Specificity of the AMP-6000 Method for Enumerating Clostridium Endospores in Milk.

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements.

Importance of Pre-Milking Udder Hygiene to Reduce Transfer of Clostridial Spores from Teat Skin to Raw Milk.

Butyric acid producing clostridia (BAPC) cause the so-called late-blowing defect, a serious quality problem in semi-hard and hard cheeses. Late-blown cheeses are characterized by undesired slits and cracks, irregular eyes, and off-flavors due to excessive amounts of gas and organic acids produced by clostridia. Clostridial transfer to raw milk can occur during milking through dirty teats. Therefore, teat cleaning before milking is a key factor in preventing clostridial contamination of the milk. However, different cleaning methods are used, and little information is available on the efficacy of routine teat cleaning in reducing clostridial endospores. The main objectives of this study were to assess the extent of udder contamination with BAPC spores and to investigate the efficacy of routine teat cleaning on BAPC spore counts in milk. In a longitudinal study, eight dairy farms were visited during five sampling events. Clostridial spore counts were quantified from teat skin before and after routine teat cleaning, in pooled quarter milk samples from individual cows, and in bulk tank milk samples using a most probable number method. In addition, farm management data were collected periodically through a survey, and average cow cleanliness was assessed by a veterinarian. On average, teat cleaning resulted in a 0.6 log unit reduction in BAPC spores on teat skin, and a strong positive correlation was found between BAPC spore concentrations on teat skin after cleaning and in pooled quarter milk samples. Seasonal variations and the potential influence of differences in farm management were also noted. Interestingly, average cow cleanliness correlated strongly with BAPC spore levels in milk, suggesting the potential for a quick and rough estimation method of clostridial contamination that could be implemented by farmers.

Biogenic Amine Contents and Microbial Characteristics of Cambodian Fermented Foods.

Naturally fermented foods are an important part of the typical diet in Cambodia. However, the food safety status of these products has not been widely studied. The aim of this study was, therefore, to provide an overview of the quality of these foods in relation to microbiology and biogenic amines. Additionally, the obtained results were compared to the habits and practices of Cambodians in handling this type of food. A total of 57 fermented foods (42 fishery and 15 vegetable products) were collected from different retail markets in the capital of Cambodia. Pathogenic Salmonella spp., Listeria spp., and Listeria monocytogenes were not detected in 25 g samples. Generally, less than 102 cfu/g of Staphylococcus aureus, Escherichia coli, Pseudomonas spp., Enterobacteriaceae, and molds were present in the fermented foods. Bacillus cereus group members (<102 to 2.3 × 104 cfu/g), lactic acid bacteria (<102 to 1.1 × 107 cfu/g), halophilic and halotolerant bacteria (<102 to 8.9 × 106 cfu/g), sulfite-reducing Clostridium spp. (<102 to 3.5 × 106 cfu/g), and yeasts (<102 to 1.1 × 106 cfu/g) were detected in this study. Still, the presence of pathogenic and spoilage microorganisms in these fermented foods was within the acceptable ranges. Putrescine, cadaverine, tyramine, and histamine were detected in 100%, 89%, 81%, and 75% of the tested products, respectively. The concentrations of histamine (>500 ppm) and tyramine (>600 ppm) were higher than the recommended maximum levels in respectively four and one of 57 fermented foods, which represents a potential health risk. The results suggest that the production process, distribution, and domestic handling of fermented foods should be re-evaluated. Further research is needed for the establishment of applicable preservation techniques in Cambodia.

Bacterial growth dynamics and corresponding metabolite levels in the extraction area of an Austrian sugar beet factory using antimicrobial treatment.

BACKGROUND: During the manufacture of sucrose from sugar beet, different microorganisms originating from the plant material as well as from the soil enter the process. Due to the formation of polysaccharide-based slimes, these contaminants may induce several adverse effects such as filtration problems during juice purification. Certain microorganisms also metabolize sucrose, leading to product losses with financial consequences. To better understand and to prevent these negative effects, the aim of the study was to investigate the evolution of relevant bacterial groups, including their metabolites appearing during the extraction process. For this purpose, one production cycle was monitored to identify the major contamination steps and to clarify how they relate to the processing conditions. Traditionally, different antimicrobial agents such as formaldehyde, sulfur dioxide, hypochlorous acid, sodium hypochlorite, and chlorine dioxide have been added to inhibit microbial growth. In the present study, a rosin-based product derived from pine trees was applied as an alternative to those substances. RESULTS: Press water, raw juice, and mid-tower juice were identified as being highly contaminated with bacteria, and processing conditions such as time, temperature and pH level significantly influenced bacterial levels and the corresponding metabolites. Among the contaminants identified, lactic acid bacteria, and mesophilic and thermophilic aerobic bacteria played a dominant role, whereas lactic acid, acetic acid, butyric acid, and ethanol were identified as typical metabolites. CONCLUSION: Bacterial growth during production could be reduced by shock dosing of the rosin-based material in the extraction area. © 2020 Society of Chemical Industry.

