Phylogeny and spatiotemporal dynamics of hepatitis E virus infections in wild boar and deer from six areas of Germany during 2013-2017.

The hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. Infections with the zoonotic HEV genotype 3, which can be transmitted from infected wild boar and deer to humans, are increasingly detected in Europe. To investigate the spatiotemporal HEV infection dynamics in wild animal populations, a study involving 3572 samples of wild boar and three deer species from six different geographic areas in Germany over a 4-year period was conducted. The HEV-specific antibody detection rates increased between 2013-2014 and 2016-2017 in wild boar from 9.5% to 22.8%, and decreased in deer from 1.1% to 0.2%. At the same time, HEV-RNA detection rates increased in wild boar from 2.8% to 13.3% and in deer from 0.7% to 4.2%. Marked differences were recorded between the investigated areas, with constantly high detection rates in one area and new HEV introductions followed by increasing detection rates in others. Molecular typing identified HEV subtypes 3c, 3f, 3i and a putative new subtype related to Italian wild boar strains. In areas, where sufficient numbers of positive samples were available for further analysis, a specific subtype dominated over the whole observation period. Phylogenetic analysis confirmed the close relationship between strains from the same area and identified closely related human strains from Germany. The results suggest that the HEV infection dynamics in wild animals is dependent on the particular geographical area where area-specific dominant strains circulate over a long period. The virus can spread from wild boar, which represent the main wild animal reservoir, to deer, and generally from wild animals to humans.

Impact of Bacteriophage-Supplemented Drinking Water on the E. coli Population in the Chicken Gut.

Among intestinal coliform microbes in the broiler gut, there are potentially pathogenic Escherichia (E.) coli that can cause avian colibacillosis. The treatment with antibiotics favors the selection of multidrug-resistant bacteria and an alternative to this treatment is urgently required. A chicken model of intestinal colonization with an apathogenic model strain of E. coli was used to test if oral phage application can prevent or reduce the gut colonization of extraintestinal pathogenic E. coli variants in two individual experiments. The E. coli strain E28 was used as a model strain, which could be differentiated from other E. coli strains colonizing the broiler gut, and was susceptible to all cocktail phages applied. In the first trial, a mixture of six phages was continuously applied via drinking water. No reduction of the model E. coli strain E28 occurred, but phage replication could be demonstrated. In the second trial, the applied mixture was limited to the four phages, which showed highest efficacy in vitro. E. coli colonization was reduced in this trial, but again, no reduction of the E. coli strain E28 was observed. The results of the trials presented here can improve the understanding of the effect of phages on single strains in the multi-strain microbiota of the chicken gut.

The qualified presumption of safety assessment and its role in EFSA risk evaluations: 15 years past.

Microorganisms are intentionally added at different stages of the food and feed chain (food or feed additive, novel food or plant protection product) and are subjected to regulation and safety assessment by the European Food Safety Authority. Safety evaluation is based on application dossiers for market authorisation to the European Commission. The qualified presumption of safety (QPS) concept was developed in 20031 to provide a harmonised generic safety pre-appraisal of the above microorganisms. Unambiguously defined biological taxonomic units (TUs) are assessed for their body of knowledge, their safety and their end use. Identified safety concerns for a certain TU can be, where reasonable in number and not universally present, reflected as 'qualifications.' Strains belonging to TUs having QPS status may benefit of a fast track evaluation. The lowest TU for which the QPS status is granted is the species level for bacteria and yeasts and the family for viruses. The QPS concept is also applicable to genetically modified microorganisms used for production purposes. Based on the current body of knowledge and/or the ambiguous taxonomic position, some TUs, such as filamentous fungi, bacteriophages, Enterococcus faecium, Escherichia coli, Streptomyces spp. and Oomycetes, are not considered liable for QPS status.

Influence of lauric acid on the susceptibility of chickens to an experimental Campylobacter jejuni colonisation.

