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Melioidosis is a potentially fatal disease caused by the saprophytic bacterium Burkholderia pseudomallei . Resistance to gentamicin is generally a hallmark of B. pseudomallei , and gentamicin is a selective agent in media used for diagnosis of melioidosis. In this study, we determined the prevalence and mechanism of gentamicin susceptibility found in B. pseudomallei isolates from Sarawak, Malaysian Borneo. We performed multilocus sequence typing and antibiotic susceptibility testing on 44 B. pseudomallei clinical isolates from melioidosis patients in Sarawak district hospitals. Whole-genome sequencing was used to identify the mechanism of gentamicin susceptibility. A novel allelic-specific PCR was designed to differentiate gentamicin-sensitive isolates from wild-type B. pseudomallei . A reversion assay was performed to confirm the involvement of this mechanism in gentamicin susceptibility. A substantial proportion (86%) of B. pseudomallei clinical isolates in Sarawak, Malaysian Borneo, were found to be susceptible to the aminoglycoside gentamicin, a rare occurrence in other regions where B. pseudomallei is endemic. Gentamicin sensitivity was restricted to genetically related strains belonging to sequence type 881 or its single-locus variant, sequence type 997. Whole-genome sequencing identified a novel nonsynonymous mutation within amrB , encoding an essential component of the AmrAB-OprA multidrug efflux pump. We confirmed the role of this mutation in conferring aminoglycoside and macrolide sensitivity by reversion of this mutation to the wild-type sequence. Our study demonstrates that alternative B. pseudomallei selective media without gentamicin are needed for accurate melioidosis laboratory diagnosis in Sarawak. This finding may also have implications for environmental sampling of other locations to test for B. pseudomallei endemicity.

In Lactococcus lactis , the interactions between oxidative defense, metal metabolism, and respiratory metabolism are not fully understood. To provide an insight into these processes, we isolated and characterized mutants of L. lactis resistant to the oxidizing agent tellurite (TeO 3 2− ), which generates superoxide radicals intracellularly. A collection of tellurite-resistant mutants was obtained using random transposon mutagenesis of L. lactis . These contained insertions in genes encoding a proton-coupled Mn 2+ /Fe 2+ transport homolog ( mntH ), the high-affinity phosphate transport system ( pstABCDEF ), a putative osmoprotectant uptake system ( choQ ), and a homolog of the oxidative defense regulator spx ( trmA ). The tellurite-resistant mutants all had better survival than the wild type following aerated growth. The mntH mutant was found to be impaired in Fe 2+ uptake, suggesting that MntH is a Fe 2+ transporter in L. lactis . This mutant is capable of carrying out respiration but does not generate as high a final pH and does not exhibit the long lag phase in the presence of hemin and oxygen that is characteristic of wild-type L. lactis . This study suggests that tellurite-resistant mutants also have increased resistance to oxidative stress and that intracellular Fe 2+ can heighten tellurite and oxygen toxicity.

The BspA protein of Lactobacillus fermentum BR11 (BR11) is a cell envelope constituent that is similar to known solute-binding proteins and putative adhesins. BspA is required for L-cystine uptake and oxidative defense and is likely to be an L-cystine-binding protein. The aim of this study was to directly measure L-cystine-BspA binding and BspA expression. De-energized BR11 cells bound radiolabelled L-cystine with a Kd of 0.2 microM. A bspA mutant could not bind L-cystine. L-cystine-BR11 binding was unaffected by large excesses of L-glutamine, L-methionine, or collagen, indicating L-cystine specificity. BR11 and the bspA mutant were identical in their abilities to bind L-cysteine, indicating that L-cysteine is not a BspA ligand. BspA expression levels were deduced from radiolabelled L-cystine binding and it was found that there are 1-2 x 10(5) BspA molecules per cell, and that expression is slightly higher under oxidizing conditions. It is proposed that BspA be renamed CyuC.

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Φadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.