Prevalence of antibiotic-resistant E. coli in broilers challenged with a multi-resistant E. coli strain and received ampicillin, an organic acid-based feed additive or a synbiotic preparation.

The aim of this study was to evaluate the effect of ampicillin, an organic acid-based feed additive and a synbiotic preparation on the prevalence of antibiotic-resistant E. coli in the ceca of broilers. A total of 2000 broiler chickens (Ross 708) were randomly assigned to 5 groups with 8 replicates. The negative control group was the only group that was not subjected to avian pathogenic E. coli challenge, while all the other 4 groups received a multi-resistant E. coli strain that was resistant to ampicillin, cephalexin, and nalidixic acid as an oral challenge. The second group served as a challenge control, and the third group received the antibiotic ampicillin via water for 5 d. The fourth group received a feed additive based on organic acids and cinnamaldehyde, and the fifth group received a synbiotic preparation via feed and water. On day 17 and 38 of the trial, cecal samples from 3 birds from each of the 40 pens were obtained, and the E. coli counts and abundances of antibiotic-resistant E. coli were determined. Oral challenge with an avian pathogenic E. coli strain did not influence the performance, and there was no significant difference in growth performance between groups. The total E. coli count was lower (P < 0.05) in the group supplemented with the synbiotic than in the challenge control group on day 38 of the trial. Administration of an antibiotic for 5 d led to a significant increase in the abundance of E. coli strains resistant to ampicillin, amoxicillin-clavulanic acid, cefoxitin, and ceftriaxone. There was no increase in the abundance of antibiotic-resistant E. coli observed in the groups that received feed supplemented with an organic acid/cinnamaldehyde-based feed additive or a synbiotic. Moreover, the effects of the tested feed additives on the prevalence of resistant E. coli are demonstrated by the lower ceftriaxone minimal inhibitory concentration values for this group than for the antibiotic group. Additionally, the synbiotic group exhibited lower ceftriaxone minimal inhibitory concentration values than the antibiotic group.

The application of antibiotics in broiler production and the resulting antibiotic resistance in Escherichia coli: A global overview.

The increase in antibiotic resistance is a global concern for human and animal health. Resistant microorganisms can spread between food-producing animals and humans. The objective of this review was to identify the type and amount of antibiotics used in poultry production and the level of antibiotic resistance in Escherichia coli isolated from broilers. Isolate information was obtained from national monitoring programs and research studies conducted in large poultry-producing regions: US, China, Brazil, and countries of EU-Poland, United Kingdom, Germany, France, and Spain. The survey results clearly display the absence of a harmonized approach in the monitoring of antibiotics per animal species and the evaluation of resistances using the same methodology. There is no public long-term quantitative data available targeting the amount of antibiotics used in poultry, with the exception of France. Data on antibiotic-resistant E. coli are available for most regions but detection of resistance and number of isolates in each study differs among regions; therefore, statistical evaluation was not possible. Data from France indicate that the decreased use of tetracyclines leads to a reduction in the detected resistance rates. The fluoroquinolones, third-generation cephalosporins, macrolides, and polymyxins ("highest priority critically important" antibiotics for human medicine according to WHO) are approved for use in large poultry-producing regions, with the exception of fluoroquinolones in the US and cephalosporins in the EU. The approval of cephalosporins in China could not be evaluated. Tetracyclines, aminoglycosides, sulfonamides, and penicillins are registered for use in poultry in all evaluated countries. The average resistance rates in E. coli to representatives of these antibiotic classes are higher than 40% in all countries, with the exception of ampicillin in the US. The resistance rates to fluoroquinolones and quinolones in the US, where fluoroquinolones are not registered for use, are below 5%, while the average of resistant E. coli is above 40% in Brazil, China, and EU, where use of fluoroquinolones is legalized. However, banning of fluoroquinolones and quinolones has not totally eliminated the occurrence of resistant populations.

Comparison of the CLSI guideline and ISO/IDF standard for antimicrobial susceptibility testing of Lactobacilli.

Lactobacilli play a crucial role as probiotics and as starter cultures in the production of fermented foods. Although lactobacilli are a technologically useful and beneficial group of bacteria, a few members of them have been rarely correlated with bacterial infections. Correspondingly, clinicians are interested in the antimicrobial susceptibility of lactobacilli. In addition, antimicrobial susceptibility testing (AST) is also relevant for commercially applied lactobacilli as bacterial strains harboring transferable antibiotic resistance genes should not be used in fermented and probiotic foods. Therefore, two methods were developed by different organizations, which were compared within this study. For this purpose, 22 Lactobacillus-type strains were tested for their antimicrobial susceptibility to 16 antibiotics following the procedures of the Clinical and Laboratory Standards Institute (CLSI) and the International Organization of Standardization (ISO)/International Dairy Federation (IDF). Crucial discrepancies between both procedures were detected mainly due to the different AST media. Hence, half of the strains tested did not consistently grow in the CLSI medium, whereas all showed evaluable growth in the ISO/IDF medium. However, some antibiotics were influenced by the latter medium. In particular, low levels of essential agreement between both methods were obtained with seven antibiotics. Accordingly, different interpretative criteria are needed for both procedures to distinguish resistant from susceptible strains.

