Lactic acid bacteria isolated from rye sourdoughs produce bacteriocin-like inhibitory substances active against Bacillus subtilis and fungi.

AIM: To screen five strains of lactic acid bacteria (LAB) isolated from rye sourdoughs for the potential production of antimicrobial substances. METHODS AND RESULTS: Lactobacillus sakei KTU05-06, Pediococcus acidilactici KTU05-7, Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 isolated from rye sourdoughs were investigated for the production of bacteriocin-like inhibitory substances (BLIS). The supernatants of analysed LAB inhibited growth of up to 15 out of 25 indicator bacteria strains as well as up to 25 out of 56 LAB strains isolated from rye sourdoughs. Moreover, these five LAB were active against ropes-producing Bacillus subtilis and the main bread mould spoilage causing fungi -Aspergillus, Fusarium, Mucor and Penicillium. Lactobacillus sakei KTU05-6 demonstrated the best antibacterial properties and is resistant towards heat treatment even at 100°C for 60 min. CONCLUSIONS: The use of LAB-producing antibacterial substances may be a good choice as a co-starter culture to ensure the stability of sourdoughs and to avoid the bacterial and fungi spoilage of the end product. SIGNIFICANCE AND IMPACT OF THE STUDY: The antimicrobial compounds designated as sakacin KTU05-6, pediocin KTU05-8 KTU05-9, KTU05-10 and AcKTU05-67 were not identical to any other known BLIS, and this finding leads up to the assumption that they might be the novel.

Dietary gluten reduces the number of intestinal regulatory T cells in mice.

It is well established that gluten-free diet reduces the incidence of type 1 diabetes mellitus (T1D) in non-obese diabetic (NOD) mice, though the mechanism is not known. However, regulatory T cells (Treg) are likely to play an important role. Also, it is known that dietary gluten induces an intestinal increase in the bacterium Lactococcus garvieae, but the importance of this phenomenon for T1D development is doubtful. Our hypothesis is that gluten is responsible for mediating its effect on T1D through the influence on Treg development independent of gluten-induced Lactococci. Four groups of female NOD and BALB/c mice of 3 week old were fed either a gluten-free diet or a standard diet. Lactococcus garvieae or saline water was administered per oral to one of each dietary group. Spleen and Peyer's patches were sampled from BALB/c mice for flow cytometric monitoring of IL-10 and Treg. NOD mice were diagnosed diabetic with blood glucose level >12 mmol/l. Dietary gluten significantly decreased the occurrence of Tregs by 10-15% (P < 0.05) in mice compared with those fed a standard diet. These results and the diabetes incidence were independent of the gluten-induced bacterial factor Lactococci. The prevalence of Treg was 5- to 10-fold more abundant in the Peyer's patches than in the spleen (P < 0.001). In conclusion, dietary gluten has a significant negative quantitative impact on the generation of Treg in mice, independent of gluten-induced Lactococcus garvieae, and Treg are far more abundant in Peyer's patches than in the spleen.

Enhanced surveillance of infectious diseases : the 2006 FIFA World Cup experience, Germany.

The 2006 FIFA World Cup was held in 12 German cities between 9 June and 9 July 2006. We identified a need to accelerate and sensitise the pre-existing surveillance system for infectious diseases in order to timely detect adverse health events during the World Cup. Enhanced surveillance, based on Germany's pre-existing system of mandatory notifications was conducted between 7 June and 11 July 2006 in the 12 World Cup cities by: accelerating frequency of electronic data transmission of case-definition based notifiable diseases from weekly to daily transmission, additional reporting of non-case definition-based infectious disease events, lay and expert press screening and intensifying communication between all stakeholders of the surveillance system. Median delay of notification data transmission from the community to the federal level was reduced from three days to one day. The enhanced reporting system detected a norovirus outbreak in the International Broadcast Centre in Munich with 61 epidemiologically linked cases within the first week after onset, as well as four single cases related to the World Cup, two of them with relevance for the International Health Regulations. After the World Cup, all surveillance stakeholders agreed that communication between local, state and federal levels had improved considerably. Unlike the majority of health planners of previous mass gatherings in the last decade we did not introduce syndromic surveillance. Nevertheless, enhancement of infectious disease surveillance successfully detected adverse health events in a timely manner during the FIFA World Cup. Additionally, it provided a valuable communication and networking exercise for potentially critical health-related events. We recommend continuing daily notification data transmission for routine infectious disease surveillance in Germany.