Among the organic acids, lauric acid has shown a high level of in vitro activity against Campylobacter jejuni. The prevalence and intensity of C. jejuni excretion at slaughter often becomes lower with increasing age. In higher-aged broilers on organic farms which often use other breeds, in turn, the prevalence of C. jejuni is sometimes higher at slaughter. The question then arises as to whether a diet with higher lauric acid concentrations, the age alone or the genetic breed might have an effect in the spread and intensity of an experimental C. jejuni infection in vivo. Therefore, two complete diets with or without 2% lauric acid from palm kernel fatty acids were offered to 450 chickens (ten subgroups à 15 birds, repetitions: n = 3) of two broiler and two layer breeds (Ross 308, Hubbard JA 757, Lohmann Dual and Lohmann Brown-Classic). All breeds were reared for 42 days, Lohmann Brown-Classic also for about 98 days. Twenty-one days before dissection, three seeder birds per subgroup were orally infected with a 1 mL inoculum of C. jejuni (4.46±0.35 log10 CFU/mL). Qualitative detection of C. jejuni in cloacal swabs was performed at days 2, 4, 7, 14 after inoculation and at dissection in all birds. Quantitative detection was performed on excreta samples of seeder birds at days 2, 11 and 17 after experimental challenge and on caecal samples of all birds at dissection. Two days after experimental inoculation, C. jejuni prevalence was higher in control birds without lauric acid supplementation (48.9% vs. 39.6%; P = 0.0462). Depending on age, two days after inoculation the C. jejuni prevalence in young Lohmann Brown-Classic chickens was significantly lower (37.8% vs. 61.1%) whereas at dissection it was higher (99% vs. 67%). At day 2 after inoculation C. jejuni counts in the excreta of young Lohmann Brown-Classic were lower in comparison to those in old ones (log10 CFU/g: 3.30±2.68 vs. 5.24±1.56). Eleven (log10 CFU/g: 5.14±1.13 vs. 4.16±0.82) and 17 days after inoculatioin (log10 CFU/g: 3.77±2.02 vs. 1.72±1.87) it was the reverse situation. At dissection, the carriage of C. jejuni in caecal content was higher in younger than in older birds (log10 CFU/g: 8.57±0.46 vs. 6.66±1.43). An effect of genetic breed on C. jejuni prevalence was seen at dissection, this being lowest in Lohmann Dual chickens (91% vs. 98.9% in other breeds). At d 17 after challenge, C. jejuni counts in the excreta of young Lohmann Brown-Classic were lower in comparison to Ross 308 and Hubbard JA 757 (log10 CFU/g: 3.77±2.02 vs. 5.21±0.85 and 5.62±0.90). Lohmann Dual chickens showed an intermediary excretion, this being only significant lower compared to Hubbard JA 757 (log10 CFU/g: 4.31±0.89). In summary, the effect of lauric acid is limited to the initial phase after experimental inoculation. A higher age at infection seems to lead to a more rapid limitation of the infection. The excretion of C. jejuni appears to decrease more rapidly in layer breeds than in broiler lines after experimental inoculation.

Susceptibility of Methicillin-Resistant and -Susceptible Staphylococcus aureus Isolates of Various Clonal Lineages from Germany to Eight Biocides.