Extended-spectrum β-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-oneKlebsiella pneumoniaeisolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV β-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried theblaSHV-2agene and seven strains carried theblaSHV-12gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBLblaSHV-11gene and one strain carried the non-ESBLblaSHV-1gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to theblaSHV-2aorblaSHV12gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed “first-nucleotide change,” involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carryingblaSHV-2afrom strains carryingblaSHV-12. In addition, this method was used to demonstrate an association between the relative copy numbers ofblaSHVgenes in individual strains and the levels of antibiotic resistance.

BspA is a basic surface-exposed protein from Lactobacillus fermentum BR11. Sequence comparisons have shown that it is a member of family III of the solute binding proteins. It is 89% identical to the collagen binding protein, Cnb, from Lactobacillus reuteri . Compared with the database of Escherichia coli proteins, BspA is most similar to the l -cystine binding protein FliY. To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid. These mutants were significantly impaired in their abilities to take up l -cystine. Uptake rates of l -glutamine, l -histidine, and l -lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected. Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress. Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat. Measurements of sulfhydryl levels showed that incubation of L. fermentum BR11 with l -cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant. The role of BspA as an extracellular matrix protein adhesin was also addressed. L. fermentum BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin. Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin. As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake.

Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from an alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types which we have designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3, and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a, and 6b) thus facilitating high resolution typing of U. urealyticum. Inclusion of the reference strains for serovars 1, 3, 6, and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however this fragment was invariant within the parvo biovar. This study has shown that the sequence of the mba can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.

Summary Chlamydia are considered to be the most important pathogen of koalas in which they cause ocular and urogenital infections. As recently as 1996 it was realised that koala chlamydial infections do not belong to the species Chlamydia psittaci but instead should be reassigned to the species C. pecorum and C. pneumoniae . We have used DNA sequence analysis of part of the chlamydial major outer membrane protein gene, omp A VD4, to compare 15 koala C. pecorum isolates. Unexpectedly, we found that the koala isolates did not cluster as a single branch in the C. pecorum tree, but instead were represented by five genetically very distinct genotypes. Two of the genotypes (which contained five koala isolates each) were koala-specific whereas one genotype contained a single koala isolate plus three sheep and two cattle isolates. For all five koala genotypes, their nearest relatives were not other koala genotypes, but sheep, cattle or pig isolates. It may be inferred from our data that C. pecorum strains infecting koalas do not form a monophyletic group with respect to other C. pecorum strains, and therefore the model which states that there was a single acquisition of a C. pecorum infection by a koala and that all C. pecorum strains now infecting koalas are descended from that founding strain is unlikely to be correct. The most plausible model is that koalas have obtained C. pecorum infections as a result of a series of cross-species transmission events, possibly from pigs and/or ruminants.

Ultra-deep Illumina sequencing was performed on whole genome amplified DNA derived from a Chlamydia trachomatis-positive vaginal swab. Alignment of reads with reference genomes allowed robust SNP identification from the C. trachomatis chromosome and plasmid. This revealed that the C. trachomatis in the specimen was very closely related to the sequenced urogenital, serovar F, clade T1 isolate F-SW4. In addition, high genome-wide coverage was obtained for Prevotella melaninogenica, Gardnerella vaginalis, Clostridiales genomosp. BVAB3 and Mycoplasma hominis. This illustrates the potential of metagenome data to provide high resolution bacterial typing data from multiple taxa in a diagnostic specimen.

A library of Actinomyces naeslundii T14V DNA was constructed in plasmid pUC18 and from this several sucrose-positive clones were isolated. Evidence was obtained that all these clones contained the same gene. One clone, which carried a plasmid that was named pPNG102, was chosen for further study. It was found that the enzyme specified by this plasmid hydrolyzed sucrose, raffinose, inulin and levan, but not dextran, and did not synthesize fructan or glucan from sucrose. The sequence of the insert in pPNG102 was determined and was found to contain a large ORF that specifies a polypeptide of 99 319 Da with similarity to other sucrases. This gene was named levJ. The deduced amino acid (aa) sequence contained both a potential signal sequence and potential C-terminal cell envelope attachment domain. Alignments revealed an internal 331-aa domain not present in other levanases and sucrases. A neighbour-joining tree showed that sucrases of eukaryotic origin form a cluster with eubacterial sucrase/fructanases, and this cluster does not include other eubacterial sucrases. It is postulated that certain eukaryotic sucrase-encoding genes are of eubacterial origin.

