Genome Sequence and Comparative Genome Analysis of Lactobacillus casei: Insights into Their Niche-Associated Evolution

Lactobacillus casei is remarkably adaptable to diverse habitats and widely used in the food industry. To reveal the genomic features that contribute to its broad ecological adaptability and examine the evolution of the species, the genome sequence of L. casei ATCC 334 is analyzed and compared with other sequenced lactobacilli. This analysis reveals that ATCC 334 contains a high number of coding sequences involved in carbohydrate utilization and transcriptional regulation, reflecting its requirement for dealing with diverse environmental conditions. A comparison of the genome sequences of ATCC 334 to L. casei BL23 reveals 12 and 19 genomic islands, respectively. For a broader assessment of the genetic variability within L. casei, gene content of 21 L. casei strains isolated from various habitats (cheeses, n = 7; plant materials, n = 8; and human sources, n = 6) was examined by comparative genome hybridization with an ATCC 334-based microarray. This analysis resulted in identification of 25 hypervariable regions. One of these regions contains an overrepresentation of genes involved in carbohydrate utilization and transcriptional regulation and was thus proposed as a lifestyle adaptation island. Differences in L. casei genome inventory reveal both gene gain and gene decay. Gene gain, via acquisition of genomic islands, likely confers a fitness benefit in specific habitats. Gene decay, that is, loss of unnecessary ancestral traits, is observed in the cheese isolates and likely results in enhanced fitness in the dairy niche. This study gives the first picture of the stable versus variable regions in L. casei and provides valuable insights into evolution, lifestyle adaptation, and metabolic diversity of L. casei

Microtiter Plate Assay for Assessment of Listeria monocytogenes Biofilm Formation

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32°C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses

Synergistic effect of combination of lactococcal phage resistance fragments of pNP40 with cloned abortive infection gene abiD

Two fragments conferring partial phage resistance were located on plasmid pNP40 from Lactococcus lactis ssp. lactis biovar diacetylactis DRC3 and cloned. A 2.3-kb PstI fragment from pNP40 containing abiF conferred partial phage resistance to prolate-headed phage c2, and a 4.8-kb PstI fragment of pNP40 containing abiE conferred partial phage resistance to small isometric-headed phage sk1. When each of the two fragments was cloned individually into a plasmid containing the abortive phage infection gene abiD from L. lactis ssp. lactis KR5, phage resistance was enhanced. When cloned with abiD, the 2.3-kb PstI fragment enhanced the resistance against prolate-headed phages, as was indicated by a 400-fold decrease in the efficiency of plating compared with that of abiD alone. When the 4.8-kb PstI fragment of pNP40 was cloned with abiD, resistance to small isometric-headed phages was enhanced, as was indicated by a greater than 50-fold decrease in efficiency of plating compared with that of abiD alone. The 4.8-kb PstI fragment of pNP40 cloned with abiD showed a large decrease (500- to 1000-fold) in efficiency of plating against prolate-headed phages, even though the 4.8-kb PstI fragment of pNP40 by itself conferred no resistance to the prolate-headed phages

Cloning and characterization of the abortive infection genetic determinant abiD isolated from pBF61 of Lactococcus lactis subsp. lactis KR5.

A 6.3-kb fragment from pBF61 in Lactococcus lactis subsp. lactis KR5 was cloned and found to confer an abortive phage infection (Abi+) phenotype exhibiting a reduction in efficiency of plating and plaque size for small isometric- and prolate-headed bacteriophages sk1 and c2, respectively, and to produce a 10-fold decrease in c2 phage burst size. Phage adsorption was not significantly reduced. An open reading frame of 1,098 bp was sequenced and designated abiD. Tn5 mutagenesis confirmed that abiD was required for the Abi+ phenotype

C5a peptidase alters clearance and trafficking of group A streptococci by infected mice.

