Influence of in ovo prebiotic and synbiotic administration on meat quality of broiler chickens.

A trial was conducted to evaluate the effect of in ovo injection of prebiotic and synbiotics on growth performance, meat quality traits (cholesterol content, intramuscular collagen properties, fiber measurements), and the presence of histopathological changes in the pectoral muscle (PS) of broiler chickens. On d 12 of incubation, 480 eggs were randomly divided into 5 experimental groups treated with different bioactives, in ovo injected: C, control with physiological saline; T1 with 1.9 mg of raffinose family oligosaccharides; T2 and T3 with 1.9 mg of raffinose family oligosaccharides enriched with different probiotic bacteria, specifically 1,000 cfu of Lactococcus lactis ssp. lactis SL1 and Lactococcus lactis ssp. cremoris IBB SC1, respectively; T4 with commercially available synbiotic Duolac, containing 500 cfu of both Lactobacillus acidophilus and Streptococcus faecium with the addition of lactose (0.001 mg/embryo). Among the hatched chickens, 60 males were randomly chosen (12 birds for each group) and were grown to 42 d in collective cages (n = 3 birds in each 4 cages: replications for experimental groups). Broilers were fed ad libitum commercial diets according to age. In ovo prebiotic and synbiotic administration had a low effect on investigated traits, but depend on the kind of bioactives administered. Commercial synbiotic treatment (T4) reduced carcass yield percentage, and the feed conversion ratio was higher in T3 and T4 groups compared with other groups. The abdominal fat, the ultimate pH, and cholesterol of the PS were not affected by treatment. Broiler chickens of the treated groups with both slightly greater PS and fiber diameter had a significantly lower amount of collagen. The greater thickness of muscle fibers (not significant) and the lower fiber density (statistically significant), observed in treated birds in comparison with those of the C group, are not associated with histopathological changes in the PS of broilers. The incidence of histopathological changes in broiler chickens from examined groups was low, which did not affect the deterioration of meat quality obtained from these birds.

Molecular, physiological and phylogenetic traits of Lactococcus 936-type phages from distinct dairy environments.

Bacteriophage infection of Lactococcus species can cause serious disruption of dairy fermentation processes. The most common isolates from the dairy environment are Siphoviridae lytic 936-type phages. To gain specific knowledge about this group of phages in Polish dairies, we examined 90 isolates from 8 different locations. Based on restriction fragment length polymorphism analysis, coupled with physiological and molecular studies, the isolated phages were divided into 8 distinct groups. Whole-genome sequencing of single representatives from each phage group provided data about their biology and genetic composition. The phages present an overall conserved genome organization. High sequence homology to another Polish isolate, Lactococcus phage bIBB29, indicates their close phylogenetic relatedness to this strain. Such similarity may be suggestive of a general genome conservation among phages persisting in Polish dairies. Comparative genome analyses with other 936-type phages revealed several discriminative traits, including the presence and position of HNH endonuclease genes, varying number of orfs in the early gene region, and a putative TpeX gene. Interestingly, host range of the sequenced phages was restricted to L. lactis subsp. lactis biovar. diacetylactis strains. The results provide new data regarding phages present in the Polish dairy environment and permit analysis of their biology, genome composition and relatedness to other Lactococcus 936-type phages.

Effect of recombinant Lactococcus lactis producing myelin peptides on neuroimmunological changes in rats with experimental allergic encephalomyelitis.

Multiple sclerosis (MS) is a human autoimmune neurodegenerative disease with an unknown etiology. Despite various therapies, there is no effective cure for MS. Since the mechanism of the disease is based on autoreactive T-cell responses directed against myelin antigens, oral tolerance is a promising approach for the MS treatment. Here, the experiments were performed to assess the impact of oral administration of recombinant Lactococcus lactis producing encephalogenic fragments of three myelin proteins: myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein, on neuroimmunological changes in rats with experimental allergic encephalomyelitis (EAE) - an animal model of MS. Lactococcus lactis whole-cell lysates were administered intragastrically at two doses (103 and 106 colony forming units) in a twenty-fold feeding regimen to Lewis rats with EAE. Spinal cord slices were subjected to histopathological analysis and morphometric evaluation, and serum levels of cytokines (IL-1b, IL-10, TNF-α and IFN-γ) were measured. Results showed that administration of the L. lactis preparations at the tested doses to rats with EAE, diminished the histopathological changes observed in EAE rats and reduced the levels of serum IL-1b, IL-10 and TNF-α, previously increased by evoking EAE. This suggests that oral delivery of L. lactis producing myelin peptide fragments could be an alternative strategy to induce oral tolerance for the treatment of MS.

