Antimicrobial Susceptibility of Lactobacillus delbrueckii subsp. lactis from Milk Products and Other Habitats

As components of many cheese starter cultures, strains of Lactobacillus delbrueckii subsp. lactis (LDL) must be tested for their antimicrobial susceptibility to avoid the potential horizontal transfer of antibiotic resistance (ABR) determinants in the human body or in the environment. To this end, a phenotypic test, as well as a screening for antibiotic resistance genes (ARGs) in genome sequences, is commonly performed. Historically, microbiological cutoffs (MCs), which are used to classify strains as either ‘sensitive’ or ‘resistant’ based on the minimal inhibitory concentrations (MICs) of a range of clinically-relevant antibiotics, have been defined for the whole group of the obligate homofermentative lactobacilli, which includes LDL among many other species. This often leads to inaccuracies in the appreciation of the ABR status of tested LDL strains and to false positive results. To define more accurate MCs for LDL, we analyzed the MIC profiles of strains originating from various habitats by using the broth microdilution method. These strains’ genomes were sequenced and used to complement our analysis involving a search for ARGs, as well as to assess the phylogenetic proximity between strains. Of LDL strains, 52.1% displayed MICs that were higher than the defined MCs for kanamycin, 9.9% for chloramphenicol, and 5.6% for tetracycline, but no ARG was conclusively detected. On the other hand, all strains displayed MICs below the defined MCs for ampicillin, gentamycin, erythromycin, and clindamycin. Considering our results, we propose the adaptation of the MCs for six of the tested clinically-relevant antibiotics to improve the accuracy of phenotypic antibiotic testing

Genomic rearrangements in the aspA-dcuA locus of Propionibacterium freudenreichii are associated with aspartase activity.

Propionibacterium freudenreichii is crucial in Swiss-type cheese manufacture. Classic propionic acid fermentation yields the nutty flavor and the typical eyes. Co-metabolism of aspartate pronounces the flavor of the cheese; however, it also increases the size of the eyes, which can induce splitting and reduce the cheese quality. Aspartase (EC 4.3.1.1) catalyzes the deamination of aspartate, yielding fumarate and ammonia. The aspartase activity varies considerably among P. freudenreichii strains. Here, the correlation between aspartase activity and the locus of aspartase-encoding genes (aspA ) and dcuA encoding the C4-dicarboxylate transporter was investigated in 46 strains to facilitate strain selection for cheese culture. Low aspartase activity was correlated with a particular genomic rearrangement: low in vitro aspartase activity always occurred in strains with gene clusters aspA - dcuA where the dcuA was frameshifted, producing a stop codon or was disrupted by an ISL3-like element. The low aspartase activity could be due to the protein sequence of the aspartase or a dysfunctional DcuA. The highest values of aspartase activity were detected in strains with aspA1 - aspA2-dcuA with a DcuA sequence sharing 99.07 - 100% identity with the DcuA sequence of strain DSM 20271 T and an additional C4-dicarboxylate transporter belonging to the DcuAB family

Influence of raw milk microflora and starter cultures in cheese on protein hydrolysis and peptide generation during digestion

Do bacterial strains in cheese have an impact on the protein hydrolysis during human digestion, and if so, does a higher microbial diversity lead to the generation of a higher number of different peptides after digestion?Cheese bacteria are responsible for the hydrolysis of proteins already during cheese ripening. These bacterial cultures are introduced at different steps of the cheese manufacturing process. First, the raw milk flora that is dependent on the milk heat treatment before the cheese manufacturing process, is a major source of a variety of bacteria. Second, starter cultures, needed for a successful acidification of the cheese curd are added to prevent side fermentations. Third, addition of cultures is made for improving flavor or accelerating the ripening process, depending on the type of cheese. Because of bacterial proteolytic activity, proteins in cheese are partially hydrolyzed, depending on the ripening time and are further digested by gastric and pancreatic proteases, after consumption, to the level of small peptides and free amino acids. In order to elucidate possible differences in proteolysis depending on the presence of different bacteria, Swiss Raclette cheeses were produced either from raw or pasteurized milk with or without addition of a proteolytic bacterial strains (Lactobacillus helveticus) and ripened during 120 days.The microbial diversity and the relative abundance of specific strains in the different cheeses was assessed after 24 hours, 80 and 120 days of cheese ripening by sequencing the hypervariable regions V1-V2 of the 16S rRNA genes. Moreover, protein hydrolysis in the different cheeses was analyzed with gel electrophoresis, mass spectrometry, and HPLC after in vitro digestion, applying a static (Infogest) and a dynamic (DIDGI®) oro-gastrointestinal in vitro digestion protocol. In order to gain information on the influence of bacterial strains on protein hydrolysis, the 16s meta-genomic and 16s meta-transcriptomic results were correlated with protein and peptide patterns

Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese.

Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively

Conversion of Methionine to Cysteine in Lactobacillus paracasei Depends on the Highly Mobile cysK-ctl-cysE Gene Cluster

Milk and dairy products are rich in nutrients and are therefore habitats for various microbiomes. However, the composition of nutrients can be quite diverse, in particular among the sulfur containing amino acids. In milk, methionine is present in a 25-fold higher abundance than cysteine. Interestingly, a fraction of strains of the species L. paracasei – a flavor-enhancing adjunct culture species – can grow in medium with methionine as the sole sulfur source. In this study, we focus on genomic and evolutionary aspects of sulfur dependence in L. paracasei strains. From 24 selected L. paracasei strains, 16 strains can grow in medium with methionine as sole sulfur source. We sequenced these strains to perform gene-trait matching. We found that one gene cluster – consisting of a cysteine synthase, a cystathionine lyase, and a serine acetyltransferase – is present in all strains that grow in medium with methionine as sole sulfur source. In contrast, strains that depend on other sulfur sources do not have this gene cluster. We expanded the study and searched for this gene cluster in other species and detected it in the genomes of many bacteria species used in the food production. The comparison to these species showed that two different versions of the gene cluster exist in L. paracasei which were likely gained in two distinct events of horizontal gene transfer. Additionally, the comparison of 62 L. paracasei genomes and the two versions of the gene cluster revealed that this gene cluster is mobile within the species

The Histidine Decarboxylase Gene Cluster of Lactobacillus parabuchneri Was Gained by Horizontal Gene Transfer and Is Mobile within the Species

Histamine in food can cause intolerance reactions in consumers. Lactobacillus parabuchneri (L. parabuchneri) is one of the major causes of elevated histamine levels in cheese. Despite its significant economic impact and negative influence on human health, no genomic study has been published so far. We sequenced and analyzed 18 L. parabuchneri strains of which 12 were histamine positive and 6 were histamine negative. We determined the complete genome of the histamine positive strain FAM21731 with PacBio as well as Illumina and the genomes of the remaining 17 strains using the Illumina technology. We developed the synteny aware ortholog finding algorithm SynOrf to compare the genomes and we show that the histidine decarboxylase (HDC) gene cluster is located in a genomic island. It is very likely that the HDC gene cluster was transferred from other lactobacilli, as it is highly conserved within several lactobacilli species. Furthermore, we have evidence that the HDC gene cluster was transferred within the L. parabuchneri species

RAMÓN, PEDRO [Material gráfico]

SRA. DECopia digital. Madrid : Ministerio de Educación, Cultura y Deporte, 201

Adhésion du corps infirmier à la prescription des médicaments antalgiques postopératoires

Les deux objectifs de cette étude prospective étaient : 1) d'évaluer l'adhésion du corps infirmier à la prescription de trois médicaments antalgiques en postopératoire: le paracétamol, les anti-inflammatoires non stéroïdiens et les opiacés, 2) de comparer cette adhésion à celle de l'héparine, comme prophylaxie anti-thrombotique. Résultats (odds ratio) : La majorité des patients souffrant de douleurs sévères reçoivent moins d'opiacés que la dose prescrite. Le jour de l'intervention, la morphine est 102 fois moins administrée que l'héparine, le paracétamol 32 fois moins et les AINS 40 fois moins. Le lendemain de l'intervention, la morphine est 196 fois moins administrée que l'héparine, le paracétamol 40 fois moins et les AINS 24 fois moins. Conclusion : le manque d'adhésion à la prescription des médicaments antalgiques est une des raisons de la prise en charge insatisfaisante de la douleur postopératoire, bien que d'autres facteurs émotionnels et culturels soient à prendre en considération

Genomic approach to studying nutritional requirements of Clostridium tyrobutyricum and other Clostridia causing late blowing defects.

Clostridium tyrobutyricum is the main microorganism responsible for the late blowing defect in hard and semi-hard cheeses, causing considerable economic losses to the cheese industry. Deeper knowledge of the metabolic requirements of this microorganism can lead to the development of more effective control approaches. In this work, the amino acids and B vitamins essential for sustaining the growth of C. tyrobutyricum were investigated using a genomic approach. As the first step, the genomes of four C. tyrobutyricum strains were analyzed for the presence of genes putatively involved in the biosynthesis of amino acids and B vitamins. Metabolic pathways could be reconstructed for all amino acids and B vitamins with the exception of biotin (vitamin B7) and folate (vitamin B9). The biotin pathway was missing the enzyme amino-7-oxononanoate synthase that catalyzes the condensation of pimeloyl-ACP and l-alanine to 8-amino-7-oxononanoate. In the folate pathway, the missing genes were those coding for para-aminobenzoate synthase and aminodeoxychorismate lyase enzymes. These enzymes are responsible for the conversion of chorismate into para-aminobenzoate (PABA). Two C. tyrobutyircum strains whose genome was analyzed in silico as well as other 10 strains isolated from cheese were tested in liquid media to confirm these observations. 11 strains showed growth in a defined liquid medium containing biotin and PABA after 6-8 days of incubation. No strain showed growth when only one or none of these compounds were added, confirming the observations obtained in silico. Furthermore, the genome analysis was extended to genomes of single strains of other Clostridium species potentially causing late blowing, namely Clostridium beijerinckii, Clostridium sporogenes and Clostridium butyricum. Only the biotin biosynthesis pathway was incomplete for C. butyricum and C. beijerincki. In contrast, C. sporogenes showed missing enzymes in biosynthesis pathways of several amino acids as well as biotin, folate, and cobalamin (vitamin B12). These observations agree with the results of growth experiments of these species in liquid media reported in the literature. The results of this study suggest that biotin and folate are potential targets for reducing late blowing in cheese and highlight the usefulness of genomic analysis for identifying essential nutrients in bacteria