Group A streptococcal C5a peptidase (SCPA) specifically cleaves the human serum chemotaxin C5a at the polymorphonuclear leukocyte (PMNL) binding site. This study tested the proposal that SCPA contributes to virulence by retarding the influx of inflammatory cells and clearance of streptococci during the first few hours after infection. To investigate the specific contribution of SCPA to the virulence of group A streptococci, scpA insertion and deletion mutants were created by directed plasmid insertion into scpA and gene replacement. The precise locations of insertion and deletion mutations were confirmed by PCR and DNA sequence analysis. The impact of mutation on virulence was investigated with a mouse air sac model of inflammation. Experiments evaluated clearance of streptococci from the air sac within 4 h after infection. SCPA- streptococci were cleared more efficiently than wild-type bacteria. Localization of streptococci in lymph nodes and spleens of infected mice revealed a significant difference between mutant and wild-type streptococci. PMNLs and other granulocytes that infiltrated the air sac were quantitated by single-color flow cytometry. The total cellular infiltrate was greater and PMNLs dominated the granulocytic infiltrates of air sacs inoculated with SCPA- mutant bacteria. The data obtained are consistent with the possibility that SCPA- streptococci are initially cleared from the site of infection primarily by PMNLs. Moreover, mutant and wild-type streptococci followed different paths of dissemination. SCPA- bacteria were transported to lymph nodes, whereas wild-type streptococci avoided transport to the lymph nodes and rapidly spread to the spleen

Inactivation of Listeria monocytogenes on a polyethylene surface modified by layer-by-layer deposition of the antimicrobial N-halamine

Modification of food contact surfaces to be antimicrobial represents an approach to address the problem of cross-contamination in the food industry. The effect of increasing levels of surface modification on low density polyethylene (LDPE) through application of N-halamines on the inactivation kinetics of Listeria monocytogenes Scott A was evaluated. Increasing levels of modification were applied through layer by layer deposition on LDPE surface (1-5 double layers of polyethyleneimine and poly(acrylic acid)). Surface modification was achieved and confirmed through Fourier Transform Infrared Spectroscopy (FTIR). From I to 5 double layers, the N-halamine content ranged from 3.42 +/- 1.2 to 27.30 +/- 3.5 nmol cm(-2). More than four logarithmic cycles (>99.99%) reduction was reached against L. monocytogenes Scott A after different contact times depending on the level of modification, that varied from 50 to 110 min (from 5 to 2 double layers). Inactivation kinetics followed a sigmoidal behavior. (c) 2013 Elsevier Ltd. All rights reserved

Effects of primers and Taq polymerase on randomly amplified polymorphic DNA analysis for typing Listeria monocytogenes from the environment of a shrimp processing plant

Ninety-nine randomly selected isolates of Listeria monocytogenes obtained during a 5 month sampling period from several processing environment locations in a shrimp processing plant were subjected to RAPD analysis with the use of 4 primers. Preliminary studies indicated that the number of DNA bands and their intensity differed greatly with respect to the commercial source of the Taq polymerase used with individual isolates. 18 composite RAPD types were discerned with the use of the 4 primers. Among these 18 composite RAPD types, type 1 comprised 14 indistinguishable isolates and type 9 comprised 49 indistinguishable isolates. Results indicated that the shrimp processing plant was dominated by these 2 RAPD types, which comprised 63.6% of the 99 randomly selected isolates

Studies on the growth of Escherichia coli O157:H7 strains at 45oC

The objectives of the present report were to examine the ability of 18 strains of Escherichia coli O157:H7 to grow in EC broth at 42.4, 43.5, 44.5, and 45.5°C, and to document the incidence of phenotypic variants present in low numbers that are capable of growth at 45.5°C in EC broth. Among the 18 strains of E. coli O157:H7 studied, only 3 were capable of producing turbid growth with gas formation in EC broth at 45.5°C with 1 × 102 initial CFU/ml. Higher initial densities of CFU resulted in turbid growth and gas formation in EC broth at 45.5° C with all strains. The presence of bile salts #3 in EC broth was found to be inhibitory at 45.5°C. All 18 strains were found to be capable of growth at 45.5°C in nonselective media. The ability of at least one sensitive strain to grow in EC broth at 45.5°C was found to be dependent on the initial number of CFU/ml. Prior growth of cells of a sensitive strain in EC broth at 45.5°C from a cell density of 2.0 × 107 to 8.0 × 107 CFU/ml followed by removal of cells and reinoculation at a cell density of 2.0 × 106 CFU/ml resulted in growth at 45.5°C that did not occur without such conditioning of the inhibitory medium. These results indicate that the ability of most strains of E. coli O157:H7 to grow in EC broth at 45.5°C is dependent on the initial density of CFU and that at low densities of CFU the ability to initiate growth is dependent on either low numbers of phenotypic variants tolerant to the presence of bile salts #3 in EC broth at 45.5°C or to conditioning of the medium with prior elevated numbers of cells