Microbial Succession in the Cheese Ripening Process-Competition of the Starter Cultures and the Microbiota of the Cheese Plant Environment.

A large variety of cheeses can be produced using different manufacturing processes and various starter or adjunct cultures. In this study, we have described the succession of the microbial population during the commercial production and subsequent ripening of smear-ripened cheese using 16S rRNA gene sequencing. The composition of the microbiota during the first 6 days of production was constant and consisted mainly of LAB (lactic acid bacteria) originating from the starter culture. From day 7, the proportion of LAB decreased as other bacteria from the production environment appeared. From the 14th day of production, the relative proportion of LAB decreased further, and at the end of ripening, bacteria from the environment wholly dominated. These adventitious microbiota included Psychrobacter, Pseudoalteromonas haloplanktis/hodoensis, Vibrio toranzoniae, and Vibrio litoralis (Proteobacteria phylum), as well as Vagococcus and Marinilactibacillus (Firmicutes phylum), Psychrilyobacter (Fusobacteria phylum), and Malaciobacter marinus (Campylobacterota phylum), all of which appeared to be characteristic taxa associated with the cheese rind. Subsequent analysis showed that the production and ripening of smear-ripened cheese could be divided into three stages, and that the microbiota compositions of samples from the first week of production, the second week of production, and supermarket shelf life all differed.

Host Species Adaptation of Obligate Gut Anaerobes Is Dependent on Their Environmental Survival.

The gut microbiota of warm-blooded vertebrates consists of bacterial species belonging to two main phyla; Firmicutes and Bacteroidetes. However, does it mean that the same bacterial species are found in humans and chickens? Here we show that the ability to survive in an aerobic environment is central for host species adaptation. Known bacterial species commonly found in humans, pigs, chickens and Antarctic gentoo penguins are those capable of extended survival under aerobic conditions, i.e., either spore-forming, aerotolerant or facultatively anaerobic bacteria. Such bacteria are ubiquitously distributed in the environment, which acts as the source of infection with similar probability in humans, pigs, chickens, penguins and likely any other warm-blooded omnivorous hosts. On the other hand, gut anaerobes with no specific adaptation for survival in an aerobic environment exhibit host adaptation. This is associated with their vertical transmission from mothers to offspring and long-term colonisation after administration of a single dose. This knowledge influences the design of next-generation probiotics. The origin of aerotolerant or spore-forming probiotic strains may not be that important. On the other hand, if Bacteroidetes and other host-adapted species are used as future probiotics, host preference should be considered.

Probiotic Lactobacilli Do Not Protect Chickens against Salmonella Enteritidis Infection by Competitive Exclusion in the Intestinal Tract but in Feed, Outside the Chicken Host.

Lactobacilli are commonly used as probiotics in poultry to improve production parameters and to increase chicken resistance to enteric infections. However, lactobacilli do not efficiently colonise the chicken intestinal tract, and also, their anti-infection effect in vivo is sometimes questionable. In this study, we therefore evaluated the potential of a mixture of four Lactobacillus species (L. salivarius, L. reuteri, L. ingluviei and L. alvi) for the protection of chickens against Salmonella Enteritidis infection. Whenever the chickens were inoculated by lactobacilli and S. Enteritidis separately, there was no protective effect of lactobacilli. This means that when lactobacilli and S. Enteritidis are exposed to each other as late as in the crop of chickens, lactobacilli did not influence chicken resistance to S. Enteritidis at all. The only positive effect was recorded when the mixture of lactobacilli and S. Enteritidis was used for the inoculation of feed and the feed was anaerobically fermented for 1 to 5 days. In this case, chickens fed such a diet remained S. Enteritidis negative. In vitro experiments showed that the protective effect was caused by acidification of feed down to pH 4.6 due to lactobacilli fermentation and was associated with S. Enteritidis inactivation. The probiotic effect of lactobacilli was thus expressed in the feed, outside the chicken host.

Vacuum-Packed Steak Tartare: Prevalence of Listeria monocytogenes and Evaluation of Efficacy of ListexTM P100.

Steak tartare is a raw, ready-to-eat meal popular in European countries, the safety of which is often discussed due to the risk of foodborne illness. The aim of this study was to determine the prevalence of Listeria monocytogenes in vacuum-packed steak tartare from retailers in the Czech Republic, characterize the strains obtained by typing methods and to evaluate the efficacy of ListexTM P100 against L. monocytogenes artificially inoculated into steak tartare samples. The prevalence of L. monocytogenes was 55% and 17 isolates belonging mostly to serotype 1/2a were obtained. Altogether 11 sequence types and 11 clonal complexes were assigned based on the whole genome sequencing (WGS) signifying the high diversity of L. monocytogenes isolates obtained. Core genome multi-locus sequence typing (cgMLST) did not confirm an epidemiological connection with human cases of listeriosis. The efficacy of ListexTM P100 treatment at concentrations of 108 and 109 PFU/g on artificially inoculated beef steak tartare samples was not efficient. Based on the results of this study, steak tartare from retailers can be considered as a source of L. monocytogenes that remains a challenge to the food industry.