Enhanced Surveillance of Infectious Diseases : the 2006 FIFA World Cup experience, Germany.

The 2006 FIFA World Cup was held in 12 German cities between 9 June and 9 July 2006. We identified a need to accelerate and sensitise the pre-existing surveillance system for infectious diseases in order to timely detect adverse health events during the World Cup. Enhanced surveillance, based on Germany's pre-existing system of mandatory notifications was conducted between 7 June and 11July 2006 in the 12 World Cup cities by: accelerating frequency of electronic data transmission of case-definition based notifiable diseases from weekly to daily transmission, additional reporting of non-case definition-based infectious disease events, lay and expert press screening and intensifying communication between all stakeholders of the surveillance system. Median delay of notification data transmission from the community to the federal level was reduced from three days to one day. The enhanced reporting system detected a norovirus outbreak in the International Broadcast Centre in Munich with 61 epidemiologically linked cases within the first week after onset, as well as four single cases related to the World Cup, two of them with relevance for the International Health Regulations. After the World Cup, all surveillance stakeholders agreed that communication between local, state and federal levels had improved considerably. Unlike the majority of health planners of previous mass gatherings in the last decade we did not introduce syndromic surveillance. Nevertheless, enhancement of infectious disease surveillance successfully detected adverse health events in a timely manner during the FIFA World Cup. Additionally, it provided a valuable communication and networking exercise for potentially critical health-related events. We recommend continuing daily notification data transmission for routine infectious disease surveillance in Germany.

Detection of lactococcal 936-species bacteriophages in whey by magnetic capture hybridization PCR targeting a variable region of receptor-binding protein genes.

AIMS: To develop PCR assays able to distinguish between groups within lactococcal 936-species bacteriophages, as defined by their different receptor-binding protein (RBP) genes. METHODS AND RESULTS: DNA sequences of RBP genes from 17 lactococcal bacteriophages of the 936-species were compared, and six phage groups were identified. For each phage group a specific primer pair targeting a variable region of the RBP genes was designed. In nine of 20 whey samples, from dairies with recorded phage problems, between one and six phage groups were identified by conventional PCR. The sensitivity and specificity of the method was improved by magnetic capture hybridization (MCH)-PCR using a capture probe targeting an 80-bp highly conserved region just upstream from the RBP gene in all the investigated phages. The MCH-PCR was performed on 100 microl whey samples and the detection limit of the assay was 10(2)-10(3) PFU ml(-1) as opposed to the detection limit of 10(4) PFU ml(-1) for conventional PCR performed on 1-microl whey samples. CONCLUSIONS: In this study, PCR assays have been developed to detect six different types of RBP genes in lactococcal 936-species bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR assays have practical applications at cheese plants for detection of 936-species phages with different RBP and thereby potentially with different host ranges. This knowledge will make it possible to improve starter culture rotation systems in the dairy industry.

Characterization and purification of acidocin CH5, a bacteriocin produced by Lactobacillus acidophilus CH5.

AIMS: To characterize and to purify a bacteriocin produced by Lactobacillus acidophilus strain with its activity restricted to Gram-positive bacteria. METHODS AND RESULTS: Native acidocin CH5, a bacteriocin produced by L. acidophilus CH5 an isolate from a dairy starter culture forms in MRS (Oxoid, Basingstoke, UK) broth high-molecular weight aggregates which can dissociate into smaller units (retained by 5 kDa membrane) with higher activity. Acidocin CH5 was purified using combinations of chromatographic methods based on hydrophobic and cation exchange principles and the N-terminal region was sequenced. CONCLUSIONS: Based on our results it is evident that acidocin CH5 belongs, according to bacteriocin classification, to the class II bacteriocins with identical N-terminal amino acid sequence described in the literature previously. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided further data on bacteriocin acidocin CH5 from class II with wide spectrum of antimicrobial activity atypical for bacteriocins produced by L. acidophilus sharing the same homology.