Summary: Analysis of the region downstream of the ftf gene of Streptococcus salivarius led to the detection of two open reading frames (ORFs). The deduced amino acid sequences of these ORFs were homologous to proteins encoded by genes not previously described and/or sequenced in Gram-positive bacteria. The deduced amino acid sequence of the first of these (orf2) showed strong homology to the product of the FUR1 gene of Saccharomyces cerevisiae, which codes for a uracil phosphoribosyltransferase. The over-expression of the product of this gene appeared to be the source of the detrimental effect observed with phagemids carrying orf2 in Escherichia coli hosts. The deduced amino acid sequence of the second ORF (orf3) was homologous to the ClpP family of proteases. Examination of the upstream region of the ftf gene led to the discovery of a new insertion sequence-like element which has been designated IS1161.

The glucosyltransferases from oral streptococci cleave sucrose and polymerize the glucose moieties. In Streptococcus salivarius ATCC 25975, two glucosyltransferase-encoding genes, gtfJ and gtfK, are closely linked and transcribed in the same direction. A procedure for the isolation of intact RNA from this organism was devised. The procedure incorporated a high-temperature mutanolysin treatment and selective precipitation by LiCl. The RNA was subject to Northern hybridization and RNase protection assays and it was concluded that the two genes are transcribed separately. A potential factor-independent transcription terminator was located in the intergenic region.

The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli . High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA -based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP. flaA HRM analysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.

Received 3 May 2009; returned 17 June 2009; revised 21 August 2009; accepted 24 August 2009 Background: Extended-spectrum b-lactamases (ESBLs) belonging to the SHV family remain a major cause of ESBL-positive phenotypes in Klebsiella pneumoniae. The blaSHV gene is a normal constituent of the K. pneumoniae chromosome. However, most ESBL-encoding blaSHV genes found in K. pneumoniae are plasmid borne. The objective was to determine the contribution of promoter variants to the expression of plasmid-borne blaSHV genes. Methods: K. pneumoniae clinical isolates were analysed for the presence of IS26 insertions characteristic of plasmid-borne blaSHV, and differences in their blaSHV promoter sequences and expression levels. A high resolution melting (HRM)-based method for rapid promoter analysis was developed. Results :A n IS26 insertion characteristic of the plasmid-borne blaSHV-1/blaSHV-2/blaSHV-5 family was 100% linked to a promoter mutated in the 210 region, a mutation previously only found on the chromosome. The mutation was shown by real-time reverse transcriptase PCR to be associated with increased blaSHV expression. Conclusions: Plasmid-borne blaSHV is associated with strong promoters. It is likely that an SHV-dependent ESBL-positive phenotype requires both a strong promoter and a coding sequence mutation. An HRM assay can indicate blaSHV expression.

Klebsiella pneumoniae isolates frequently contain complex mixtures of bla SHV alleles. A high-resolution melting-based method for interrogating the extended-spectrum activity conferring codon 238 and 240 polymorphisms was developed. This detects minority extended-spectrum β-lactamase-encoding alleles, allows estimation of allele ratios, and discriminates between single and double mutants.

Non-multiresistant methicillin-resistant Staphylococcus aureus (nmMRSA) infections are emerging worldwide and are often community-associated. This prospective case–cohort study compares features of 96 nmMRSA clinical isolates with 96 matched multiresistant MRSA (mMRSA) and 192 matched methicillin-susceptible S. aureus (MSSA) clinical isolates. Seventy-four percent of nmMRSA infections were healthcare-associated. nmMRSA infections were much more likely to involve skin and soft tissue (skin and soft tissue infections; SSTIs) and were much less likely to be treated appropriately with antibiotics than MSSA or mMRSA infections. Panton-Valentine leukocidin (PVL) genes were detected in 55% of nmMRSA, 16% of MSSA and 2% of mMRSA isolates. Independent of the methicillin-resistance phenotype, 59% of PVL-positive SSTIs presented as furunculosis compared to only 10% of PVL-negative SSTIs. Patients with PVLpositive infections were much younger than patients with PVL-negative infections. The proportion of PVL-positive infections peaked in the 10–29 years old age group, followed by a linear decline.

