Cold-tolerant microorganisms causing spoilage of vacuum-packed beef under time-temperature abuse determined by culture and qPCR.

Vacuum packaging and storage conditions at chilled temperatures are commonly used in order to prolong the shelf life of meat. Under these conditions and time-temperature abuse, cold-tolerant (facultatively) anaerobic spoilage microorganisms can continue growing. This study investigated growth of six relevant spoilage microorganisms in vacuum-packed beef (n = 12, 72 subsamples, stored at 10 °C for 28 days) using culture and qPCR methods. Correspondingly, six qPCRs were newly developed/modified (for total bacteria, lactic acid bacteria (LAB), Enterobacterales, total fungi, Kazachstania psychrophila, and cold-tolerant Clostridium spp.). Besides microbial quantification, four spoilage appearances of meat (gas production, spoilage odor, % drip loss, and meat color) were observed. Results obtained from culture and qPCR show that total bacteria, LAB, and Enterobacterales reached their stationary phase at day 7 when spoilage parameters such as gas production were statistically increased and a deviation of odor was detected. Fastidious cold-tolerant Clostridium spp. and K. psychrophila could be detected from day 7. Based on microbiological and sensory analysis results, the maximum shelf life of vacuum-packed beef stored at 10 °C is 7 days. The developed qPCR has the potential to be used as an alternative method to culturing for determination of microbial growth.

Unknown cold-tolerant Clostridium spp.: Characteristics and potential to cause meat spoilage.

Clostridium spp. are ubiquitous bacteria and often found in foods and animals. Some species are pathogenic, others food spoiling or commensals. In this study, 65 cold-tolerant Clostridium spp. strains isolated from variable samples (beef, lamb, venison, feces/skin of wild boars) were investigated. Fifty strains were lecithinase positive; six additionally produced ß-hemolysis. By applying specific qPCR, 16S rRNA gene analysis, RFLP method, and MALDI-TOF MS, they were classified into two major groups: 29 strains were identified as C. tagluense-like, while the other 36 remained unidentified. Subsequently, twenty-two vacuum-packed beef samples were spiked with a single strain from both groups and stored at 4 °C for 8 weeks. The odor of challenged samples was variable (from unchanged, sour/musty, to sulfurous), while color, meat consistency and drip loss were similar to the control group. The ability to produce gas of all tested strains was lower than of C. estertheticum. Even though both groups of cold-tolerant clostridia exhibited similar 16S rRNA genes and biochemical activities, RFLP methods and MALDI-TOF MS are sufficient to differentiate them. In terms of food safety, strains producing lecithinase and hemolysin should be further investigated for their potential to produce substances affecting human and animal health.

Rapid and selective detection of macrocyclic trichothecene producing Stachybotrys chartarum strains by loop-mediated isothermal amplification (LAMP).

Cytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay's detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing.

Differentiation of S. chartarum (Ehrenb.) S. Hughes Chemotypes A and S via FT-IR Spectroscopy.

Stachybotrys (S.) chartarum is a cellulolytic mould with the ability to produce highly cytotoxic macrocyclic trichothecenes. Two chemotypes are defined according to their ability to produce either atranones or satratoxins. S. chartarum has been well known as the causative agent of the lethal disease stachybotryotoxicosis in horses. Further investigations revealed that this disease is strictly correlated with the presence of macrocyclic trichothecenes. Furthermore, their occurrence in water-damaged buildings has been linked to adverse health effects such as the sick building syndrome. As the chemotypes cannot be characterized via phenotypic criteria, different methods such as PCR, MALDI-TOF MS, LC-MS/MS, thin-layer chromatography and cytotoxicity assays have been used so far. Fourier-transform-infrared spectroscopy (FT-IR) is commonly used for the differentiation of bacteria and yeasts, but this technique is also applicable to filamentous fungi. Hence, this study aimed at evaluating to which extent a reliable differentiation of S. chartarum chemotypes A and S is possible. Besides, another objective was to verify if the recently introduced third genotype of S. chartarum can be identified. Therefore, 28 strains including the two chemotypes and the third genotype H were cultivated on malt extract agar (MEA) and potato dextrose agar in three biological replicates. Each sample was applied to FT-IR measurements on day 7, 14 and 21 of cultivation. In this study, we achieved a distinction of the chemotypes A and S via FT-IR spectroscopy after incubation for 7 days on MEA. In terms of genotype differentiation, the PCR detecting satratoxin- and atranone-gene clusters remained the only applicable method.