Improvement of phage defence in Lactococcus lactis by introduction of the plasmid encoded restriction and modification system LlaAI.

AIMS: To study the ability of the plasmid-encoded restriction and modification (R/M) system LlaAI to function as a bacteriophage resistance mechanism in Lactococcus lactis during milk fermentations. METHODS AND RESULTS: Plasmid pAIcat4, carrying the R/M system LlaAI and a chloramphenicol resistance cassette, was introduced into the plasmid-free strain L. lactis MG1614 and the industrial strain L. lactis 964. By measuring changes in conductivity the influence of different phage on the growth was determined. CONCLUSIONS: The plasmid-encoded R/M system LlaAI significantly improves the bacteriophage resistance of L. lactis during milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: It is essential to determine the potential of a phage defence mechanism in L. lactis starter culture strains during growth in milk before steps are taken to improve starter cultures. This study shows that LlaAI is useful for improvement of starter cultures.

The ability of the plasmid-encoded restriction and modification system LlaBIII to protect Lactococcus lactis against bacteriophages.

AIMS: To investigate the potential of the plasmid-encoded restriction and modification (R/M) system LlaBIII to protect Lactococcua lactis against bacteriophages during milk fermentations. METHODS AND RESULTS: The R/M system LlaBIII on plasmid pJW566 was cloned with a chloramphenicol cassette, resulting in plasmid pJK1. When introduced into L. lactis strains, pJK1 conferred increased phage resistance against the three most common lactococcal phage species 936, c2, and P335 and three unclassified industrial phages. The growth of the strains in RSM was not affected by the presence of plasmid pJK1. CONCLUSIONS: The plasmid-encoded R/M system LlaBIII has great ability to protect L. lactis strains against bacteriophages in milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evaluates the ability of the LlaBIII R/M system to function as a phage defence mechanism which is an essential step prior to considering utilizing it for improving starter cultures.

The LlaGI restriction and modification system of Lactococcus lactis W10 consists of only one single polypeptide.

The naturally occurring 12.1-kb plasmid, pEW104, in Lactococcus lactis ssp. cremoris W10 was found to confer decreased bacteriophage sensitivity to its host. Plasmid pEW104 encodes a non-classic restriction and modification (R/M) system, named LlaGI, consisting of only one single polypeptide. Analysis of the amino acid sequence revealed the presence of a catalytic motif and seven helicase-like motifs (DEAD-box motifs) characteristic of type I and III endonucleases, followed by four conserved methylase motifs characteristic of adenine-methylases. A comparison between LlaGI and the very similar R/M system, LlaBIII, suggests that the C-terminal region of LlaGI, apparently containing no known motifs, could possibly specify target DNA recognition. Conceivably, the LlaGI gene is included in the operon of the plasmid replication machinery. Finally, it is proposed that LlaGI represents a variant of the type I R/M systems.

Characterization of a novel plasmid-encoded HsdS subunit, S.LlaW12I, from Lactococcus lactis W12.

A novel type I restriction-modification specificity subunit, S. LlaW12I, has been identified on the naturally occurring 8.0-kb plasmid pAW122 in the lactic acid bacterium Lactococcus lactis subsp. cremoris W12. Presence of the HsdS protein together with a complete type I restriction-modification system conferred increased phage restriction to the host, indicating exchange of specificity subunits. Sequence analysis showed that the S.LlaW12I subunit is most probably of type IC. Presumably, the hsdS gene is organized together with the repB gene on one transcriptional unit.

TPW22, a lactococcal temperate phage with a site-specific integrase closely related to Streptococcus thermophilus phage integrases.

The temperate phage TPW22, induced from Lactococcus lactis subsp. cremoris W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on L. lactis subsp. cremoris 3107, which could also be lysogenized by site-specific integration. The attachment site (attP), 5'-TAAGGCGACGGTCG-3', of phage TPW22 was present on a 7.5-kb EcoRI fragment, a 3.4-kb EcoRI-HindIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (int). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing attP and int was constructed, and integration in L. lactis subsp. cremoris MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages phiLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the phiLC3 lactococcal integrase with known Streptococcus thermophilus integrases.