The principal objective of this study was to determine if Campylobacter jejuni genotyping methods based upon resolution optimised sets of single nucleotide polymorphisms (SNPs) and binary genetic markers were capable of identifying epidemiologically linked clusters of chicken-derived isolates. Eighty-eight C. jejuni isolates of known flaA RFLP type were included in the study. They encompassed three groups of ten isolates that were obtained at the same time and place and possessed the same flaA type. These were regarded as being epidemiologically linked. Twenty-six unlinked C. jejuni flaA type I isolates were included to test the ability of SNP and binary typing to resolve isolates that were not resolved by flaA RFLP. The remaining isolates were of different flaA types. All isolates were typed by real-time PCR interrogation of the resolution optimised sets of SNPs and binary markers. According to each typing method, the three epidemiologically linked clusters were three different clones that were well resolved from the other isolates. The 26 unlinked C. jejuni flaA type I isolates were resolved into 14 SNP-binary types, indicating that flaA typing can be unreliable for revealing epidemiological linkage. Comparison of the data with data from a fully typed set of isolates associated with human infection revealed that abundant lineages in the chicken isolates that were also found in the human isolates belonged to clonal complex (CC) -21 and CC-353, with the usually rare C-353 member ST-524 being especially abundant in the chicken collection. The chicken isolates selected to be diverse according to flaA were also diverse according to SNP and binary typing. It was observed that CC-48 was absent in the chicken isolates, despite being very common in Australian human infection isolates, indicating that this may be a major cause of human disease that is not chicken associated.

In Klebsiella pneumoniae , it is common for plasmid-located and chromosome-located bla SHV copies to coexist within single cells. The plasmid-borne genes are mainly derived from two separate IS 26 -mediated mobilizations of bla SHV . The objective of this study was to test the hypothesis that the presence of a non-extended-spectrum β-lactamase (non-ESBL) encoding plasmid-borne form of bla SHV facilitates the cefotaxime (CTX)-mediated selection of ESBL-expressing mutants, even when there is a chromosomal copy of the same gene. Twenty-one diverse ESBL-negative, bla TEM -negative K. pneumoniae clinical isolates were tested for the IS 26 insertions characteristic of the two mobilization events. The isolates were then tested for their ability to be selected for ESBL-mediated CTX resistance by serial subculturing with a doubling of the CTX concentration at every subculture. Fourteen isolates possessed neither of the IS 26 insertions. None of these became ESBL positive, and all died during the course of the experiment, despite possessing chromosomal bla SHV copies. The other isolates all became ESBL positive and grew abundantly up to a CTX concentration of 128 μg/ml. Similar results were obtained with ceftazidime. ESBL expression was associated with the appearance of the expected G→A mutation at position 1 of codon 238 and also with bla SHV copy number amplification. It was concluded that plasmid-borne bla SHV greatly facilitates the selection of ESBL expression, even when the same gene is on the chromosome, and that gene dosage effects are likely to contribute to this phenomenon.

BackgroundSingle nucleotide polymorphisms (SNPs) and genes that exhibit presence/absence variation have provided informative marker sets for bacterial and viral genotyping. Identification of marker sets optimised for these purposes has been based on maximal generalized discriminatory power as measured by Simpson's Index of Diversity, or on the ability to identify specific variants. Here we describe the Not-N algorithm, which is designed to identify small sets of genetic markers diagnostic for user-specified subsets of known genetic variants. The algorithm does not treat the user-specified subset and the remaining genetic variants equally. Rather Not-N analysis is designed to underpin assays that provide 0% false negatives, which is very important for e.g. diagnostic procedures for clinically significant subgroups within microbial species.ResultsThe Not-N algorithm has been incorporated into the "Minimum SNPs" computer program and used to derive genetic markers diagnostic for multilocus sequence typing-defined clonal complexes, hepatitis C virus (HCV) subtypes, and phylogenetic clades defined by comparative genome hybridization (CGH) data forCampylobacter jejuni,Yersinia enterocoliticaandClostridium difficile.ConclusionNot-N analysis is effective for identifying small sets of genetic markers diagnostic for microbial sub-groups. The best results to date have been obtained with CGH data from several bacterial species, and HCV sequence data.