Correction to: Analysis of the Fungal Flora in Environmental Dust Samples by PCR-SSCP Method.

The original version of this article contained a mistake in the co-author's conflict of interest statement. Erika von Mutius wishes to add the following disclosures: "E. von Mutius is listed as an inventor on the following patents: publication number EP 1411977, composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases, granted on 18 April 2007; publication number EP1637147, stable dust extract for allergy protection, granted on 10 December 2008; publication number EP 1964570, pharmaceutical compound to protect against allergies and inflammatory diseases, granted on 21 November 2012. E. von Mutius is listed as inventor and has received royalties on the following patent: publication number EP2361632, specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders, granted on 19 March 2014".

Differentiation of live and dead Mycobacterium tuberculosis complex in meat samples using PMA qPCR.

The causative agents of zoonotic bovine tuberculosis (bTB), Mycobacterium bovis and M. caprae, are members of the M. tuberculosis complex (MTC). Wildlife such as red deer infected with bTB are often without pathological findings, thus meat thereof may be classified as safe for human consumption. The culturing of MTC is time consuming and inappropriate to be applied with fresh meat and food. Therefore, a rapid method "PMA qPCR" to differentiate living and dead cells of MTC was developed in this study. By treating with 50 µM PMA™ dye, dead M. bovis BCG (≤104 cells/ml meat suspension) could be completely discriminated and was not detected by specific MTC PCR. The limit of detection of MTC without treatment with PMA™ dye was 10 cells/ml. All 50 venison samples obtained for field study purposes were negative for MTC. However, 40% were slightly PCR positive for non-TBC mycobacteria. By culturing using selective enrichment, one single colony of M. avium was isolated. This is the first report on the isolation of M. avium from venison. Considering the difficulties of diagnosing mycobacteria in various matrices, the developed PMA qPCR is applicable for the differentiation of dead and living cells of MTC in meat samples.

Unraveling the Role of Vegetables in Spreading Antimicrobial-Resistant Bacteria: A Need for Quantitative Risk Assessment.

In recent years, vegetables gain consumer attraction due to their reputation of being healthy in combination with low energy density. However, since fresh produce is often eaten raw, it may also be a source for foodborne illness. The presence of antibiotic-resistant bacteria might pose a particular risk to the consumer. Therefore, this review aims to present the current state of knowledge concerning the exposure of humans to antibiotic-resistant bacteria via food of plant origin for quantitative risk assessment purposes. The review provides a critical overview of available information on hazard identification and characterization, exposure assessment, and risk prevention with special respect to potential sources of contamination and infection chains. Several comprehensive studies are accessible regarding major antimicrobial-resistant foodborne pathogens (e.g., Salmonella spp., Listeria spp., Bacillus cereus, Campylobacter spp., Escherichia coli) and other bacteria (e.g., further Enterobacteriaceae, Pseudomonas spp., Gram-positive cocci). These studies revealed vegetables to be a potential-although rare-vector for extended-spectrum beta-lactamase-producing Enterobacteriaceae, mcr1-positive E. coli, colistin- and carbapenem-resistant Pseudomonas aeruginosa, linezolid-resistant enterococci and staphylococci, and vancomycin-resistant enterococci. Even if this provides first clues for assessing the risk related to vegetable-borne antimicrobial-resistant bacteria, the literature research reveals important knowledge gaps affecting almost every part of risk assessment and management. Especially, the need for (comparable) quantitative data as well as data on possible contamination sources other than irrigation water, organic fertilizer, and soil becomes obvious. Most crucially, dose-response studies would be needed to convert a theoretical "risk" (e.g., related to antimicrobial-resistant commensals and opportunistic pathogens) into a quantitative risk estimate.

Endogenous microbial contamination of melons (Cucumis melo) from international trade: an underestimated risk for the consumer?