Intestinal MMC-related electric fields and pancreatic juice control the adhesion of Gram-positive and Gram-negative bacteria to the gut epithelium--in vitro study.

The adhesion of six different Lactobacillus and Lactococcus and three pathogenic Escherichia and Salmonella strains was studied using Caco-2 cell line. In this in vitro model system the influence of weak electric field (EF) on bacterial adhesion was tested. The EF source was the in vitro reconstruction of spiking potentials recorded in the duodenum of a healthy calf during one myoelectrical migration complex (MMC) cycle. The ability to adhere to Caco-2 cells of bacteria belonging to two groups, Gram-positive lactobacilli and lactococci, and Gram-negative Escherichia and Salmonella differed considerably. The pathogenic bacteria adhered better to well-differentiated Caco-2 cells whereas lactobacilli and lactococci displayed better adhesion to non-differentiated Caco-2 cells. In the presence of MMC-related EF an increased adhesion of Lactobacillus and Lactococcus but not of Salmonella enterica s. Enteritidis and E. coli 269 to Caco-2 cells was observed. Two later strains adhered even less in the presence of EF. The same tendency was found in the presence of pancreatic juice in a cell medium. In conclusion, the myoelectric component of the small intestinal motility, the MMC-related EF, and pancreatic juice may increase the ability of lactic acid bacteria to adhere to GI epithelial cells, creating better environmental conditions for colonization of the intestine and competition with Gram-negative pathogens.

Mosaic structure of p1658/97, a 125-kilobase plasmid harboring an active amplicon with the extended-spectrum beta-lactamase gene blaSHV-5.

Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum beta-lactamase (ESBL) of the SHV type. The beta-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the blaSHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum beta-lactamase gene blaCTX-M-3.

Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum beta-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla(CTX-M-3), it also bears the bla(TEM-1), aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of beta-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla(CTX-M-3), accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

Preliminary attempts at bacteriocin typing of lactic streptococci.

Preliminary attempts at typing Streptococcus lactis, S. lactis subsp. diacetylactis and Streptococcus cremoris strains by bacteriocins (lactostrepcins) are presented. Among 106 strains used about 85% were sensitive to lactostreptocins. The highest proportion of bacteriocin-typing strains was observed in S. lactis species. Lactostrepcin-sensitive strains could be divided into 6 types. The results confirm some individual features of S. diacetylactis compared with S. lactis.

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis.

A fragment of the Lactococcus lactis chromosome containing an open reading frame of 265 codons, denoted bglR, has been characterized. The polypeptide encoded by bglR shares 36 to 30% sequence identity with a family of regulatory proteins including ArbG from Erwinia chrysanthemi, BglG from Escherichia coli, and SacT and SacY from Bacillus subtilis. These regulatory proteins are involved in positive control of the utilization of different sugars by transcription antitermination. For some of these regulatory proteins it has been demonstrated that antitermination is exerted by binding to a conserved RNA sequence, partially overlapping the transcription terminator and thus preventing transcription termination. Upstream of bglR, we identified a transcription terminator whose 5' end was overlapped by a 32-bp sequence, highly homologous to the RNA-binding site that is conserved in other regulatory systems. Constitutive expression of bglR in E. coli increased the expression of a bglG::lacZ transcriptional fusion. The fact that that the expression of BglG is autoregulated in E. coli suggests that BglG and BglR are functionally equivalent. In L. lactis, we observed that (i) the expression of a bglR::lacZ fusion is increased by beta-glucoside sugars, (ii) disruption of bglR impairs growth on some beta-glucosides, and (iii) the expression of bglR is positively autoregulated. Because of these structural and functional similarities between BglR and the transcription antiterminators of the BglG family, we propose that BglR may be the lactococcal counterpart of the E. coli BglG regulator of beta-glucoside utilization.

Tryptophan biosynthesis genes in Lactococcus lactis subsp. lactis.