The antimicrobial activity of acidocin CH5 in MRS broth and milk with added NaCl, NaNO3 and lysozyme.

The ability of acidocin CH5, a bacteriocin from Lactobacillus acidophilus CH5 in the form of neutralized and heated supernatant, to prevent the growth of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30 alone or together with other antimicrobials was investigated. The inhibitory activity of acidocin CH5 was higher in MRS broth than in reconstituted skim milk (RSM). In MRS broth and RSM, 1.92 and 32 AU acidocin CH5/ml, respectively, caused 97 and 89% inhibition of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30. The presence of 5 and 10% milk fat in RSM decreased the inhibitory activity of acidocin CH5 to 20 and 11%, respectively. The inhibitory activity of acidocin CH5 was also reduced in the presence of NaCl, NaNO3 and lysozyme. In RSM the inhibition was weaker with both acidocin CH5 and NaCl added compared with NaCl alone. In MRS broth the inhibition was stronger with both acidocin CH5 and NaCl added compared with NaCl alone. The inhibition of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30 was stronger with both NaNO3 and acidocin CH5 in MRS broth (but not in RSM) than with only NaNO3 present, but the strongest level was obtained with acidocin CH5 alone. Addition of acidocin CH5 and more than 30 mg/ml lysozyme to MRS broth increased the level of inhibition above the level obtained by acidocin CH5 alone. The indicator Lactobacillus delbrueckii subsp. lactis LTI 30 was also sensitive to NaCl, NaNO3 and lysozyme in both MRS broth and RSM.

Cloning and characterization of the lactococcal plasmid-encoded type II restriction/modification system, LlaDII.

The LlaDII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp. cremoris W39. A 2.4-kb PstI-EcoRI fragment inserted into the Escherichia coli-L. lactis shuttle vector pCI3340 conferred to L. lactis LM2301 and L. lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The LlaDII endonuclease was partially purified and found to recognize and cleave the sequence 5'-GC decreases NGC-3', where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu4HI. Sequencing of the 2.4-kb PstI-EcoRI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the LlaDII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the LlaDII and Bsp6I R/M systems are identical. Both methylases have two recognition sites (5'-GCGGC-3' and 5'-GCCGC-3') forming a putative stemloop structure spanning part of the presumed -35 sequence and part of the intervening region between the -35 and -10 sequences. Alignment of the LlaDII and Bsp6I methylases with other m5C methylases showed that the protein primary structures possessed the same organization.

Characterization of LlaCI, a new restriction-modification system from Lactococcus lactis subsp. cremoris W15.

The genes encoding the restriction-modification (R/M) system LlaCI have been found on the naturally occurring 7.0 kb plasmid pAW153 in L. lactis subsp. cremoris W15. The R/M system was isolated on a chloramphenicol resistant derivative of the wild type plasmid (pAW153cat). Plasmid pAW153cat and a 2.4 kb HincII-SphI fragment cloned into a high- and a low-copy vector conferred decreased sensitivity in L. lactis LM2301 and L. lactis SMQ86 against small isometric-headed phages of the 936 or P335 species, respectively. Increased plasmid copy number enhanced the level of phage restriction. Sequencing the 2.4 kb HincII-SphI fragment revealed two open reading frames arranged convergently with a 94 bp separation. IlaCIM showed 66% identity to hindIIIM, and IlaCIR showed 45% identity to hindIIIR. The organization of the LlaCI operon differs from the HindIII operon, where the endonuclease and methylase genes overlap and are transcribed in the same direction. The LlaCI methylase is predicted to be 296 amino acids long, with 63% identity to the HindIII methylase, while the LlaCI endonuclease is predicted to consist of 324 or 332 amino acids, depending on the position of the start codon. It shows 24% identity to the HindIII endonuclease.

Determination of the recognition sequence of the type II restriction endonuclease, LlaCI, from Lactococcus lactis W15.

A new type II restriction endonuclease, called LlaCI, was partially purified from Lactococcus lactis subsp. cremoris W15. The characterisation of the LlaCI endonuclease showed it to be an isoschizomer of HindIII, recognising the sequence 5-'A decreases AGCTT-3'. The cleavage site is indicated by the arrow.

Replication regions of two pairs of incompatible lactococcal theta-replicating plasmids.