Few studies have been conducted on the susceptibility of bacteria to biocides. A total of 182 methicillin-resistant and -susceptible Staphylococcus aureus isolates collected from healthy or diseased humans and animals in Germany were included in the present study. Sixty-three isolates of animal origin and 119 human isolates were tested for their MICs to eight biocides or heavy metals by the broth microdilution method. The MIC50 and MIC90 values of human and animal isolates were equal or differed by not more than 1 dilution step, and statistical analysis revealed that differences between MICs of human and animal isolates were not significant. However, when taking into account the multilocus sequence type (MLST), a strong tendency (P = 0.054) to higher MICs of silver nitrate was detected for clonal complex 398 (CC398) isolates from humans compared to those from animals. Furthermore, a comparison of MIC values from isolates belonging to different clonal lineages revealed that important human lineages such as CC22 and CC5 exhibited significantly (P < 0.05) higher MICs for the biocides chlorhexidine, benzethonium chloride, and acriflavine than the main animal lineage sequence type 398 (ST398). Isolates with elevated MIC values were tested for the presence of biocide and heavy metal tolerance-mediating genes by PCR assays, and the following genes were detected: mepA (n [no. of isolates containing the gene] = 44), lmrS (n = 36), norA (n = 35), sepA (n = 22), mco (n = 5), czrC (n = 3), smr (n = 2), copA (n = 1), qacA and/or -B (n = 1), qacG (n = 2), and qacJ (n = 1). However, only for some compounds was a correlation between the presence of a biocide tolerance gene and the level of MIC values detected.IMPORTANCE Biocides play an essential role in controlling the growth of microorganisms and the dissemination of nosocomial pathogens. In this study, we determined the susceptibility of methicillin-resistant and -susceptible S. aureus isolates from humans and animals to various biocides and heavy metal ions and analyzed differences in susceptibilities between important clonal lineages. In addition, the presence of biocide or heavy metal tolerance-mediating genes was investigated. We demonstrated that important human lineages such as CC22 and CC5 had significantly higher MIC values for chlorhexidine, benzethonium chloride, and acriflavine than the main farm animal lineage, ST398. In addition, it was shown that for some combinations of biocides and tolerance genes, significantly higher MICs were detected for carriers. These findings provide new insights into S. aureus biocide and heavy metal tolerance.

Inactivation of Salmonella Typhimurium and Listeria monocytogenes on ham with nonthermal atmospheric pressure plasma.

The application of cold atmospheric pressure plasma (CAP) for decontamination of sliced ready-to-eat (RTE) meat products (in this case, rolled fillets of ham), inoculated with Salmonella (S.) Typhimurium and Listeria (L.) monocytogenes was investigated. Cold atmospheric plasma (CAP) is an ionised gas that includes highly reactive species and ozone, interacting with cell membranes and DNA of bacteria. The mode of action of CAPs includes penetration and disruption of the outer cell membrane or intracellular destruction of DNA located in the cytoplasm. Inoculated ham was treated for 10 and 20 min with CAP generated by a surface-micro-discharge-plasma source using cost-effective ambient air as working gas with different humidity levels of 45-50 and 90%. The chosen plasma modes had a peak-to-peak voltage of 6.4 or 10 kV and a frequency of 2 and 10 kHz. Under the tested conditions, the direct effectiveness of CAP on microbial inactivation was limited. Although all treated samples showed significant reductions in the microbial load subsequent to plasma treatment, the maximum inactivation of S. Typhimurium was 1.14 lg steps after 20 min of CAP-treatment (p<0.05), and L. monocytogenes was reduced by 1.02 lg steps (p<0.05) using high peak-to-peak voltage of 10 kV and a frequency of 2 kHz regardless of moisture content. However, effective inactivation was achieved by a combination of CAP-treatment and cold storage at 8°C ± 0.5°C for 7 and 14 days after packaging under sealed high nitrogen gas flush (70% N2, 30% CO2). Synergistic effects of CAP and cold storage for 14 days led to a clearer decrease in the microbial load of 1.84 lg steps for S. Typhimurium (p<0.05) and 2.55 lg steps for L. monocytogenes (p<0.05). In the case of L. monocytogenes, subsequent to CAP-treatment (10 kV, 2 kHz) and cold storage, microbial counts were predominantly below the detection limit. Measurement showed that after CAP-treatment, surface temperature of ham did not exceed the room temperature of 22°C ± 2°C. With the application of humidity levels of 45-50%, the colour distance ΔE increased in CAP treated samples due to a decrease in L* values. In conclusion, effectiveness of CAP-treatment was limited. However, the combination of CAP-treatment and cold storage of samples under modified-atmospheric-conditions up to 14 days could significantly reduce microorganisms on RTE ham. Further investigations are required to improve effectiveness of CAP-treatment.

Genomic characterization of malonate positive Cronobacter sakazakii serotype O:2, sequence type 64 strains, isolated from clinical, food, and environment samples.