Eggshell and Feed Microbiota Do Not Represent Major Sources of Gut Anaerobes for Chickens in Commercial Production.

In this study, we addressed the origin of chicken gut microbiota in commercial production by a comparison of eggshell and feed microbiota with caecal microbiota of 7-day-old chickens, using microbiota analysis by 16S rRNA sequencing. In addition, we tested at which timepoint during prenatal or neonatal development it is possible to successfully administer probiotics. We found that eggshell microbiota was a combination of environmental and adult hen gut microbiota but was completely different from caecal microbiota of 7-day-old chicks. Similarly, we observed that the composition of feed microbiota was different from caecal microbiota. Neither eggshell nor feed acted as an important source of gut microbiota for the chickens in commercial production. Following the experimental administration of potential probiotics, we found that chickens can be colonised only when already hatched and active. Spraying of eggs with gut anaerobes during egg incubation or hatching itself did not result in effective chicken colonisation. Such conclusions should be considered when selecting and administering probiotics to chickens in hatcheries. Eggshells, feed or drinking water do not act as major sources of gut microbiota. Newly hatched chickens must be colonised from additional sources, such as air dust with spores of Clostridiales. The natural colonisation starts only when chickens are already hatched, as spraying of eggs or even chickens at the very beginning of the hatching process did not result in efficient colonisation.

The distribution of antibiotic resistance genes in chicken gut microbiota commensals.

Antibiotic resistance in bacterial pathogens or several indicator bacteria is commonly studied but the extent of antibiotic resistance in bacterial commensals colonising the intestinal tract is essentially unknown. In this study, we aimed to investigate the presence of horizontally acquired antibiotic resistance genes among chicken gut microbiota members in 259 isolates with known whole genomic sequences. Altogether 124 isolates contained at least one gene coding for antibiotic resistance. Genes coding for the resistance to tetracyclines (detected in 101 isolates), macrolide-lincosamide-streptogramin B antibiotics (28 isolates) and aminoglycosides (25 isolates) were the most common. The most frequent tetracycline resistance genes were tet(W), tet(32), tet(O) and tet(Q). Lachnospiraceae and Ruminococcaceae frequently encoded tet(W). Lachnospiraceae commonly coded also for tet(32) and tet(O). The tet(44) gene was associated with Erysipelotrichaceae and tet(Q) was detected in the genomes of Bacteroidaceae and Porphyromonadaceae. Without any bias we have shown that antibiotic resistance is quite common in gut commensals. However, a comparison of codon usage showed that the above-mentioned families represent the most common current reservoirs but probably not the original host of the detected resistances.

Systematic Culturomics Shows that Half of Chicken Caecal Microbiota Members can be Grown in Vitro Except for Two Lineages of Clostridiales and a Single Lineage of Bacteroidetes.

Epidemiological data show that the composition of gut microbiota influences host health, disease status, and even behaviour. However, to confirm these epidemiological observations in controlled experiments, pure cultures of gut anaerobes must be obtained. Since the culture of gut anaerobes is not a simple task due to the large number of bacterial species colonising the intestinal tract, in this study we inoculated 174 different culture media with caecal content from adult hens, and compared the microbiota composition in the original caecal samples and in bacterial masses growing in vitro by 16S rRNA sequencing. In total, 42% of gut microbiota members could be grown in vitro and since there were some species which were not cultured but for which the culture conditions are known, it is likely that more than half of chicken gut microbiota can be grown in vitro. However, there were two lineages of Clostridiales and a single lineage of Bacteroidetes which were common in chicken caecal microbiota but resistant to culture. Of the most selective culture conditions, nutrient broths supplemented with mono- or di-saccharides, including those present in fruits, positively selected for Lactobacillaceae. The addition of bile salts selected for Veillonellaceae and YCFA (yeast casitone fatty acid agar) enriched for Desulfovibrionaceae. In addition, Erysipelotrichaceae were positively selected by colistin, trimethoprim, streptomycin and nalidixic acid. Culture conditions tested in this study can be used for the selective enrichment of desired bacterial species but also point towards the specific functions of individual gut microbiota members.

Antibiotic Resistance, Core-Genome and Protein Expression in IncHI1 Plasmids in Salmonella Typhimurium.