BACKGROUND: Fruits and vegetables have increasingly been related to foodborne outbreaks. Besides surface contamination, a possible internalization of microorganisms into edible parts of plants during growth has already been observed. To examine an actual risk for the consumer, microbial contamination of the rind and pulp of 147 muskmelons from international trade was assessed using cultural and biochemical methods, polymerase chain reaction and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. RESULTS: One hundred percent of the rind samples [3.69-8.92 log colony forming units (CFU) g-1 ] and 89.8% of the pulp samples (maximum load 3.66 log CFU g-1 ) were microbiologically contaminated. Among the 432 pulp isolates, opportunistic and potentially pathogenic bacteria were identified, mainly Staphylococcus spp. (48.9%), Clostridium spp. (42.9%) and Enterobacteriaceae (27.9%). Salmonella spp., Escherichia coli and isolates of the Bacillus cereus group were found on the rind (1.4%, 0.7% and 42.9%, respectively) and in the pulp (0.7%, 1.4% and 4.7%). Clostridium perfringens was isolated from the rind of seven melons. CONCLUSION: The present study revealed a regularly occurring internal contamination of melons. Possible health risks for consumers because of an occurrence of microorganisms in melon pulp should be considered in future food safety assessments. © 2018 Society of Chemical Industry.

Identification of fungal candidates for asthma protection in a large population-based study.

BACKGROUND: Exposure to molds has been related to asthma risk both positively and negatively, depending on the environmental setting. The pertinent results are based on generic markers or culturing methods although the majority of present fungi cannot be cultured under laboratory conditions. The aim of the present analysis was to assess environmental dust samples for asthma-protective fungal candidates with a comprehensive molecular technique covering also non-cultivable and non-viable fungi. METHODS: Mattress dust samples of 844 children from the GABRIELA study were analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) of the fungus-specific internal transcribed spacer (ITS) region. Known asthma candidate species were tested for their associations with asthma, and further gel positions were sought to explain the above. As a second, data-driven, analysis, we tested the association of each individual gel position with asthma. RESULTS: In the hypothesis-driven approach, Penicillium chrysogenum emerged with an odds ratio of 0.80 (95% confidence interval 0.66-0.96; p = 0.020). The effect size was changed by 39% toward the null when adjusting for the two bands 683 (DNA of Metschnikowia sp., Aureobasidium spp.) and 978 (DNA of Epicoccum spp., Galactomyces spp., uncultured Penicillium). The data-driven approach yielded an additional band (containing DNA of Pseudotaeniolina globosa) with reduced risk of asthma (OR = 0.80 [0.66-0.96], p = 0.012). CONCLUSIONS: A large population-based study revealed several fungal taxa with inverse associations with childhood asthma. Molds produce a variety of bioactive compounds with detrimental but also beneficial immunoregulatory capacities, which renders them promising targets for further asthma research.

Hepatitis E Virus in Wild Boar in Northwest Poland: Sensitivity of Methods of Detection.

In northwest Poland, 163 blood and 53 fecal samples of wild boars were collected in winter 2012/13 and 2013/14. All blood samples were tested for the presence of hepatitis E virus (HEV) ribonucleic acid (RNA) by two reverse transcription-polymerase chain reaction (RT-PCR) based methods and by anti-HEV IgG enzyme-linked immunosorbent assay (ELISA). About 17.2% of blood samples were seropositive. One-step nested RT-PCR turned out to be too insensitive (11.6% were positive). Therefore a two-step nested RT-PCR was applied where 25.8% of the blood samples were tested positive for HEV RNA. About 50.0% of blood samples positive in ELISA were also positive in two-step nested RT-PCR. The prevalence of HEV RNA in feces was 9.4%. Based on the results of blood (ELISA, PCR) and fecal (PCR) tests, the overall prevalence of HEV in wild boars in northwest Poland was 36.8%. There was no correlation between the ELISA results and the presence of HEV RNA in plasma or in feces. According to the sequencing results of 348 bp PCR products of HEV, there were four different subtypes identified. Reports on the prevalence of HEV in wild boar populations are varying due to different sensitivities of the detection methods. However, this study reveals based on a highly sensitive method that HEV is widely spread in wild boar populations in the northwestern region of Poland and posing a potential risk to the consumer of game meat.

Identification of Stachybotrys spp. by MALDI-TOF mass spectrometry.