Incompatibility tests were performed employing 12 replicons belonging to a family of homologous lactococcal theta-replicating plasmids. Two pairs of incompatible plasmids were found, namely, pFV1001 and pFV1201, and pJW565 and pFW094. The replicons of plasmids pFV1001, pFV1201, pJW565, pJW566, and pFW094 were sequenced. Alignments were made of the replicational origins (repA) and putative replication proteins (RepB) of these and 11 related plasmid sequences. Comparison of the alignments with the incompatibility data indicated that the incompatibility determinant could be contained within the 22-bp tandem repeats DRII and/or the inverted repeat IR1 in repA. In support, the incompatibility determinant of pJW563 was localized to a 743-bp fragment encompassing repA. A stretch of 13 amino acids of RepB was proposed to be responsible for the plasmid-specific initiation of replication. This stretch is part of a domain containing features that are highly conserved within the proposed DNA binding regions of the initiation proteins from several well-characterized plasmids from Gram-negative bacteria, including pSC101, R6K, and mini-F.

Cloning and analysis of the restriction-modification system LlaBI, a bacteriophage resistance system from Lactococcus lactis subsp. cremoris W56.

The genes coding for the type II restriction-modification (R/M) system LlaBI, which recognized the sequence 5'-C decreases TRYAG-3', have been cloned from a plasmid in Lactococcus lactis subsp. cremoris W56 and sequenced. The DNA sequence predicts an endonuclease of 299 amino acids (33 kDa) and a methylase of 580 amino acids (65 kDa). A 4.0-kb HindIII fragment in pSA3 was able to restrict bacteriophages, showing that the cloned R/M system can function as a phage defense mechanism in L. lactis.

Characterization of the replicon from the lactococcal theta-replicating plasmid pJW563.

The replication region of the lactococcal plasmid pJW563 was localized to a 2.3-kb EcoRI fragment. This DNA fragment was sequenced ans a 1155-bp open reading frame, repB563, encoding a putative protein RepB563 of 385 amino acids was found. An AT-rich noncoding region, repA563, was found upstream of repB563. This segment included several direct and inverted repeats. A downstream 591-bp open reading frame, ORF X, which was not necessary for replication, was putatively translationally coupled to repB563, RepB563 supplied in trans could support replication of a plasmid containing repA563 and a truncated repB563. This observation suggests that RepB563 is a trans-acting replication protein, and repA563 the cis-acting origin of replication, repA563, repB563, and the beginning of ORF X showed high homology to similar regions in a family of lactococcal theta-replicating plasmids. The repA DNA sequences and the RepB amino acid sequences of the plasmids were aligned and the consensus sequences generated. The comparison revealed highly conserved areas among this family of plasmids. In addition, variable domains emerged, presumably having a plasmid specific function, pVS40 and pC1305 were plasmids with replication proteins showing high homology to RepB563. Despite this homology, replication from repA563 could not be supported by the pVS40 or pC1305 replication protein supplied in trans. Likewise the pJW563 protein could not support replication from the pVS40 origin. pJW563 was found to be compatible with the pVS40 and pC1305 replicons. The results indicate that pJW563 belongs to the widespread family of lactococcal theta-replicating pladmids. Despite the high homology between their replicons, the interaction between the replication origin and the protein is highly specific in many cases rendering the plasmids compatible.

Restriction-modification systems in Lactococcus lactis.

Several restriction-modification (R-M) systems have been identified in Lactococcus lactis. Most of the systems have been plasmid encoded and function as phage-resistance mechanisms. At least five different type-II R-M systems, LlaAI, LlaBI, LlaCI, LlaDI and LlaEI, were identified in isolates from a mixed Cheddar starter culture. LlaAI and LlaBI recognized the DNA sequences 5'- decreases GATC-3' and 5'-C decreases TRYAG-3', respectively. The genes coding for the LlaAI and LlaBI R-M systems have been cloned and sequenced. The LlaAI R-M system had two genes coding for methyltransferases (MTases) and one gene coding for a restriction endonuclease (ENase). The MTases showed high homology to the MTases from DpnII. The LlaBI R-M system had one gene coding for a MTase and one gene coding for an ENase.