Lactobacillus reuteri inhibits Staphylococcus aureus growth on Baird-Parker agar. This activity required the presence of tellurite and was not shared with other lactic acid bacteria or an L. reuteri mutant defective in cystine metabolism. Secreted products generated from L. reuteri cystine metabolism and thiols were shown to augment tellurite toxicity.

Current treatments for inflammatory bowel disease (IBD) are relatively ineffective. Recently, probiotics have emerged as a potential treatment modality for numerous gastrointestinal disorders, including IBD. Few probiotics, however, have undergone appropriate preclinical screening in vivo. The current study compared the effects of four candidate probiotics on development of dextran sulfate sodium (DSS)-induced colitis in rats. Sprague Dawley rats were gavaged 1 mL of the potential probiotic (1 x 10(10) CFU/mL), or vehicle, twice daily for 14 days. Strains tested were Lactobacillus rhamnosus GG (LGG), Streptococcus thermophilus TH-4 (TH-4), Bifidobacterium lactis Bb12 (Bb12) and Lactobacillus fermentum BR11 (BR11). Colitis was induced from day 7 to 14 via administration of 2% DSS in drinking water. Disease activity index (DAI) was monitored daily until rats were killed at day 14. DAI decreased in DSS+Bb12 and DSS+BR11 compared to DSS+Vehicle. Colon length increased in DSS+BR11 (10%) and DSS+LGG (10%) compared to DSS+Vehicle. DSS+Bb12 and DSS+BR11 prevented the distal colon crypt hyperplasia evident in DSS+Vehicle, DSS+LGG and DSS+TH-4. BR11 was most effective at reducing colitic symptoms. Bb12 had minimal effects, whilst TH-4 did not prevent DSS-colitis and LGG actually exacerbated some indicators of colitis. Further studies into the potential benefits of L. fermentum BR11 are indicated.

Opposing effects of the prebiotic, fructooligosaccharide, have been reported in experimental colitis. We compared the effects of the prebiotic, fructooligosaccharide, alone and in synbiotic combination with Lactobacillus fermentum BR11, on the development of dextran sulfate sodium-induced colitis in rats. Rats consumed an 18 percent casein-based diet or diet supplemented with 6 percent fructooligosaccharide or maltodextrin for 14 days. The synbiotic group was gavaged 1 ml of L. fermentum BR11 (1 × 109 cfu/ml) twice daily. From Days 7 to 14, colitis was induced via 3 percent dextran sulfate sodium in drinking water. Disease activity was assessed daily, and at killing, gastrointestinal organs were measured, weighed, and examined by quantitative histology, proliferating cell nuclear antigen immunohistochemistry, and colonic myeloperoxidase activity. Administration of dextran sulfate sodium resulted in an increased colitic disease activity, and an increased colon and cecum weight compared with normal controls. Colon and cecum weights were further increased in dextran sulfate sodium+fructooligosaccharide (colon: 19 percent; cecum: 48 percent) and dextran sulfate sodium+fructooligosaccharide/L. fermentum BR11-treated rats (16 and 62 percent) compared with dextran sulfate sodium+vehicle-treatment. Dextran sulfate sodium+fructooligosaccharide-treated rats displayed an 81 percent increase in colonic myeloperoxidase activity compared with dextran sulfate sodium-treated controls. Histologic damage severity scores increased in dextran sulfate sodium+vehicle, dextran sulfate sodium+fructooligosaccharide, and dextran sulfate sodium+fructooligosaccharide/L. fermentum BR11-treated rats compared with normal controls (P