Stachybotrys (S.) spp. are omnipresent cellulolytic molds. Some species are highly toxic owing to their ability to synthesize various secondary metabolites such as macrocyclic trichothecenes or hemolysins. The reliable identification of Stachybotrys at species level is currently limited to genome-based identification. This study aimed to establish a fast and reliable MALDI-TOF MS identification method by optimizing the pre-analytical steps for protein extraction for subsequent generation of high-quality fingerprint mass spectra. Eight reference strains of the American Type Culture Collection and the Technical University of Denmark were cultivated in triplicate (biological repetitions) for 2 days in malt extract broth. The mycelia (1.5 ml) were first washed with 75 % ethanol and an additional washing step with dimethyl sulfoxide (10 %) was added to remove unspecific low weight masses. Furthermore, mycelia were broken with roughened glass beads in formic acid (70 %) and acetonitrile. The method was successfully applied to a total of 45 isolates of Stachybotrys originating from three different habitats (indoor, feed, and food samples; n = 15 each): Twenty-seven isolates of S. chartarum and 18 isolates of S. chlorohalonata could be identified by MALDI-TOF MS. The data obtained exactly matched those obtained by genome-based identification. The mean score values for S. chartarum ranged from 2.509 to 2.739 and from 2.148 to 2.622 for S. chlorohalonata with a very good reproducibility: the relative standard deviations were between 0.3 % and 6.8 %. Thus, MALDI-TOF MS proved to be a fast and reliable alternative to identification of Stachybotrys spp. by nucleotide amplification and sequencing.

Changes in the Microbiota from Fresh to Spoiled Meat, Determined by Culture and 16S rRNA Analysis.

Growth of meat microbiota usually results in spoilage of meat that can be perceived by consumers due to sensory changes. However, a high bacterial load does not necessarily result in sensory deviation of meat; nevertheless, this meat is considered unfit for human consumption. Therefore, the aims of this study were to investigate changes in the microbiota from fresh to spoiled meat and whether the proportions of certain bacteria can probably be used to indicate the hygiene status of meat. For this purpose, 12 fresh pork samples were divided into two groups, and simultaneously aerobically stored at 4°C and 22°C. At each time-temperature point (fresh meat, days 6, 13, and 20 at 4°C, and days 1, 2, 3, and 6 at 22°C), 12 meat subsamples were investigated. Sequences obtained from next-generation sequencing (NGS) were further analyzed down to species level. Plate counting of six bacterial groups and NGS results showed that Pseudomonas spp. and lactic acid bacteria (LAB) were found in a high proportion in all stored meat samples and can therefore be considered as important "spoilage indicator bacteria". On the contrary, sequences belonging to Staphylococcus epidermidis were found in a relatively high proportion in almost all fresh meat samples but were less common in stored meat. In this context, they can be considered as "hygiene indicator bacteria" of meat. Based on these findings, the proportion of the "hygiene indicator bacteria" in relation to the "spoilage indicator bacteria" was calculated to determine a "hygiene index" of meat. This index has a moderate to strong correlation to bacterial loads obtained from culture (p < 0.05), specifically to Pseudomonas spp., LAB and total viable counts (TVCs). Knowledge of the proportions of hygiene and spoilage indicator bacteria obtained by NGS could help to determine the hygiene status even of (heat-) processed composite meat products for the first time, thus enhancing food quality assurance and consumer protection.

Viable Campylobacter jejuni on Eggshells and Its Potential to Cross-contaminate Egg White and Yolk When Using a Manual Separation Technique, Determined by Culture and Propidium Monoazide (PMA) qPCR.

Manual separation of egg yolk from egg white using the eggshell is common practice in private households. For this, the egg is cracked and both components are separated by passing the egg yolk back and forth between the two halves of the eggshell, allowing the egg white to drip down while the egg yolk remains in the shell. During this process, the egg content naturally gets in contact with the outside of the eggshell, which might lead to a cross-contamination with its microorganisms, thus was correspondingly assessed in this study. Campylobacter jejuni is one of the most important zoonotic pathogens that can be found on eggshells. Therefore, this bacterium was used to artificially contaminate the eggshells (n = 22) with concentrations of 3.1 ± 0.6 log10 cfu/g. After separating the egg yolk from the egg white, cross-contamination was determined using culture and qPCR. Altogether, cross-contaminations with C. jejuni were found in 15 egg white (68%) and in three egg yolk (14%) samples. Afterward, 90 eggs from 30 egg packs from different producers in and around Munich (Germany) were obtained for field study purposes. To address the problem of culturing due to a possible viable but nonculturable (VBNC) status of C. jejuni, a method to differentiate viable and dead C. jejuni on eggshell using 10 µM propidium monoazide (PMA) and qPCR was developed. As a result, seven egg packs (23%) were positive for C. jejuni. Of these, only one (3%) was contaminated with viable cells, but still in a concentration of 3.3 log10 cells/g shell. According to these results and considering that eggshells might also be naturally contaminated with other pathogens, the authors recommend avoiding the manual separation technique of egg white and yolk by the eggshell. Especially if raw egg white or yolk is used for preparation of not sufficiently heated foods, where contaminating pathogens are not inactivated during processing, this technique might be a safety hazard for the consumer.

