Cellular Injuries in Cronobacter sakazakii CIP 103183T and Salmonella enterica Exposed to Drying and Subsequent Heat Treatment in Milk Powder.

Because of the ability of foodborne pathogens to survive in low-moisture foods, their decontamination is an important issue in food protection. This study aimed to clarify some of the cellular mechanisms involved in inactivation of foodborne pathogens after drying and subsequent heating. Individual strains of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii were mixed into whole milk powder and dried to different water activity levels (0.25 and 0.58); the number of surviving cells was determined after drying and subsequent thermal treatments in closed vessels at 90 and 100°C, for 30 and 120 s. For each condition, the percentage of unculturable cells was estimated and, in parallel, membrane permeability and respiratory activity were estimated by flow cytometry using fluorescent probes. After drying, it was clearly observable that the percentage of unculturable cells was correlated with the percentage of permeabilized cells (responsible for 20-40% of the total inactivated bacteria after drying), and to a lesser degree with the percentage of cells presenting with loss of respiratory activity. In contrast, the percentages of unculturable cells observed after heat treatment were strongly correlated with the loss of respiratory activity and weakly with membrane permeability (for 70-80% of the total inactivated bacteria after heat treatment). We conclude that cell inactivation during drying is closely linked to membrane permeabilization and that heat treatment of dried cells affects principally their respiratory activity. These results legitimize the use of time-temperature scales and allow better understanding of the cellular mechanisms of bacterial death during drying and subsequent heat treatment. These results may also allow better optimization of the decontamination process to ensure food safety by targeting the most deleterious conditions for bacterial cells without denaturing the food product.

Caco-2 Invasion by Cronobacter sakazakii and Salmonella enterica Exposed to Drying and Heat Treatments in Dried State in Milk Powder.

Due to the ability of foodborne pathogens to survive in low moisture food, the decontamination of milk powder is an important issue in food protection. The safety of food products is, however, not always insured and the different steps in the processing of food involve physiological and metabolic changes in bacteria. Among these changes, virulence properties may also be affected. In this study, the effect of drying and successive thermal treatments on the invasion capacity of Salmonella Typhimurium, Salmonella Senftenberg, and Cronobacter sakazakii was assessed. Bacteria were dried on milk powder at three different water activity levels (0.25, 0.58, and 0.80) and heated at two different temperatures (90°C and 100°C) for 30 and 120 s. After recovery, stressed bacterial populations were placed in contact with Caco-2 cells to estimate their invasion capacity. Our results show that drying increases the invasion capacity of foodborne pathogens, but that heat treatment in the dried state did not exert a selective pressure on bacterial cells depending on their invasion capacity after drying. Taken together, our findings add to the sum of knowledge on food safety in dried food products and provide insight into the effects of food processing.

Modeling the heat inactivation of foodborne pathogens in milk powder: High relevance of the substrate water activity.

Due to the ability of foodborne pathogens to survive in low moisture foods, the decontamination of these products is an important issue in food hygiene. Up to now, such decontamination has mostly been achieved through empirical methods. The intention of this work is to establish a more rational use of heat treatment cycles. The effects of thermal treatment cycles on the inactivation of dried Salmonella Typhimurium, Salmonella Senftenberg, Cronobacter sakazakii and Escherichia coli were assessed. Bacteria were mixed with whole milk powder and dried down to different water activity levels (0.11, 0.25, 0.44 and 0.58). The rate of inactivated bacteria was determined after thermal treatment at 85°C, 90°C, 95°C and 100°C, from 0s to 180s in closed vessels, in order to maintain aw during treatment. In a first step, logarithmic bacterial inactivation was fitted by means of a classical loglinear model in which temperature and aw have a significant effect (p<0.05). DT,aw values were estimated for each T, aw condition and the results clearly showed that aw is a major parameter in the thermal decontamination of dried foods, a lower aw involving greater thermal resistance. In a second step, Bigelow's law was used to determine zT, a classical parameter relative to temperature, and yaw values, a new parameter relative to aw resistance. The values obtained for zT and yaw showed that the bacterium most resistant to temperature variations is Salmonella Typhimurium, while the one most resistant to aw variations is Escherichia coli. These data will help design decontamination protocols or processes in closed batches for low moisture foods.

Drying parameters greatly affect the destruction of Cronobacter sakazakii and Salmonella Typhimurium in standard buffer and milk.

