Cause and Effect Analysis between Influencing Factors Related to Environmental Conditions, Hunting and Handling Practices and the Initial Microbial Load of Game Carcasses.

Environmental, hunting and handling factors affect the microbial load of hunted game and the resulting meat products. The aim of this study was to systematically investigate the influence of several factors on the initial microbial load (IML) of game carcasses during the early hunting chain. Eviscerated roe deer body cavities (n = 24) were investigated in terms of total viable count and the levels of Pseudomonas spp., Lactobacillus spp., Enterobacteriaceae and Escherichia coli (E. coli). Furthermore, a risk analysis based on the obtained original IML data, literature search and a Failure Mode and Effects Analysis (FMEA) was performed. The IML could be explained in a regression model by factors including the higher body weight (BW), damaged gastrointestinal tract by the shot, ambient temperature or rain. The levels of Lactobacillus spp. (p = 0.0472), Enterobacteriaceae (p = 0.0070) and E. coli (p = 0.0015) were lower on the belly flap surface when gloves were used during evisceration. The literature search revealed that studies examining influencing factors (IF) on the IML of game carcasses found contradictory effects of the comparable IF on IML. Potential handling failures may lead to a higher IML of game carcasses during the early hunting chain ranked by FMEA. Several handling practices for game carcasses are recommended, such as ensuring efficient cooling of heavier BW carcasses to limit bacterial growth or eviscerating heavier carcasses before lighter ones.

Impact of On-Farm Interventions against CTX-Resistant Escherichia coli on the Contamination of Carcasses before and during an Experimental Slaughter.

Cefotaxime (CTX)-resistant Enterobacteriaceae are still an ongoing challenge in human and veterinary health. High prevalence of these resistant bacteria is detected in broiler chickens and the prevention of their dissemination along the production pyramid is of major concern. The impact of certain on-farm interventions on the external bacterial contamination of broiler chickens, as well as their influence on single processing steps and (cross-) contamination, have not yet been evaluated. Therefore, we investigated breast skin swab samples of broiler chickens before and during slaughter at an experimental slaughter facility. Broiler chickens were previously challenged with CTX-resistant Escherichia coli strains in a seeder-bird model and subjected to none (control group (CG)) or four different on-farm interventions: drinking water supplementation based on organic acids (DW), slow growing breed Rowan x Ranger (RR), reduced stocking density (25 kg/sqm) and competitive exclusion with Enterobacteriales strain IHIT36098(CE). Chickens of RR, 25 kg/sqm, and CE showed significant reductions of the external contamination compared to CG. The evaluation of a visual scoring system indicated that wet and dirty broiler chickens are more likely a vehicle for the dissemination of CTX-resistant and total Enterobacteriaceae into the slaughterhouses and contribute to higher rates of (cross-) contamination during processing.

Management Strategies for Prevention of Campylobacter Infections Through the Poultry Food Chain: A European Perspective.

Numerous studies point out that at present, a complete elimination of Campylobacter species in the poultry food chain is not feasible. Thus, the current aim should be to establish control measures and intervention strategies to minimize the occurrence of Campylobacter spp. in livestock (esp. poultry flocks) and to reduce the quantitative Campylobacter burden along the food chain in animals and subsequently in foods. The most effective measures to mitigate Campylobacter focus on the primary production stage. Nevertheless, measures applied during slaughter and processing complement the general meat hygiene approaches by reducing fecal contamination during slaughtering and processing and as a consequence help to reduce Campylobacter in poultry meat. Such intervention measures at slaughter and processing level would include general hygienic improvements, technological innovations and/or decontamination measures that are applied at single slaughter or processing steps. In particular, approaches that do not focus on a single intervention measure would need to be based on a thorough process of evaluation, and potential combinatory effects have to be modeled and tested. Finally, the education of all stakeholders (including retailers, food handlers and consumers) is required and will help to increase awareness for the presence of foodborne pathogens in raw meat and meat products and can thus aid in the development of the required good kitchen hygiene.

Draft Genome Sequences of Pseudomonas sp. Isolates Recovered from Ghanaian Fish Food Samples in 2018.

The genus Pseudomonas represents a broad diversity of opportunistic and pathogenic species that are able to colonize a wide range of ecological niches. Here, we report on draft genome sequences of 35 Pseudomonas sp. isolates that were recovered from small processed Ghanaian fishes offered at food markets in 2018.

Composition of nutrients, heavy metals, polycyclic aromatic hydrocarbons and microbiological quality in processed small indigenous fish species from Ghana: Implications for food security.