Reduced detectability of Listeria monocytogenes in the presence of Listeria innocua.

Recent foodborne crises have demonstrated the importance of monitoring food safety. In terms of microbiological criteria, food safety requires the reliable detection of pathogens such as Listeria monocytogenes along the food chain by appropriate analytical methods. However, indications exist that accompanying Listeria innocua strains suppress the growth of L. monocytogenes during selective enrichment, which may cause reduced or even inhibited detection. To study these effects, the limit of detection of L. monocytogenes was investigated in the presence of L. innocua using the International Organization for Standardization standard method ISO 11290-1 and the VIDAS LDUO system, an automated method based on enzyme-linked fluorescence technology. The challenge was to provide low initial Listeria concentrations at sufficient precision to quantify the influence on the probability of detection of L. monocytogenes. The application of reference materials appropriate for quantitative test methods and a standardized dilution procedure were necessary to ensure accurate CFU levels of defined proportions of mixtures of both Listeria species. During selective enrichment, overgrowth of L. monocytogenes by L. innocua could be confirmed, leading to high rates of false-negative results. Moreover, with both methods, a significant decrease in the detectability of L. monocytogenes could be quantified at ratios of 2:1 at very low concentrations representative of natural contamination levels often found in foods and environments. It is concluded that there is a need to improve existing procedures with respect to selective enrichment, as well as the detection techniques.

Application of plant-based antimicrobials for the growth inhibition of clostridia in pressed beet pulp silage.

BACKGROUND: In this study the inhibition of hop beta acids on the growth of clostridia in soil-contaminated pressed sugar beet pulp silages was investigated. Hop beta acids are natural substances which display their effect at low concentrations. Fresh pressed beet pulp material was mixed with soil to artificially contaminate it with clostridia. Laboratory silos were filled with the substrate, stored at 25 °C and opened for sampling at 0, 2, 8, 15, 30, 60, and 90 days. The impact on clostridial growth during silage fermentation was monitored by determination of the pH value and dry matter content, as well as chemical analysis of the fermentation products. Throughout the experiments, the effect of a commercial silage inoculant based on lactic acid bacteria (LAB) and hop-resistant LAB were examined with and without the combination of plant-based antimicrobials. RESULTS: Results indicate that in contaminated silage samples without any additives high butyric acid contents occurred due to clostridial growth. This spoilage could not be suppressed by the application of LAB, whereas the combined application of LAB and hop beta acids significantly improved silage quality, which was reflected by favourable organic acid composition (P < 0.05). CONCLUSION: The experimental data indicate that the application of hop beta acids improves the preservation effect of LAB in suppressing clostridial growth in silages and thus demonstrates some potential for the combined use of plant-based antimicrobials and LAB.

Comparison of broth microdilution, Etest, and agar disk diffusion methods for antimicrobial susceptibility testing of Lactobacillus acidophilus group members.

In recent years, the absence of acquired antimicrobial resistance has become an important criterion to evaluate the biosafety of lactobacilli used as industrial starter or probiotic cultures. At present, however, standards for susceptibility testing of Lactobacillus strains or approved guidelines for interpreting the test results are not available. Hence, this study was carried out to contribute to the establishment of a standardized procedure for antimicrobial susceptibility testing of lactobacilli. The results obtained by testing 104 strains of the Lactobacillus acidophilus group were compared based on broth microdilution, disk diffusion, and Etest. Except for some specific agent-related effects, agreement between MICs resulting from the broth microdilution method and the Etest was good. In addition, inhibition zone diameters determined with disk diffusion correlated well with MICs from Etest and broth microdilution.

Antibiotic susceptibility testing of Bifidobacterium thermophilum and Bifidobacterium pseudolongum strains: broth microdilution vs. agar disc diffusion assay.

There is urgent need for having available suitable methods and data regarding the susceptibility levels of antibiotic resistant and sensitive strains of bifidobacteria. Based on a defined standard operation procedure, agar disc diffusion and broth microdilution were compared in order to evaluate the antimicrobial susceptibility profiles of 82 B. pseudolongum and 80 B. thermophilum strains mainly originating from the meat production chain. The methods that were assessed showed interpretable agreement within this study. The disc diffusion zone diameters are highly reproducible making the method a useful alternative to broth microdilution for antimicrobial susceptibility screening of bifidobacteria.