The Lactococcus lactis chromosomal region containing the seven structural genes required for tryptophan biosynthesis was characterized by cloning and sequencing. All of the trp genes were identified by the homology of their products with known Trp proteins from other organisms. The identification was confirmed for five genes by their ability to complement trp mutations in Escherichia coli. The seven structural genes are present in the order trpEGDCFBA and span a 7,968-bp segment. Each gene is preceded by a putative ribosome binding site complementary to the 3' end of the L. lactis 16S rRNA. Three pairs of genes (trpG-trpD, trpC-trpF, and trpB-trpA) overlap, and there is intercistronic spacing of 124, 46, and 585 bp between the trpE-trpG, trpD-trpC, and trpF-trpB gene pairs, respectively. No gene fusion was found. Upstream of the trp genes, a 457-bp noncoding DNA segment contains several regions fitting the consensus for gram-positive promoters and one region strongly resembling a transcription terminator. However, it seems unlikely that an attenuation mechanism similar to the one found in E. coli regulates tryptophan biosynthesis in L. lactis, since no potential leader peptide was detected. We propose that a mechanisms resembling that described in Bacillus spp. can regulate trp genes expression in L. lactis.

Lactostrepcins--acid bacteriocins produced by lactic streptococci.

All 47 non-nisin producing strains of Streptococcus lactis and 12/13 strains of Str. lactis subsp. diacetylactis examined produced bacteriocins, for which the term lactostrepcins is suggested. Seven strains of Str. cremoris examined produced no bacteriocins active against 3 lactic streptococci strains used as indicators. The strains examined were divided into 3 groups: I, those producing lactostrepcins active against only one streptomycin resistant mutant of Str. lactis 60 indicator strain; II, those producing lactostrepcins active against all 3 indicator strains; III, those not producing lactostrepcins active against the indicator strains employed. The lactostrepcins were sensitive to various proteolytic enzymes and to phospholipase D, but retained full or partial activity after dialysis. Most of the bacteriocins studied were fully active only within the pH range 4.2--5.0 and were reversibly inactivated at pH 7.0 or 8.0. Results suggested occurrence of 4 different lactostrepcins. The lactostrepcins produced by all group I strains were the same, but there were differences among the lactostrepcins produced by group II strains. Lactostrepcins killed some beta-haemolytic streptococci and some strains of Lactobacillus helveticus. One of the lactostrepcins was also active against certain Leuconostoc strains, but not against other Leuconostoc strains, nor against L. helveticus or other Gram-positive bacteria.

Chromosomal localization of resistance to fosfomycin and aminocyclitol antibiotics in hospital strains of Staphylococcus aureus.

The chromosomal localization of fosfomycin resistance genes in three hospital Staphylococcus aureus strains and in the standard strain NCTC 8507 was shown. Moreover, the chromosomal locus of the gene determining resistance to three aminocyclitol antibiotics: gentamycin, kanamycin and tobramycin in the one of hospital strains was determined. The possibility of transducing this resistance and the absence of plasmid DNA in the obtained transductants, suggest transposomal nature of the gene determining gentamycin, kanamycin and tobramycin resistance in the investigated strain.

Multiple transcriptional control of the Lactococcus lactis trp operon.

The Lactococcus lactis trpEGDCFBA operon is preceded by a noncoding leader region. Transcriptional studies of the trp operon revealed three transcripts with respective sizes of 8 kb (encompassing the entire operon), 290 bases, and 160 bases (corresponding to parts of the leader region). These transcripts most likely result from initiation at the unique Ptrp promoter, transcription termination at either T1 (upstream of the trp operon) or T2 (downstream of the trp operon), and/or processing. Three parameters were shown to differentially affect the amount of these transcripts: (i) following tryptophan depletion, the amount of the 8-kb transcript increases 300- to 500-fold; (ii) depletion in any amino acid increased transcription initiation about fourfold; and (iii) upon entry into stationary phase the amount of the 8-kb transcript decreases abruptly. The tryptophan-dependent transcription control is exerted through transcription antitermination.

Gene inactivation in Lactococcus lactis: branched-chain amino acid biosynthesis.

The Lactococcus lactis subsp. lactis strains isolated from dairy products are auxotrophs for branched-chain amino acids (leucine, isoleucine, and valine), while most strains isolated from nondairy media are prototrophs. We have cloned and sequenced the leu genes from one auxotroph, IL1403. The sequence is 99% homologous to that of the prototroph NCDO2118, which was determined previously. Two nonsense mutations and two small deletions were found in the auxotroph sequence, which might explain the branched-chain amino acid auxotrophy. Nevertheless, the leu genes from the auxotroph appear to be transcribed and regulated similarly to those from the prototroph.