Salmonella Typhimurium and Cronobacter sakazakii are two foodborne pathogens involved in neonatal infections from milk powder and infant formula. Their ability to survive in low-moisture food and during processing from the decontamination to the dried state is a major issue in food protection. In this work, we studied the effects of the drying process on Salmonella Typhimurium and Cronobacter sakazakii, with the aim of identifying the drying parameters that could promote greater inactivation of these two foodborne pathogens. These two bacteria were dried under different atmospheric relative humidities in milk and phosphate-buffered saline, and the delays in growth recovery and cultivability were followed. We found that water activity was related to microorganism resistance. C. sakazakii was more resistant to drying than was S. Typhimurium, and milk increased the cultivability and recovery of these two species. High drying rates and low final water activity levels (0.11-0.58) had a strong negative effect on the growth recovery and cultivability of these species. In conclusion, we suggest that effective use of drying processes may provide a complementary tool for food decontamination and food safety during the production of low-moisture foods.

Recovery Estimation of Dried Foodborne Pathogens Is Directly Related to Rehydration Kinetics.

Drying is a common process which is used to preserve food products and technological microorganisms, but which is deleterious for the cells. The aim of this study is to differentiate the effects of drying alone from the effects of the successive and necessary rehydration. Rehydration of dried bacteria is a critical step already studied in starter culture but not for different kinetics and not for pathogens. In the present study, the influence of rehydration kinetics was investigated for three foodborne pathogens involved in neonatal diseases caused by the consumption of rehydrated milk powder: Salmonella enterica subsp. enterica serovar Typhimurium, Salmonella enterica subsp. enterica serovar Senftenberg and Cronobacter sakazakii. Bacteria were dried in controlled relative humidity atmospheres and then rehydrated using different methods. Our results showed that the survival of the three pathogens was strongly related to rehydration kinetics. Consequently, rehydration is an important step to consider during food safety assessment or during studies of dried foodborne pathogens. Also, it has to be considered with more attention in consumers' homes during the preparation of food, like powdered infant formula, to avoid pathogens recovery.

A single mutation in the gene responsible for the mucoid phenotype of Bifidobacterium animalis subsp. lactis confers surface and functional characteristics.

Exopolysaccharides (EPS) are extracellular carbohydrate polymers synthesized by a large variety of bacteria. Their physiological functions have been extensively studied, but many of their roles have not yet been elucidated. We have sequenced the genomes of two isogenic strains of Bifidobacterium animalis subsp. lactis that differ in their EPS-producing phenotype. The original strain displays a nonmucoid appearance, and the mutant derived thereof has acquired a mucoid phenotype. The sequence analysis of their genomes revealed a nonsynonymous mutation in the gene Balat_1410, putatively involved in the elongation of the EPS chain. By comparing a strain from which this gene had been deleted with strains containing the wild-type and mutated genes, we were able to show that each strain displays different cell surface characteristics. The mucoid EPS synthesized by the strain harboring the mutation in Balat_1410 provided higher resistance to gastrointestinal conditions and increased the capability for adhesion to human enterocytes. In addition, the cytokine profiles of human peripheral blood mononuclear cells and ex vivo colon tissues suggest that the mucoid strain could have higher anti-inflammatory activity. Our findings provide relevant data on the function of Balat_1410 and reveal that the mucoid phenotype is able to alter some of the most relevant functional properties of the cells.

Sequence analysis of plasmid pSP02 from Bifidobacterium longum M62 and construction of pSP02-derived cloning vectors.

Replicons from bifidobacteria species are required for the construction of general- and special-purpose vectors that would allow the undertaking of molecular studies of these bacteria. In this work, pSP02, a cryptic plasmid from Bifidobacterium longum M62, was cloned, sequenced and characterized. pSP02 was found to consist of 4896bp with four ORFs coding for proteins over 50 amino acids long. Among the deduced protein sequences only a replicase (RepA) and a mobilization-like protein (MobA) showed known functional domains. Similar to previously described bifidobacterial plasmids, the organization of the putative ori region of pSP02 resembles that of the theta-replicating plasmids of Gram-positives. In spite of this, hybridization experiments detected single stranded (ss)-DNA as an intermediate product in the pSP02 replication, demonstrating it follows the rolling-circle (RC) replication mode. The ori region of pSP02 was used to construct a series of first generation cloning vectors able to replicate in many bifidobacterial species. Real time quantitative PCR established the copy number of pSP02 and its derived vectors to be around 12 copies per chromosome equivalent. pSP02-derivatives showed full segregational and structural stability even in the absence of antibiotic selection.