BACKGROUND: Malonate utilization, an important differential trait, well recognized as being possessed by six of the seven Cronobacter species is thought to be largely absent in Cronobacter sakazakii (Csak). The current study provides experimental evidence that confirms the presence of a malonate utilization operon in 24 strains of sequence type (ST) 64, obtained from Europe, Middle East, China, and USA; it offers explanations regarding the genomic diversity and phylogenetic relatedness among these strains, and that of other C. sakazakii strains. RESULTS: In this study, the presence of a malonate utilization operon in these strains was initially identified by DNA microarray analysis (MA) out of a pool of 347 strains obtained from various surveillance studies involving clinical, spices, milk powder sources and powdered infant formula production facilities in Ireland and Germany, and dried dairy powder manufacturing facilities in the USA. All ST64 C. sakazakii strains tested could utilize malonate. Zebrafish embryo infection studies showed that C. sakazakii ST64 strains are as virulent as other Cronobacter species. Parallel whole genome sequencing (WGS) and MA showed that the strains phylogenetically grouped as a separate clade among the Csak species cluster. Additionally, these strains possessed the Csak O:2 serotype. The nine-gene, ~ 7.7 kbp malonate utilization operon was located in these strains between two conserved flanking genes, gyrB and katG. Plasmidotyping results showed that these strains possessed the virulence plasmid pESA3, but in contrast to the USA ST64 Csak strains, ST64 Csak strains isolated from sources in Europe and the Middle East, did not possess the type six secretion system effector vgrG gene. CONCLUSIONS: Until this investigation, the presence of malonate-positive Csak strains, which are associated with foods and clinical cases, was under appreciated. If this trait was used solely to identify Cronobacter strains, many strains would likely be misidentified. Parallel WGS and MA were useful in characterizing the total genome content of these Csak O:2, ST64, malonate-positive strains and further provides an understanding of their phylogenetic relatedness among other virulent C. sakazakii strains.

The safety and quality of pork and poultry meat imports for the common European market received at border inspection post Hamburg Harbour between 2014 and 2015.

Though imports of products of animal origin into the European Union (EU) have to comply with legal requirements and quality standards of the community, food consignment rejections at external EU borders have been increasing in recent years. This study explored microbiological metrics according to national target and critical values valid for samples at consumer level of 498 fresh poultry meat and 136 fresh pork filets from consignments subjected to physical checks during clearing at the border inspection post Hamburg harbour between January 2014 and December 2015 with ISO standard methods. Quantitative results indicated that critical thresholds for aerobic counts, Enterobacteriaceae, and E. coli were never surpassed. Merely for staphylococci, one poultry sample (0.2%) and 10 pork samples (9.3%) exceeded the critical limit (3.7 log cfu/g). However, qualitative analyses revealed that, Staphylococcus aureus was present in 16% and 10% of all poultry and pork samples, respectively, though no methicillin-resistant Staphylococcus aureus could be confirmed. Moreover, E. coli was present in 50% and 67% of all pork and poultry samples, respectively, and thereof 33 isolates were confirmed as extended-spectrum ß-lactamase-producing E. coli. Only 1.2% of the poultry samples were unacceptable due to the presence of Salmonella spp., whereas they were not detected in any pork sample. Campylobacter spp. were not detected in any sample. Though imported pork and poultry meat complies mostly with national market requirements, it might pose a potential risk to public health, especially for a direct or indirect foodborne transmission of imported, uncommon strains of zoonotic bacteria.

Prevalence and characterization of extended-spectrum ß-lactamase (ESBL) and AmpC ß-lactamase producing Enterobacteriaceae in fresh pork meat at processing level in Germany.