The triple burden of malnutrition is an incessant issue in low- and middle-income countries, and fish has the potential to mitigate this burden. In Ghana fish is a central part of the diet, but data on nutrients and contaminants in processed indigenous fish species, that are often eaten whole, are missing. Samples of smoked, dried or salted Engraulis encrasicolus (European anchovy), Brachydeuterus auritus (bigeye grunt), Sardinella aurita (round sardinella), Selene dorsalis (African moonfish), Sierrathrissa leonensis (West African (WA) pygmy herring) and Tilapia spp. (tilapia) were collected from five different regions in Ghana. Samples were analyzed for nutrients (crude protein, fat, fatty acids, several vitamins, minerals, and trace elements), microbiological quality (microbial loads of total colony counts, E. coli, coliforms, and Salmonella), and contaminants (PAH4 and heavy metals). Except for tilapia, the processed small fish species had the potential to significantly contribute to the nutrient intakes of vitamins, minerals, and essential fatty acids. High levels of iron, mercury and lead were detected in certain fish samples, which calls for further research and identification of anthropogenic sources along the value chains. The total cell counts in all samples were acceptable; Salmonella was not detected in any sample and E. coli only in one sample. However, high numbers of coliform bacteria were found. PAH4 in smoked samples reached high concentrations up to 1,300 µg/kg, but in contrast salted tilapia samples had a range of PAH4 concentration of 1 µg/kg to 24 µg/kg. This endpoint oriented study provides data for the nutritional value of small processed fish as food in Ghana and also provides information about potential food safety hazards. Future research is needed to determine potential sources of contamination along the value chains in different regions, identify critical points, and develop applicable mitigation strategies to improve the quality and safety of processed small fish in Ghana.

Antimicrobial activity of organic acids against Campylobacter spp. and development of combinations-A synergistic effect?

Contaminated poultry meat is considered to be the main source of human infection with Campylobacter spp., a pathogen that asymptomatically colonizes broiler chickens during fattening and contaminates carcasses during slaughter. To prevent or reduce the colonization of broiler flocks with Campylobacter spp., applying different organic acids, especially in combinations, via feed or drinking water seems to be a promising approach. However, only very few combinations of organic acids have been tested for their antibacterial efficacy against Campylobacter spp. Therefore, the in vitro susceptibility of 30 Campylobacter spp. isolates (20 C. jejuni and ten C. coli) to ten organic acids and ten combinations was determined. The testing of minimum inhibitory concentration (MIC) values was performed at pH 6.0 and 7.3 by using the broth microdilution method and included the following organic acids: Caprylic acid, sorbic acid, caproic acid, benzoic acid, ascorbic acid, propionic acid, acetic acid, formic acid, fumaric acid and tartaric acid and combinations thereof. The lowest MIC values were seen for caprylic acid (MIC range at pH 7.3: 0.5-2 mmol/L) and sorbic acid (MIC range at pH 7.3: 1-4 mmol/L). One to two dilution steps lower MIC values were determined at the lower pH value of 6.0. Furthermore, ten combinations consisting of three to five organic acids were developed. In addition to the tested antibacterial activity, other criteria were included such as approval as feed additives, reported synergistic effects and chemical properties. For nine of ten combinations, the MIC90 values of the organic acids decreased 1.25- to 241.5-fold compared to the MIC90 values for the individual substances. Furthermore, nine of ten combinations exhibited synergistic activities against two or more of the tested C. jejuni and C. coli isolates. A combination of caprylic acid, sorbic acid and caproic acid exhibited synergistic activities against the largest number of Campylobacter spp. isolates (six C. jejuni and four C. coli) with fractional inhibitory concentration (FIC) indices (∑FIC) ranging from 0.33 to 1.42. This study shows in vitro synergistic activities of different organic acids in combinations against the major Campylobacter species and could therefore be a promising basis for reducing Campylobacter spp. in vivo.

Evaluation of antibacterial properties of lactic acid bacteria from traditionally and industrially produced fermented sausages from Germany.

With regards to the frequently reported findings of spoilage bacteria and pathogens in various foods there is a need to explore new ways to control hazards in food production and to improve consumer safety. Fermented sausages from traditional and industrial production in Germany were screened for lactic acid bacteria with antibacterial effects towards important foodborne pathogens (Escherichia coli DSM 1103, Listeria innocua DSM 20649, Listeria monocytogenes DSM 19094, Pseudomonas aeruginosa DSM 939, Staphylococcus aureus DSM 799 and Salmonella Typhimurium DSM 19587). The obtained isolates and their cell-free supernatants were tested for their antibacterial activity by agar well diffusion assay. Isolates with an inhibitory effect were examined for the underlying antibacterial mechanism. Among the 169 collected isolates, 12.4% showed antibacterial effects only against Listeria innocua DSM 20649 and Listeria monocytogenes DSM 19094. In 6.5% of the isolates, bacteriocins were responsible for the effect. On the remaining test strains, the lactic bacteria isolates exerted no antibacterial effect. Two isolates were selected based on their antibacterial potential against Listeria spp. and the thermostability of the deriving cell free supernatants, traditional product: Pediococcus pentosaceus LMQS 331.3, industrial product: Pediococcus acidilactici LMQS 154.1, were investigated further and confirmed for the presence of bacteriocin structural genes by real-time PCR. Enriched crude bacteriocin preparations were obtained by ammonium sulfate precipitation and were found to remain stable under different pH milieus and to be active towards an extended set of Listeria spp. strains. Fermented meat products from German production are a promising source for bacteriocin-producing lactic acid bacteria. Two bacteriocin-producing isolates were identified which have the potential to contribute to product and consumer safety.