Genome sequence of the lantibiotic bacteriocin producer Streptococcus salivarius strain K12.

Streptococcus salivarius is a prevalent commensal species of the oropharyngeal tract. S. salivarius strain K12 is an isolate from the saliva of a healthy child, used as an oral probiotic. Here, we report its genome sequence, i.e., the full sequence of the 190-kb megaplasmid pSsal-K12 and a high-quality draft 2.2-Gb chromosomal sequence.

A two-component regulatory system controls autoregulated serpin expression in Bifidobacterium breve UCC2003.

This work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded by serRK, which is believed to control the expression of the ser(2003) locus in Bifidobacterium breve UCC2003. The ser(2003) locus consists of two genes, Bbr_1319 (sagA) and Bbr_1320 (serU), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region of ser(2003), and the probable recognition sequence of SerR was determined by a combinatorial approach of in vitro site-directed mutagenesis coupled to transcriptional fusion and electrophoretic mobility shift assays (EMSAs). The importance of the serRK 2CRS in the response of B. breve to protease-mediated induction was confirmed by generating a B. breve serR insertion mutant, which was shown to exhibit altered ser(2003) transcriptional induction patterns compared to the parent strain, UCC2003. Interestingly, the analysis of a B. breve serU mutant revealed that the SerRK signaling pathway appears to include a SerU-dependent autoregulatory loop.

A conserved two-component signal transduction system controls the response to phosphate starvation in Bifidobacterium breve UCC2003.

This work reports on the identification and molecular characterization of the two-component regulatory system (2CRS) PhoRP, which controls the response to inorganic phosphate (P(i)) starvation in Bifidobacterium breve UCC2003. The response regulator PhoP was shown to bind to the promoter region of pstSCAB, specifying a predicted P(i) transporter system, as well as that of phoU, which encodes a putative P(i)-responsive regulatory protein. This interaction is assumed to cause transcriptional modulation under conditions of P(i) limitation. Our data suggest that the phoRP genes are subject to positive autoregulation and, together with pstSCAB and presumably phoU, represent the complete regulon controlled by the phoRP-encoded 2CRS in B. breve UCC2003. Determination of the minimal PhoP binding region combined with bioinformatic analysis revealed the probable recognition sequence of PhoP, designated here as the PHO box, which together with phoRP is conserved among many high-GC-content Gram-positive bacteria. The importance of the phoRP 2CRS in the response of B. breve to P(i) starvation conditions was confirmed by analysis of a B. breve phoP insertion mutant which exhibited decreased growth under phosphate-limiting conditions compared to its parent strain UCC2003.

Controlled gene expression in bifidobacteria by use of a bile-responsive element.

The promoter activity of the upstream region of the bile-inducible gene betA from Bifidobacterium longum subsp. longum NCC2705 was characterized. DNA fragments were cloned into the reporter vector pMDYAbfB, and the arabinofuranosidase activity was determined under different in vitro conditions. A segment of 469 bp was found to be the smallest operational unit that retains bile inducibility. The reporter activity was strongly affected by the presence of ox gall, cholate, and conjugated cholate, but not by other bile salts and cell-surface-acting compounds. Remarkably, this bile-inducible system was also active in other bifidobacteria containing betA homologs.

Candida cabralensis sp. nov., a yeast species isolated from traditional Spanish blue-veined Cabrales cheese.

Three yeast strains, 1AD8(T), 3AD15 and 3AD23, belonging to a previously unknown yeast species were isolated from two independent batches of the Spanish blue-veined Cabrales cheese, a traditional cheese manufactured without the addition of starter and mould cultures. Physiological characterization revealed that the unknown yeast is not fermentative and does not assimilate lactose; rather it assimilates dl-lactic acid and ethanol, major end products of lactic acid bacteria metabolism in cheese. The novel yeast is anamorphic. Phylogenetic tree reconstruction based on nucleotide sequence comparison of the D1/D2 region of the 26S rRNA gene showed that Pichia terricola and Pichia fermentans are the closest relatives of the unknown species. The name Candida cabralensis sp. nov. is proposed, and the isolate 1AD8(T) (=CECT 13027(T) =CBS 11679(T)) is the type strain of this novel taxon.