Analysis of the fungal flora in environmental dust samples by PCR-SSCP method.

Conventional microbiological techniques yield only limited information on the composition of fungal communities in dust. The aim of this study was to establish and optimize PCR-single strand conformation polymorphism (PCR-SSCP) analysis for investigation of fungal diversity in rural dust samples. Three different DNA extraction protocols were tested on 38 fungal cultures. A total of six known universal fungal primer pairs were tested targeting the 18S rRNA gene, the 28S rRNA gene and the ITS region, respectively. Objective evaluation was performed with respect to the following parameters: efficiency to amplify all 38 strains; separation of seven species from different phylogenetic groups on the SSCP gel; additional bands in PCR-SSCP analysis; possibility to classify the amplified gene fragments to species level. Primer ITS1/ITS4 and PowerSoil™ DNA isolation showed the best performance in most cases and were chosen for further analysis. The detection limit of the developed system was 200 CFU/g dust. Moreover, the reproducibility of the system could be demonstrated, leading to average profile similarities of 94.94% [SD = 2.51] within gels, 93.03% [SD = 4.69] between different days and 87.66% [SD = 6.62] between different gels when testing shed and mattress dust samples. Sequencing allowed identification on species level, in detail: Alternaria alternata, Cladosporium sphaerospermum, Cladosporium cladosporioides as well as the yeasts Candida cabralensis and Candida catenulata. This demonstrates the adaptability of the method. In this study, a standardized system for fungal community analysis was developed that provides reproducible results applicable for epidemiological purposes.

Lactobacillus (Limosilactobacillus) reuteri: a probiotic candidate to reduce neonatal diarrhea in calves.

Background: Diarrhea in newborn calves is considered life-threatening and results in large economic losses in dairy farms. Lactobacilli generally play an important role in intestinal health, and Lactobacillus (Limosilactobacillus; L.) reuteri is the dominant Lactobacillus species in the feces of healthy calves during the first week of life. In calves with diarrhea on day 2 postpartum, lactobacilli are significantly reduced even up to 24 h before the onset of clinical signs. Since the probability of occurrence of diarrheal disease decreases as the L. reuteri count in the feces increases, oral administration of this species might have a protective effect against diarrhea. Objective: These studies were designed to demonstrate whether oral administration of preselected L. reuteri isolates can reduce the incidence of diarrhea in newborn calves on dairy farms. Microorganisms: 46 L. reuteri isolates from 2-day-old healthy calves were available from a previous study. Animals: 170 newborn calves of Simmental breed of 10 dairy farms in Bavaria (Germany), were included in the study; of 166 animals the data could be evaluated. Methods: Microbiological (antibiotic sensitivity test, acid and bile salt stability test, antimicrobial activity of the supernatants), molecular biological (PCR, RAPD-PCR) and toxicological methods (MTT test) were used to select and to characterize suitable L. reuteri isolates. The administration of a suspension of two selected L. reuteri isolates (6-8 × 108 colony forming units per day) to calves was performed from day 2 to day 5 after birth in a double-blinded placebo-controlled study. Clinical monitoring of the calves continued until the 14th day of life. Results: Out of 46 L. reuteri isolates, only 2 met the set criteria and were used in the feeding trial. In the placebo group, 44 of 83 calves developed diarrhea within the first 2 weeks of life, whereas in the L. reuteri group this was only the case in 31 of 83 animals (p < 0.05). Conclusion: L. reuteri appears to be of particular importance for the intestinal health of newborn calves. The diarrhea protective effect could be even more pronounced if an improved administration regimen is developed in terms of start, frequency, and duration.

Treatment of Ready-To-Eat Cooked Meat Products with Cold Atmospheric Plasma to Inactivate Listeria and Escherichia coli.