ESBL or AmpC ß-lactamase producing Enterobacteriaceae is an increasing concern in human medicine. A distribution via the food chain is discussed, but less is known about these bacteria on fresh pork meat. The aim of this study was to investigate the prevalence of ESBL/AmpC Enterobacteriaceae bacteria in fresh pork meat at processing level in Germany. The analysis comprised microbiological hygiene parameters and further pheno- and genotypical characterization of ESBL/AmpC isolates. The examination included three pools of meat and one corresponding meat juice sample from each of the tested pork meat batches (n=63). ESBL/AmpC producers were found in 42.9% (36.5% confirmed by genotype, gt) of the investigated batches, either in meat or meat juice. Meat juice was more often (28.6%) contaminated with ESBL/AmpC bacteria than meat (20.6%). Hygiene parameters were satisfactory in all samples and were thus not a suitable tool for predicting the presence of ESBL/AmpC producers. Most of the 37 confirmed ESBL/AmpC bacteria were identified as Escherichia coli (n=18) or Serratia fonticola (n=13). Susceptibility testing identified 32 of the 37 isolates to be multidrug-resistant. The most common resistance genes TEM, SHV, and CTX-M were found in 19 of the ESBL/AmpC isolates, mostly E. coli. A single detected AmpC ß-lactamase producing E. coli carried a CMY-2 gene. Multilocus sequence typing (MLST) investigations of the ESBL/AmpC E. coli revealed 11 different sequence types. In conclusion, fresh pork meat can harbor highly diverse multidrug-resistant ESBL Enterobacteriaceae, even though at low rates. The study suggests that fresh pork meat might be a source for multidrug-resistant ESBL/AmpC Enterobacteriaceae of various origins. Therefore these data contribute to the epidemiological understanding of the distribution of resistant bacteria and the impact of the food chain on public health.

Implementation of a Risk-Orientated Hygiene Analysis for the Control of Salmonella JAVA in the Broiler Production.

This field study aimed to establish a risk-orientated hygiene analysis on two broiler farms in Northwestern Germany for the practical use in broiler housing to evaluate the success of disinfection procedures for eradicating S. Java. The risk-orientated hygiene analysis enables fast, reproducible and cost-effective testing of broiler farms and in turn helps minimize the public health risk ensuing from S. Java. Farms were tested before and after cleaning as well as after disinfection according to a risk-orientated hygiene analysis for the presence of Salmonella DNA with qPCR. Positive PCR samples were confirmed by classical microbiology. Before cleaning, all checkpoints were tested positive for Salmonella DNA. Salmonella reduction of ca 66% of the sampled points could be achieved by intensive cleaning. A first disinfection on farm A and B failed to completely eradicate S. Java. A second disinfection followed and finally achieved a Salmonella-free status of the barns. During nine rearing periods, farms were tested weekly with boot swabs for Salmonella and at slaughter carcasses were tested for Salmonella status. No Salmonella were detected in these examinations. The two studied broiler farms have, to date, remained free of Salmonella.

Arcanobacterium wilhelmae sp. nov., isolated from the genital tract of a rhinoceros (Rhinoceros unicornis).

A taxonomic study using a polyphasic approach was performed on an unidentified Arcanobacterium-like Gram-stain-positive bacterium isolated from the genital tract of a rhinoceros. Comparative 16S rRNA gene sequencing showed that the bacterium belonged to the genus Arcanobacterium and was most closely related to the type strains of Arcanobacterium canis (98.8 % 16S rRNA gene sequence similarity), Arcanobacterium phocisimile (97.8 %), Arcanobacterium phocae (97.7 %), Arcanobacterium haemolyticum (97.4 %), Arcanobacterium hippocoleae (96.6 %), Arcanobacterium pinnipediorum (96.4 %) and Arcarnobacterium pluranimalium (95.4 %). DNA-DNA hybridization values between strain 647T and Arcanobacterium canisDSM 25104T were very low, 13.4 % (reciprocal 15.9 %). The genomic DNA G+C content of strain 647T was 58.7 mol%. The presence of the major menaquinone MK-9(H4) supported the affiliation of this strain to the genus Arcanobacterium. The polar lipid profile consisted of the major components diphosphatidylglycerol, phosphatidylcholine and an unidentified phosphoglycolipid. The results of physiological and biochemical testing clearly distinguished the unknown bacterium from other species of the genus Arcanobacterium. Based on these tests, it is proposed that the unknown bacterium should be classified as a representative of a novel species of the genus Arcanobacterium named Arcanobacterium wilhelmaesp. nov. The type strain is 647T (=DSM 102162T=LMG 29418T).

Infection Dynamics and Antimicrobial Resistance Profile of Salmonella Paratyphi B d-tartrate Positive (Java) in a Persistently Infected Broiler Barn.