Diversity and evolution of the microbial populations during manufacture and ripening of Casín, a traditional Spanish, starter-free cheese made from cow's milk.

Classical culturing and denaturing gradient gel electrophoresis (DGGE) techniques have been used for studying the microbial diversity and dynamics of the traditional Spanish Casín cheese during manufacturing and ripening. As with other starter-free cheeses made from raw milk, the microbial diversity of Casín was shown to be high by both culturing and DGGE analyses. The culture technique showed that lactic acid bacteria (LAB) species constituted the majority of the microbial populations. Of the 14 bacterial species identified, Lactococcus garvieae was predominant in the three-day-old cheese sample, although it was replaced by Lactococcus lactis subsp. lactis at day 30. As expected, the DGGE profiles obtained were complex, consisting, depending on the sample, in five to ten different amplification bands. Among these, a band corresponding to Streptococcus thermophilus was observed throughout the whole manufacturing process. This species had never been identified from traditional Spanish cheeses previously. Culturing and molecular methods showed high populations of undesirable microorganisms, arguing for a required improvement in the hygiene of Casín manufacture. Random amplification of polymorphic DNA (RAPD) profiling suggested that the L. garvieae and L. lactis populations were composed of one and five strains, respectively. In addition, only a single L. lactis RAPD pattern was stably maintained from day three to day 30, indicating high succession of strains along ripening. After a thoroughly characterisation, strains of the two Lactococcus species could be used in designing specific starter cultures for Casín. Additional species (such as Lactobacillus plantarum and Corynebacterium variabile) might be included as adjunct cultures.

Analysis of tetracycline resistance tet(W) genes and their flanking sequences in intestinal Bifidobacterium species.

OBJECTIVES: The tet(W) gene provides tetracycline resistance to a wide range of anaerobic intestinal and ruminal bacteria, but little is known about the molecular organization of the tet(W) gene. The aim of this study was to gain new insights into the molecular organization of the tet(W) gene in bifidobacteria strains from humans. METHODS: A segment of DNA encompassing the whole tet(W) gene and its immediate upstream and downstream sequences was analysed in 10 representative strains of four Bifidobacterium species, of which two have been shown to be tetracycline-susceptible. The non-conserved flanking regions of the tet(W) gene were further analysed in six strains. RESULTS: All 10 strains share a core DNA domain of 2154 bp [starting 250 bp upstream of the tet(W) gene start codon and ending 13 bp before the stop codon] with 98% to 100% DNA identity. Except for Bifidobacterium animalis E43, all other strains further share 408 bp upstream and 70 bp downstream of the tet(W) gene. An insertion-like element of 736 bp was found to interrupt the tet(W) coding sequence in Bifidobacterium longum M21, which may be the reason for its tetracycline susceptibility. However, genetic events explaining the susceptible phenotype of B. longum LMG 13197(T) were not observed. CONCLUSIONS: The tet(W) genes from all 10 strains shared 98% to 100% DNA and amino acid identity, though large variation was found in their flanking regions.

Improved cloning vectors for bifidobacteria, based on the Bifidobacterium catenulatum pBC1 replicon.

This study reports the development of several cloning vectors for bifidobacteria based on the replicon of pBC1, a cryptic plasmid from Bifidobacterium catenulatum L48 thought to replicate via the theta mode. These vectors, in which antibiotic resistance genes encoding either erythromycin or tetracycline resistance acted as selection markers, were able to replicate in a series of eight Bifidobacterium species at frequencies ranging from 4.0 x 10(1) to 1.0 x 10(5) transformants microg(-1) but not in Lactococcus lactis or Lactobacillus casei. They showed a relative copy number of around 30 molecules per chromosome equivalent and a good segregational stability, with more than 95% of the cells retaining the vectors after 80 to 100 generations in the absence of selection. Vectors contain multiple cloning sites of different lengths, and the lacZalpha peptide gene was introduced into one of the molecules, thus allowing the easy selection of colonies harboring recombinant plasmids in Escherichia coli. The functionality of the vectors for engineering Bifidobacterium strains was assessed by cloning and examining the expression of an alpha-l-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in alpha-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.

Functional analysis of the pBC1 replicon from Bifidobacterium catenulatum L48.