Ready-to-eat meat products have been identified as a potential vehicle for Listeria monocytogenes. Postprocessing contamination (i.e., handling during portioning and packaging) can occur, and subsequent cold storage together with a demand for products with long shelf life can create a hazardous scenario. Good hygienic practice is augmented by intervention measures in controlling post-processing contamination. Among these interventions, the application of 'cold atmospheric plasma' (CAP) has gained interest. The reactive plasma species exert some antibacterial effect, but can also alter the food matrix. We studied the effect of CAP generated from air in a surface barrier discharge system (power densities 0.48 and 0.67 W/cm2) with an electrode-sample distance of 15 mm on sliced, cured, cooked ham and sausage (two brands each), veal pie, and calf liver pâté. Colour of samples was tested immediately before and after CAP exposure. CAP exposure for 5 min effectuated only minor colour changes (ΔE max. 2.7), due to a decrease in redness (a*), and in some cases, an increase in b*. A second set of samples was contaminated with Listeria (L.) monocytogenes, L. innocua and E. coli and then exposed to CAP for 5 min. In cooked cured meats, CAP was more effective in inactivating E. coli (1 to 3 log cycles) than Listeria (from 0.2 to max. 1.5 log cycles). In (non-cured) veal pie and calf liver pâté that had been stored 24 h after CAP exposure, numbers of E. coli were not significantly reduced. Levels of Listeria were significantly reduced in veal pie that had been stored for 24 h (at a level of ca. 0.5 log cycles), but not in calf liver pâté. Antibacterial activity differed between but also within sample types, which requires further studies.

Heavy metals in liquid pig manure in light of bacterial antimicrobial resistance.

Heavy metals are regularly found in liquid pig manure, and might interact with bacterial antimicrobial resistance. Concentrations of heavy metals were determined by atomic spectroscopic methods in 305 pig manure samples and were connected to the phenotypic resistance of Escherichia coli (n=613) against 29 antimicrobial drugs. Concentrations of heavy metals (/kg dry matter) were 0.08-5.30 mg cadmium, 1.1-32.0 mg chrome, 22.4-3387.6 mg copper, <2.0-26.7 mg lead, <0.01-0.11 mg mercury, 3.1-97.3 mg nickel and 93.0-8239.0 mg zinc. Associated with the detection of copper and zinc, resistance rates against ß-lactams were significantly elevated. By contrast, the presence of mercury was significantly associated with low antimicrobial resistance rates of Escherichia coli against ß-lactams, aminoglycosides and other antibiotics. Effects of subinhibitory concentrations of mercury on bacterial resistance against penicillins, cephalosporins, aminoglycosides and doxycycline were also demonstrated in a laboratory trial. Antimicrobial resistance in the porcine microflora might be increased by copper and zinc. By contrast, the occurrence of mercury in the environment might, due to co-toxicity, act counter-selective against antimicrobial resistant strains.

Symbiotic Husbandry of Chickens and Pigs Does Not Increase Pathogen Transmission Risk.

A symbiotic or mixed animal husbandry (e.g., pigs and chickens) is considered to have a positive effect for animal welfare and sustainable agriculture. On the other hand, a risk of infection and transmission of microorganisms, especially of zoonotic pathogens, between animal species may potentially occur and thus might increase the risk of foodborne illnesses for consumers. To prove these assumptions, two groups of animals and their environmental (soil) samples were investigated in this study. Animals were kept in a free-range system. In the first group, pigs and chickens were reared together (pasture 1), while the other group contained only pigs (pasture 2). During a one-year study, fecal swab samples of 240 pigs and 120 chickens, as well as 120 ground samples, were investigated for the presence of Campylobacter spp., Salmonella spp. and E. coli. Altogether, 438 E. coli and 201 Campylobacter spp. strains were isolated and identified by MALDI-TOF MS. Salmonella spp. was not isolated from any of the sample types. The prevalences of Campylobacter coli and C. jejuni in pigs were 26.7% and 3.3% in pasture 1 and 30.0% and 6.7% in pasture 2, while the prevalences of C. coli and C. jejuni in chickens from pasture 1 were 9.2% and 78.3%, respectively. No correlation between the rearing type (mixed vs. pigs alone) and the prevalence of Campylobacter spp. was observed. All swab samples were positive for E. coli, while the average prevalences in soil samples were 78.3% and 51.7% in pasture 1 and 2, respectively. Results of similarity analysis of the MALDI-TOF MS spectra (for C. coli, C. jejuni and E. coli) and FT-IR spectra (for E. coli) of the same bacterial species showed no recognizable correlations, no matter if strains were isolated from chickens, pig or soil samples or isolated at different sampling periods. The results of the study indicate that the symbiotic husbandry of pigs and chickens neither results in an increased risk of a transmission of Campylobacter spp. or E. coli, nor in a risk of bacterial alteration, as shown by MALDI-TOF MS and FT-IR spectra. In conclusion, the benefits of keeping pigs and chickens together are not diminished by the possible transmission of pathogens.