Conjugative plasmids from the IncHI1 incompatibility group play an important role in transferring antibiotic resistance in Salmonella Typhimurium. However, knowledge of their genome structure or gene expression is limited. In this study, we determined the complete nucleotide sequences of four IncHI1 plasmids transferring resistance to antibiotics by two different next generation sequencing protocols and protein expression by mass spectrometry. Sequence data including additional 11 IncHI1 plasmids from GenBank were used for the definition of the IncHI1 plasmid core-genome and pan-genome. The core-genome consisted of approximately 123 kbp and 122 genes while the total pan-genome represented approximately 600 kbp. When the core-genome sequences were used for multiple alignments, the 15 tested IncHI1 plasmids were separated into two main lineages. GC content in core-genome genes was around 46% and 50% in accessory genome genes. A multidrug resistance region present in all 4 sequenced plasmids extended over 20 kbp and, except for tet(B), the genes responsible for antibiotic resistance were those with the highest GC content. IncHI1 plasmids therefore represent replicons that evolved in low GC content bacteria. From their original host, they spread to Salmonella and during this spread these plasmids acquired multiple accessory genes including those coding for antibiotic resistance. Antibiotic-resistance genes belonged to genes with the highest level of expression and were constitutively expressed even in the absence of antibiotics. This is the likely mechanism that facilitates host cell survival when antibiotics suddenly emerge in the environment.

Complete sequence of multidrug resistance p9134 plasmid and its variants including natural recombinant with the virulence plasmid of Salmonella serovar Typhimurium.

In this study we determined the complete nucleotide sequence of multidrug-resistance plasmid p9134, and its variants p9134dT and p9134dAT which spontaneously lost either tetracycline or both tetracycline and ampicillin resistance, respectively. The plasmids were 133,802 bp, 109,512 bp and 127,291 bp in size, respectively, and their basic backbone was similar to that of IncI plasmids. Genes coding for ampicillin (blaTEM), chloramphenicol (catA1), streptomycin (strA, strB), tetracycline (tetA(A)) and gentamicin (aac(3)-IV) resistance were confirmed in wild-type p9134. Moreover, a gene for hygromycine resistance (hph) and a putative gene for apramycin resistance were newly determined. In p9134dAT, a continuous sequence coding for ampicillin and tetracycline resistances was lost. Genetic rearrangements in p9134dT were more complex and 2 recombination events must have occurred. During the first one, the tetracycline resistance locus was replaced with rck, srgB, srgA, orf7 and pefI originating from Salmonella virulence plasmid pSLT. During the second one, ydjA, pifA and repC genes from p9134 were replaced with repA2, PSLT025 and PSLT026 genes from pSLT. Our findings indicate that recombination event between unrelated plasmids might be quite common and may lead to the generation and selection of plasmids both transferring antibiotic resistance and increasing virulence of their host.

Chicken faecal microbiota and disturbances induced by single or repeated therapy with tetracycline and streptomycin.

BACKGROUND: In this study, we characterised the microbiota present in the faeces of 15- and 46-week-old egg laying hens before and after tetracycline or streptomycin therapy. In the first experiment, the layers were subjected to 7 days of therapy. In the second experiment, the hens were subjected to two days of therapy, which was repeated for an additional two days after 12 days of antibiotic withdrawal. This enabled us to characterise dynamics of the changes after antibiotic administration and withdrawal, and to identify genera repeatedly resistant to tetracycline and streptomycin. RESULTS: Real-time PCRs specific for Enterobacteriales, Lactobacillales, Clostridiales and Bifidobacteriales showed that changes in the microbiota in response to antibiotic therapy and antibiotic withdrawal were quite rapid and could be observed within 24 hours after the change in therapy status. Pyrosequencing of PCR amplified V3/V4 variable regions of 16S rRNA genes showed that representatives of the orders Clostridiales, Lactobacillales, Bacteroidales, Bifidobacteriales, Enterobacteriales, Erysipelotrichales, Coriobacteriales, Desulfovibrionales, Burkholderiales, Campylobacterales and Actinomycetales were detected in the faeces of hens prior to the antibiotic therapy. Tetracycline and streptomycin therapies decreased the prevalence of Bifidobacteriales, Bacteroidales, Clostridiales, Desulfovibrionales, Burkholderiales and Campylobacterales in faecal samples in both experiments. On the other hand, Enterobacteriales and Lactobacillales always increased in prevalence in response to both therapies. Within the latter two orders, Escherichia and Enterococcus were the genera prevalence of which increased after all the antibiotic treatments. CONCLUSIONS: The changes in microbiota composition induced by the antibiotic therapy were rapid and quite dramatic and only representatives of the genera Enterococcus and Escherichia increased in response to the therapy with both antibiotics in both experiments.