The infection dynamics of S. Java were examined in three consecutive rearing periods on a broiler farm in Northwestern Germany which had been persistently infected with S. Java for more than five years. The barn was investigated for Salmonella occurrence after cleaning and disinfection to verify the persistent contamination of the broiler house with S. Java before the start of the first rearing cycle. Confirmation of Salmonella absence in day-old chicks (time-point 1) as well as early establishment of infection between days 5-7 (time-point 2) were confirmed by caecal swabs prepared for qPCR and classical microbiological methods. At three time-periods (between days 11-15 (time-point 3), days 25-28 (time-point 4), and days 38-40 (time-point 5)) caecal content was examined for colony forming units (CFU) Salmonella/g. In general, there was an increase in Salmonella Java load at time-point 4 compared to time-points 3 and 5. Therefore, we observed a bell-shaped course of infection resulting in higher rates of Salmonella CFU/g prior to prethinning than at final slaughter. The antimicrobial susceptibility testing revealed resistance to tetracycline, fluorquinolones, trimethoprim, and cefoxitin.

Antimicrobial susceptibility and genetic characterization of Escherichia coli recovered from frozen game meat.

The increasing number of antimicrobial resistant Enterobacteriaceae both in veterinary and human medicine, the dissemination of these bacteria in several environments and their possible repercussions on human health is causing concern. Game meat is usually seen as free of antimicrobial resistant bacteria. The objective of this study was to evaluate the current antimicrobial susceptibility status in generic Escherichia coli isolated from packed frozen game meat from a game handling establishment in Germany. A total of 229 E. coli isolates were obtained from cuts of red deer, roe deer and wild boar. The susceptibility to 12 antimicrobial agents was evaluated by a broth microdilution method according to ISO 20776-1:2006. Minimal Inhibitory Concentration (MIC) values were compared to breakpoints and cut-off values published by the EUCAST. Isolates showing MICs above the reference values were further studied for associated resistance determinants and phylogrouping by PCR. Overall, 16 E. coli isolates (7.0%) showed resistance (microbiological or clinical) to at least one antimicrobial agent tested. Clinical resistance was recorded to ampicillin (5/229) and chloramphenicol (4/229), whereas the MIC of 9 isolates exceeded the epidemiological cut-off value for doxycycline. One of the ampicillin-resistant isolates showed resistance to the ß-lactam antibiotic derivatives tested, cephalosporines and aztreonam. Three of 9 non-wild-type isolates for doxycycline were positive for tet (B) genes. The ß-lactam-resistant isolate was found to harbour blaCTX-M-1 gene. These data show a low prevalence of resistant E. coli in packed game meat compared to studies on conventional meat. Although isolates obtained in this study may also be originating from the processing environment and not necessarily from animals, based on our results, it is important to monitor the development of antimicrobial resistance in game animals and products in order to identify future threats for the consumers.

Detection of a Novel qnrB19-Carrying Plasmid Variant Mediating Decreased Fluoroquinolone Susceptibility in Salmonella enterica Serovar Hadar.

Thirty Salmonella enterica subsp. enterica serovar Hadar isolates of avian origin collected between 2007 and 2010 from chicken carcasses in five geographically spread abattoirs in Germany were investigated for plasmid-mediated quinolone resistance determinants. Four isolates were identified by PCR analysis and hybridization experiments to carry qnrB genes. The isolates were indistinguishable by their XbaI macrorestriction patterns and did not exhibit a mutation in the quinolone resistance-determining regions of the DNA gyrase and topoisomerase IV genes. The qnrB genes were found to be located on small plasmids of ∼2.6 kb, which mediated decreased susceptibility only to quinolones. The plasmids were assigned to the same type, pHAD28, and transformation studies into an Escherichia coli recipient strain confirmed their transferability. Sequence analysis of the complete plasmid pHAD28 revealed the presence of a qnrB19 gene. The gene was found on a novel variant of qnrB19-harboring plasmids with high similarity to plasmids pPAB19-3 from E. coli and pPAB19-4 from Salmonella sp. M9397. A presumptive recombination side was detected, suggesting that interplasmid recombination events might have played a role in the development of this plasmid variant.

Scientific Opinion on the update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA.