A simple method for the isolation of cold-tolerant Clostridium spp. from meat samples.

Pure isolates of Clostridium spp. are required when further investigations are needed, such as the characterisation of the colonies, sporulation and germination, biochemical analysis, growth rate and growth conditions, toxin production, spoilage potential and genomic profile. However, the isolation of cold-tolerant Clostridium spp. from suspicious meat samples is challenging due to their slow growth rates and overgrowth with less fastidious meat microbiota. Therefore, this study aimed to establish a practical method for the isolation of psychrophilic and psychrotolerant Clostridium spp. Primarily, meat drip samples (n = 3), enriched in Peptone Yeast Glucose Starch (PYGS) broth, were heated with different temperatures and durations, before they were subcultured on Columbia blood agar (CBA). The treatment procedure of heating samples at 80 °C for 5 min was evaluated as effective and was further validated using pure cultures of lactic acid bacteria (n = 5), bacteria belonging to the family of Enterobacteriaceae (n = 5), and cold-tolerant Clostridium spp. (n = 34). Almost all other meat microbiota was inactivated by heating at 80 °C for 5 min, while 28 strains of cold-tolerant Clostridium spp. survived. Finally, the treatment procedure was applied with drip of meat samples (n = 41) previously tested positive for 49 cold-tolerant Clostridium spp. using specific multiplex qPCR. All meat drip samples were enriched in PYGS broth by incubating them at 4 °C for 3 weeks, to ensure growth of Clostridium spp. before the enrichments were proceeded to heat treatment and to culturing on CBA. A total of 35 (71 %) from 49 Clostridium spp. strains were isolated using this culturing procedure. The accuracy of the recovery rates obtained from two replicates was 68 %. The method of detection and isolation applied in this study is easy and resulted in a high isolation rate of cold-tolerant Clostridium spp.

Abundance of selected bacterial groups in healthy calves and calves developing diarrhea during the first week of life: Are there differences before the manifestation of clinical symptoms?

Background: Diarrhea is still the most common and economically most significant disease of newborn calves. Objective: Analysis of the development of selected bacterial groups in the feces of neonatal calves and its significance regarding diarrhea. Animals: A total of 150 newborn Simmental calves reared in 13 Bavarian farms were included in the study. Methods: Fecal samples of calves taken at 0/6/12/24/48/72/168 hours (h) since birth were analyzed qualitatively and quantitatively for aerobic and anaerobic bacteria, such as Enterobacteriaceae, E. coli, enterococci, and lactobacilli, using cultural, biochemical, and molecular-biological methods. Concurrently, the health status of the animals was recorded. The bacterial levels of healthy and diarrheic animals were compared using statistical methods. In addition, feces samples from calves that developed diarrhea were examined by ELISA for the presence of rotaviruses, coronaviruses, E. coli F5, and Cryptosporidium (Cr.) parvum. Results: Fifty-seven out of 150 calves (37.3 %) that were examined developed diarrhea within the first week of life. In the feces of calves with diarrhea on day 1 of life, the levels of aerobes, Enterobacteriaceae, and E. coli were significantly increased (p < 0.05), while no significant differences in enterococci and lactobacilli were found. In animals with the onset of diarrhea on day 2 after birth, the load of lactobacilli was significantly reduced up to 24 h before the manifestation of clinical symptoms compared to healthy calves. For enterococci, this was only the case on the day of the onset of diarrhea. In addition, the ratios of aerobic and anaerobic bacteria, Enterobacteriaceae or E. coli to lactobacilli, of calves with diarrhea starting on day 2 after birth are significantly higher than those of healthy calves. The detection frequency of specific pathogens in diarrheic calves increased over the first week of life. Conclusion: The results suggest that the incidence of neonatal diarrhea in calves is favored by low levels of lactobacilli in the feces. From this, the hypothesis can be derived that, in addition to an optimal supply of colostrum, the earliest possible administration of lactobacilli might reduce neonatal diarrhea in calves. However, this must be verified in a subsequent feeding experiment.