]. Pigment extractions and Fourier transform Raman spectra confirm that all of the mutants contain a heterodimer. The bands in the resonance Raman spectra arising from the BPhe molecule, which is selectively enhanced, exhibit the shifts expected for the addition of a hydrogen bond to the 9-keto and 2-acetyl carbonyl groups. The oxidation--reduction midpoint potential of the donor is increased by approximately 85 mV by the addition of a hydrogen bond to the BChl molecule but is only increased by approximately 15 mV by the addition of a hydrogen bond to the BPhe molecule. An increase in the rate of charge recombination from the primary quinone is correlated with an increase in the midpoint potential. The yield of electron transfer to the primary quinone is 5-fold reduced for the mutants with a hydrogen bond to the BPhe molecule. Room- and low-temperature optical absorption spectra show small differences from the features that are typical for the heterodimer, except that a large increase in absorption is observed around 860-900 nm for the donor Qy band in the mutant that adds a hydrogen bond to the BChl molecule. The changes in the optical spectra and the yield of electron transfer are consistent with a model in which the addition of a hydrogen bond to the BChl molecule increases the energy of an internal charge transfer state while the addition to the BPhe molecule stabilizes this state. The results show that the properties of the heterodimer are different depending on which side is hydrogen-bonded and suggest that the hydrogen bonds alter the energy of the internal charge transfer state in a well-defined manner.

ENDOR studies of the primary donor cation radical in mutant reaction centers of Rhodobacter sphaeroides with altered hydrogen-bond interactions.

The electronic structure of the cation radical of the primary electron donor was investigated in genetically modified reaction centers of Rhodobacter sphaeroides. The site-directed mutations were designed to add or remove hydrogen bonds between the conjugated carbonyl groups of the primary donor, a bacteriochlorophyll dimer, and histidine residues of the protein and were introduced at the symmetry-related sites L168 His-->Phe, HF(L168), and M197 Phe-->His, FH(M197), near the 2-acetyl groups of the dimer and at sites M160 Leu-->His, LH(M160), and L131 Leu-->His, LH(L131), in the vicinity of the 9-keto carbonyls of the dimer. The single mutants and a complete set of double mutants were studied using EPR, ENDOR, and TRIPLE resonance spectroscopy. The changes in the hydrogen bond situation of the primary donor were accompanied by changes in the dimer oxidation midpoint potential, ranging from 410 to 710 mV in the investigated mutants [Lin, X., Murchison, H. A., Nagarajan, V., Parson, W. W., Williams, J. C. & Allen, J. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10265-10269]. It was found that the addition or removal of a hydrogen bond causes large shifts of the spin density between the two halves of the dimer. Measurements on double mutants showed that the unpaired electron can be gradually shifted from a localization on the L-half of the dimer to a localization on the M-half, depending on the hydrogen bond situation. As a control, the effects of the different hydrogen bonds on P.+ in the mutant HL(M202), which contains a BChlL-BPheM heterodimer as the primary donor with localized spin on the BChl aL [Bylina, E. J., & Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7226-7230; Schenck, C. C., Gaul, D., Steffen M., Boxer S. G., McDowell L., Kirmaier C., & Holten D. (1990) in Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle M. E., Ed.) pp 229-238, Springer, Berlin] were studied. In this mutant only small local changes of the spin densities (< or = 10%) in the vicinity of the hydrogen bonds were observed. The effects of the introduced hydrogen bonds on the spin density distribution of the dimer in the mutants are discussed in terms of different orbital energies of the two BChl a moieties which are directly influenced by hydrogen bond formation. The observed changes of the spin density distribution for the double mutants are additive with respect to the single mutations.(ABSTRACT TRUNCATED AT 400 WORDS)