EFSA is requested to assess the safety of a broad range of biological agents in the context of notification for market authorisation as sources of food and feed additives, food enzymes and plant protection products. The qualified presumption of safety (QPS) assessment was developed to provide a harmonised generic pre-assessment to support safety risk assessments performed by EFSA's scientific Panels. The safety of unambiguously defined biological agents (at the highest taxonomic unit appropriate for the purpose for which an application is intended), and the completeness of the body of knowledge are assessed. Identified safety concerns for a taxonomic unit are, where possible and reasonable in number, reflected as 'qualifications' in connection with a recommendation for a QPS status. The list of QPS recommended biological agents was reviewed and updated in the current opinion and therefore becomes the valid list. The 2016 update reviews previously assessed microorganisms including bacteria, yeasts and viruses used for plant protection purposes following an Extensive Literature Search strategy. The taxonomic units related to the new notifications received since the 2013 QPS opinion, were periodically evaluated for a QPS status and the results published as Statements of the BIOHAZ Panel. Carnobacterium divergens, Lactobacillus diolivorans, Microbacterium imperiale, Pasteuria nishizawae, Pediococcus parvulus, Bacillus flexus, Bacillus smithii, Xanthomonas campestris and Candida cylindracea were recommended for the QPS list. All taxonomic units previously recommended for the 2013 QPS list had their status reconfirmed as well their qualifications with the exception of Pasteuria nishizawae for which the qualification was removed. The exclusion of filamentous fungi and enterococci from the QPS evaluations was reconsidered but monitoring will be maintained and the status will be re-evaluated in the next QPS Opinion update. Evaluation of bacteriophages should remain as a case-by-case procedure and should not be considered for QPS status.

Hepatitis E Virus in Wild Boars and Spillover Infection in Red and Roe Deer, Germany, 2013-2015.

To determine animal hepatitis E virus (HEV) reservoirs, we analyzed serologic and molecular markers of HEV infection among wild animals in Germany. We detected HEV genotype 3 strains in inner organs and muscle tissues of a high percentage of wild boars and a lower percentage of deer, indicating a risk for foodborne infection of humans.

Microbiology of cooked and dried edible Mediterranean field crickets (Gryllus bimaculatus) and superworms (Zophobas atratus) submitted to four different heating treatments.

To increase the shelf life of edible insects, modern techniques (e.g. freeze-drying) add to the traditional methods (degutting, boiling, sun-drying or roasting). However, microorganisms become inactivated rather than being killed, and when rehydrated, many return to vegetative stadia. Crickets (Gryllus bimaculatus) and superworms (Zophobas atratus) were submitted to four different drying techniques (T1 = 10' cooking, 24 h drying at 60℃; T2 = 10' cooking, 24 h drying at 80℃; T3 = 30' cooking, 12 h drying at 80℃, and 12 h drying at 100℃; T4 = boiling T3-treated insects after five days) and analysed for total bacteria counts, Enterobacteriaceae, staphylococci, bacilli, yeasts and moulds counts, E. coli, salmonellae, and Listeria monocytogenes (the latter three being negative throughout). The microbial counts varied strongly displaying species- and treatment-specific patterns. T3 was the most effective of the drying treatments tested to decrease all counts but bacilli, for which T2 was more efficient. Still, total bacteria counts remained high (G. bimaculatus > Z. atratus). Other opportunistically pathogenic microorganisms (Bacillus thuringiensis, B. licheniformis, B. pumilis, Pseudomonas aeruginosa, and Cryptococcus neoformans) were also encountered. The tyndallisation-like T4 reduced all counts to below detection limit, but nutrients leakage should be considered regarding food quality. In conclusion, species-specific drying procedures should be devised to ensure food safety.

Towards a Standardized Method for Broth Microdilution Susceptibility Testing of Haemophilus parasuis.