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.

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.

Comparative analysis of the susceptibility to biocides and heavy metals of extended-spectrum ß-lactamase-producing Escherichia coli isolates of human and avian origin, Germany.

A total of 174 extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates collected from humans (n=140) and healthy broiler chickens (n = 34) was included in the study. The MIC values of alkyl diaminoethyl glycin hydrochloride, benzethonium chloride, benzalkonium chloride, chlorhexidine, acriflavine, copper sulfate, silver nitrate and zinc chloride were determined by the broth microdilution method. Significant differences in MIC distributions were found between human and avian isolates and between CTX-M-, SHV- and TEM-type ESBL E. coli for chlorhexidine, silver nitrate, zinc chloride and copper sulfate by statistical analysis. Isolates with reduced susceptibility were investigated for the presence and localization of tolerance-mediating genes by PCR analysis and Southern blotting. The genes emrE, mdfA, sugE(c), cueO, copA, zntA and zitB were commonly present in isolates with elevated MICs, while the genes qacE∆1, qacF, qacH, sugE(p), cusC and pcoA, were less prevalent. In several isolates, a plasmid localization of the genes qacE∆1, qacF, qacH and sugE(p) on large plasmids >20 kb was detected.

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.

Mitigation strategies for Campylobacter spp. in broiler at pre-harvest and harvest level.

In contrast to other foodborne zoonotic agents an elimination of Campylobacter spp. from animal production, especially poultry production, seems not to be feasible. Therefore mitigation strategies focus on reduction of the Campylobacter spp. concentration in primary production and further minimalisation during processing. In primary production biosecurity measures (incl. hygiene barriers and restricted access) are the methods applied most commonly and most effectively so far. Experimental approaches and few field trials also showed that bacteriophages, electrolyzed oxidizing water, organic acids or medium chain fatty acids (applied via drinking water) are also effective in reducing Campylobacter prevalence and/or concentration However this reduction cannot be transferred in all cases to the situation in the slaughterhouse. Therefore additional measures have to be taken in account in the slaughterhouse to prevent cross-contamination. Logistic or scheduled slaughter can prevent cross-contamination but cannot further reduce Campylobacter concentration. Process parameters like elevated scalding temperature can contribute to such a reduction, but may also alter the product quality. Therefore no single pre- or harvest measure is sufficient for the reduction of Campylobacter concentration, but a combination of measures in both production levels is needed.

Effects of fibrinopurulent polyserositis in broilers on post-harvest microbiological parameters relevant to public health of broiler meat.

Fibrinopurulent polyserositis is of utmost importance in commercial broiler production worldwide. This multifactorial endemic disease is marked by severe clinical alterations post-mortem, yet its effects on food safety and processing hygiene criteria remain unclear. Current considerations presume that bacteraemia lead to meat being unfit for consumption. In the present study, we evaluated some microbiological criteria of affected broiler carcasses in comparison to unaffected control broiler carcasses. The results thereof indicated that the lesions did not result in higher bacterial counts or in an increased percentage of contaminated meat. The carry-over of associated zoonotic pathogens into the food chain seems to be not more prevalent in birds affected with non-systemic affections of polyserositis.

Extended-spectrum ß-lactamase- and AmpC-producing enterobacteria in healthy broiler chickens, Germany.

During 2010, we evaluated the presence of extended-spectrum ß-lactamase- and AmpC-producing enterobacteria in broiler chickens at slaughter. Samples (70 carcasses and 51 ceca) from 4 flocks were analyzed by direct plating and after enrichment. Extended-spectrum ß-lactamase producers were found in 88.6% and 72.5% of carcasses and ceca, respectively; AmpC producers were found in 52.9% and 56.9% of carcasses and ceca, respectively. Most isolates were identified as Escherichia coli; Enterobacter cloacae (cecum) and Proteus mirabilis (carcass) were found in 2 samples each. Molecular characterization revealed the domination of CTX-M genes; plasmidic AmpC was CIT-like. Phylogenetic grouping of E. coli showed types A (31.5%), B1 (20.2%), B2 (13.5%), and D (34.8%). These findings provide evidence that healthy broilers in Germany are a source for the dissemination of transmissible resistance mechanisms in enterobacteria brought from the rearing environment into the food chain during slaughtering.