To determine the minimal replicon of pBC1 (a 2.5-kb cryptic plasmid of Bifidobacterium catenulatum L48) and to check the functionality of its identified open reading frames (ORFs) and surrounding sequences, different segments of pBC1 were amplified by polymerase chain reaction (PCR) and cloned into pBif, a replication probe vector for bifidobacteria. The largest fragment tested in this manner encompassed most of the pBC1 sequence, while the shortest just included the repB gene and its immediate upstream sequences. Derivatives were all shown to allow replication in bifidobacteria. Surprisingly, both the transformation frequency and segregational stability in the absence of antibiotic selection decreased with reducing plasmid length. The relative copy number of the constructs (ranging from around 3 to 23 copies per chromosome equivalent, as compared to 30 copies for the original pBC1) was shown to be strain dependent and to decrease with reducing plasmid length. These results suggest that, although not essential, the copG-like and orfX-like genes of pBC1 play important roles in pBC1 replication. Interruption of repB produced a construct incapable of replicating in bifidobacteria. The analysis of pBC1 will allow its use in the construction of general and specific cloning vectors.

Acquired macrolide resistance in the human intestinal strain Lactobacillus rhamnosus E41 associated with a transition mutation in 23S rRNA genes.

Restriction fragment length polymorphism and DNA sequencing of polymerase chain reaction (PCR) products showed that a Lactobacillus rhamnosus strain of human origin resistant to macrolides, from which no resistance determinants have been detected by specific PCR and microarray screening, contained a heterozygous A-->G transition mutation at position 2058 (Escherichia coli numbering) of its 23S rRNA genes.

Reagentless identification of human bifidobacteria by intrinsic fluorescence.

A new identification method for bifidobacteria species from the human gastrointestinal tract was developed based on the measurement and statistical analysis of the intrinsic fluorescence of aromatic amino acids (AAA) and nucleic acids (NA), following their excitation at 250 nm. The model was constructed by recording the fluorescence spectra of 53 Bifidobacterium strains of 10 different species, including the corresponding type strains, and validated by analyzing the spectra data from nine further problem strains. Principal components analysis (PCA) and factorial discriminant analysis (FDA) of the results showed the technique to distinguish between the isolates at the species level; the Bifidobacterium pseudolongum subspecies (globosum and pseudolongum) could also be distinguished. The proposed method provides a powerful, inexpensive and convenient means of rapidly identifying intestinal bifidobacteria, which could be of help for large probiotic surveys.

Screening for plasmids among human bifidobacteria species: sequencing and analysis of pBC1 from Bifidobacterium catenulatum L48.

Analysis of 72 bifidobacterial isolates for plasmid DNA identified six different plasmid profiles, two profiles consisted of a single plasmid and four contained at least two. A plasmid identified in a Bifidobacterium catenulatum strain (pBC1) was chosen for further characterization based on its small size and stability. The plasmid was shown to be a circular molecule of 2540 base pairs with an overall G+C content of 64%. At the putative origin of replication a direct repeat of 24 nucleotides repeated three and a half times was observed, as well as five inverted repeats, which resembled the organization of theta-type replicating plasmids. Three open reading frames encoding peptides larger than 50 amino acids were also identified: repB, encoding a replicase of 315 amino acids, a transcriptionally coupled gene (orfX-like), similar to the orfX of some theta-replicating lactococcal plasmids, and copG-like in the complementary strand, which showed a conserved domain present in proteins of the CopG family. Comparison of the deduced RepB protein of pBC1 to other replication proteins in databases, identified pMB1 from Bifidobacterium longum as its closest relative (81% amino acid identity). The pBC1 replicon proved to be functional in several Bifidobacterium species, including B. animalis, B. longum, and B. pseudocatenulatum. Hybridization experiments showed the replicon was uncommon among bifidobacteria. The relative copy number of pBC1 was estimated to be 30.9+/-4.62 by quantitative real-time polymerase chain reaction.

Molecular analysis of tet(W) gene-mediated tetracycline resistance in dominant intestinal Bifidobacterium species from healthy humans.

tet(W) was found responsible for tetracycline resistance (MICs, 4 to > or =32 microg ml(-1)) in dominant bifidobacterial species from the gastrointestinal tracts of healthy humans. The gene from Bifidobacterium longum H66 proved to be identical over a 2.6-kbp region to the recently described tet(W) determinant of Butyrivibrio fibrisolvens.