Currently, there is no agreed method available for broth microdilution susceptibility testing of Haemophilus parasuis, one of the most important bacterial pathogens in pig production. Therefore, the aim of this study was to develop a method that could be easily performed by diagnostic laboratories and that appears suitable for a harmonized susceptibility testing. Growth determinations using one type strain and three field isolates revealed no visible growth of H. parasuis in media which have proven to be suitable for susceptibility testing of fastidious organisms. Therefore, a new medium, cation-adjusted Mueller-Hinton broth (CAMHB) plus NADH and sterile filtered heat-inactivated chicken serum, was developed. The reproducibility of MICs obtained in this medium was evaluated and statistically analyzed, considering a model with two different variables (precondition of five identical MICs and MIC mode accepting a deviation of ±1 dilution step, respectively). No significant differences for both variables were seen between two time points investigated and between results obtained with the recently proposed test medium broth (TMB). Nearly all MICs of quality control strains were in the acceptable range. Subsequently, 47 H. parasuis isolates representing 13 serovars were tested with the newly developed medium and TMB. Statistical analysis of all isolates and 15 antimicrobial agents and antimicrobial combinations showed no significant difference between MICs obtained in supplemented CAMHB and TMB. Because of a simplified implementation in routine diagnostic and a lower chance of interference between medium components and antimicrobial agents, supplemented CAMHB is recommended with an incubation time of 24 h.

Microbiology of processed edible insect products - Results of a preliminary survey.

Little is known of the microbiology of processed insect products. The present survey analysed a total of n=38 samples of deep-fried and spiced (Acheta domesticus, Locusta migratoria, and Omphisa fuscidentalis), cooked in soy sauce ("tsukudani"; Oxya yezoensis, Vespula flaviceps, and Bombyx mori), dried (A. domesticus, L. migatoria, Alphitobius diaperinus, Tenebrio molitor, B. mori, Hermetia illucens, and Musca domestica), powdered (H. illucens, T. molitor) and other (incl. deep-frozen B. mori and honeybee pollen) insect products microbiologically (total bacterial count [TBC], Enterobacteriaceae, staphylococci, bacilli, and yeasts and moulds counts, salmonellae, Listeria monocytogenes, and Escherichia coli). Although each product type revealed a microbiological profile of its own, dried and powdered insects ("class I") displayed markedly higher counts than the deep-fried and cooked ones ("class II"). Thresholds between class I and II products were estimated at 4.0 (TBC), 1.0 (Enterobacteriaceae, yeasts and moulds), 2.5 (staphylococci), and 3.0lgcfu/g (bacilli). All samples were negative for salmonellae, L. monocytogenes, E. coli and Stapyhlococcus aureus, but dried and powdered insects, as well as pollen, contained B. cereus, coliforms, Serratia liquefaciens, Listeria ivanovii, Mucor spp., Aspergillus spp., Penicillium spp., and Cryptococcus neoformans. Comparing the results with the hygiene criteria for edible insects proposed by Belgium and the Netherlands, class I products failed to comply with many bacterial count limits despite the absence of classical food pathogens. Therefore, class I products should always be consumed after another heating step as indicated by the manufacturer, until drying techniques are able to ensure lower bacterial counts.

Variety of Antimicrobial Resistances and Virulence Factors in Staphylococcus aureus Isolates from Meat Products Legally and Illegally Introduced to Germany.

Food products of animal origin can serve as a vehicle for Staphylococcus (S.) aureus, a facultative pathogen involved in a variety of diseases. As a result, international trade and illegal transportation of foodstuffs can facilitate the distribution of S. aureus over long distances. In this study, we investigated S. aureus isolates recovered from meat products confiscated from passengers returning from non-EU countries at two German airports and from samples of legally imported meats from non-EU countries. The aim was to characterize isolates in regard to their genetic relatedness as well as their antimicrobial resistance profiles and major virulence factors in order to assess potential risks associated with these products. The isolates were characterized by spa typing, MLST, macrorestriction analysis, microarray analysis and antimicrobial susceptibility testing. MRSA isolates were further characterized by dru typing. The characteristics of the majority of the isolates indicated a human origin, rather than an association with livestock. The results further revealed a considerable heterogeneity among the MRSA isolates, despite their common origin. Overall, a plenitude of major virulence factors and antimicrobial resistances was detected among the isolates, highlighting the potential risks associated with contaminated meat products and the transportation of such products among different countries.