The effects of Campylobacter numbers in caeca on the contamination of broiler carcasses with Campylobacter.

For the presence and number of Campylobacter, 18 broiler flocks were sampled over a period of 18 months. A total of 70% of the flocks were positive for Campylobacter, with higher prevalence found in summer and autumn, compared to winter and spring. Positive flocks showed contamination rates above 90%, in negative flocks this was lower, mostly below 50%. The enumeration showed a decrease in Campylobacter during processing of positive flocks. The numbers were highest in carcasses after scalding/defeathering (mean 5.9 log10 cfu/carcass) and dropped by 0.7 log10 cfu/carcass after chilling. A positive correlation was observed between the number of Campylobacter present in the caeca and the number of bacteria present on carcasses and cut products. When a negative flock was slaughtered after Campylobacter positive flocks, the number of positive samples was higher compared to the case when a negative flock had been slaughtered previously. C. jejuni was isolated from 73.6% of the poultry samples.

Microbiological quality of sushi from sushi bars and retailers.

Sushi is a traditional Japanese food, mostly consisting of rice and raw fish. Fish is considered a healthy food, but as with other animal products, consumption of raw muscle incurs potential health risks such as ingestion of pathogenic bacteria or parasites. In this study, 250 sushi samples were analyzed for their microbiological status and the prevalence of pathogenic bacteria. A comparison was made between frozen sushi from supermarkets and fresh sushi from sushi bars. Aerobic mesophilic bacteria counts differed for sushi from these two sources, with means of 2.7 log CFU/g for frozen sushi and 6.3 log CFU/g for fresh sushi. The prevalence of Escherichia coli and Staphylococcus aureus was higher in the fresh samples. Salmonella was found in four (1.6%) of the sushi samples, and Listeria monocytogenes was found in three (1.2%) of the samples. These results indicate that the microbiological quality of industrially processed sushi is higher than that of freshly prepared sushi. The quality of freshly prepared sushi strongly depends on the skills and habits of the preparation cooks, which may vary.

Incidence of Arcobacter spp. in poultry: quantitative and qualitative analysis and PCR differentiation.

Arcobacter is part of the family Campylobacteraceae. As with the genus Campylobacter, Arcobacter is found responsible for human gastrointestinal infection, and it is assumed to originate from poultry meat sources. Samples from poultry slaughtering originating from a broiler slaughterhouse and a turkey slaughterhouse were analyzed for Arcobacter. Five broiler flocks and five turkey flocks were analyzed in the course of slaughtering and processing for the prevalence of Arcobacter. The prevalence in broilers was 43.0%, while turkey samples were contaminated with 18.2% of positive samples. The numbers of Arcobacter present on turkey skin samples ranged between 1.7 and 2.4 log CFU/cm2. The prevalence changes during processing showed an increase after chilling in broilers, whereas there was a constant decrease in turkey processing. Species identification showed that all three Arcobacter spp. of relevance in human infection could be isolated, with A. butzleri being found at higher prevalence, which was followed by A. skirrowii and A. cryaerophilus.

Quantification of thermophilic Campylobacter spp. in broilers during meat processing.

Campylobacter spp. is a common cause of gastrointestinal illness. Since animal products, especially poultry meat, are an important source of human outbreaks of campylobacteriosis, tracing back to processing and initial production is of great interest. Samples were collected at a German poultry slaughterhouse for the estimation of the prevalence of Campylobacter at different processing steps. Quantification of Campylobacter in each of the samples was also performed. Out of 99 samples examined, 51 (51.5%) were positive for Campylobacter, with bacterial counts ranging from log(10) 6.5 cfu sample(-1) for carcasses to log 3.6 cfu ml(-1) for scalding water. The Campylobacter isolates (n = 51) were subtyped by pulsed-field gel electrophoresis using SmaI and KpnI restriction enzymes. Molecular typing showed a multitude of strains with different molecular patterns. Strains found in cloacal swabs before processing could also be isolated from carcasses at different processing steps.

Prevalence of Campylobacter spp. in turkey meat from a slaughterhouse and in turkey meat retail products.

One hundred and forty-four samples of chilled turkey meat from six flocks, taken directly from the slaughterhouse, and 100 samples of turkey meat retail products were examined. Over one-quarter (29.2%) of the tested samples from the slaughterhouse were Campylobacter positive, showing high variability in the flocks. The lowest percentage of Campylobacter-positive samples was found in flocks I and III (8.3%), whereas, in flock VI, 91.7% of the samples were Campylobacter positive. Turkey meat retail products showed a prevalence of 34% for Campylobacter. Heat-treated meat was negative for Campylobacter. Quantitative studies of the samples taken at the slaughterhouse revealed a mean log range of 1.9-2.5 CFU g(-1)Campylobacter spp. Results from the quantification of retail products gave a mean log value of 2.1 CFU g(-1).