The expression and secretion signals of the Sep protein from Lactobacillus fermentum BR11 were used to direct export of two peptidoglycan hydrolases by Lb. fermentum BR11, Lactobacillus rhamnosus GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis MG1363. The production levels, hydrolytic and bacteriocidal activities of the Listeria monocytogenes bacteriophage N-acetylmuramoyl-l-alanine amidase endolysin Ply511 and the glycylglycine endopeptidase lysostaphin were examined. Buffering of the growth media to a neutral pH allowed detection of Ply511 and lysostaphin peptidoglycan hydrolytic activity from all lactic acid bacteria. It was found that purified Ply511 has a pH activity range similar to that of lysostaphin with both enzymes functioning optimally under alkaline conditions. Supernatants from lactobacilli expressing lysostaphin reduced viability of methicillin resistant Staphylococcus aureus (MRSA) by approximately 8 log(10) CFU/ml compared to controls. However, supernatants containing Ply511 were unable to control L. monocytogenes growth. In coculture experiments, both Lb. plantarum and Lb. fermentum synthesizing lysostaphin were able to effectively reduce MRSA cell numbers by >7.4 and 1.7 log(10)CFU/ml, respectively, while lactic acid bacteria secreting Ply511 were unable to significantly inhibit the growth of L. monocytogenes. Our results demonstrate that lysostaphin and Ply511 can be expressed in an active form from different lactic acid bacteria and lysostaphin showed superior killing activity. Lactobacilli producing lysostaphin may have potential for in situ biopreservation in foodstuffs or for prevention of S. aureus infections.

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with “Minimum SNPs” software. “Minimum SNPs” selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity ( D ). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution ( D = 1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli . By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE , the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.

This investigation describes the development of a generally applicable, bioinformatics-driven, single-nucleotide polymorphism (SNP) genotyping assay for the common bacterial gastrointestinal pathogenCampylobacter jejuni. SNPs were identifiedin silicousing the program ‘Minimum SNPs’, which selects for polymorphisms providing the greatest resolution of bacterial populations based on Simpson's index of diversity (D). The high-DSNPs identified in this study were derived from the combinedC. jejuni/Campylobacter colimultilocus sequence typing (MLST) database. Seven SNPs were found that provided aDof 0.98 compared with full MLST characterization, based on 959 sequence types (STs). The seven high-DSNPs were interrogated using allele-specific real-time PCR (AS kinetic PCR), which negates the need for expensive labelled primers or probes and requires minimal assay optimization. The total turnaround time of the SNP typing assay was approximately 2 h. Concurrently, 69C. jejuniisolates were subjected to MLST and flagellin A short variable region (flaASVR) sequencing and combined with a population of 84C. jejuniandC. coliisolates previously characterized by these methods. Within this collection of 153 isolates, 19flaASVR types (D=0.857) were identified, compared with 40 different STs (D=0.939). When MLST andflaASVR sequencing were used in combination, the discriminatory power was increased to 0.959. In comparison, SNP typing of the 153 isolates alone provided aDof 0.920 and was unable to resolve a small number of unrelated isolates. However, addition of theflaASVR locus to the SNP typing procedure increased the resolving power to 0.952 and clustered isolates similarly to MLST/flaASVR. This investigation has shown that a seven-memberC. jejuniSNP typing assay, used in combination with sequencing of theflaASVR, efficiently discriminatesC. jejuniisolates.

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae , while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri . It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum . The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His 6 ). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His 6 and CFTR peptides or His 6 peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.

Extended-spectrum β-lactamases (ESBLs) emerge by point mutation from non-extended-spectrum precursors. The aims of this study were to reveal the basis for variations in resistance levels found in a collection of 21 Klebsiella pneumoniae clinical isolates from Brisbane, Australia. Previous studies have shown that 20 of these isolates possess bla SHV-11 , bla SHV-2a , and/or bla SHV-12 , and there is an association between the copy numbers of the ESBL-encoding genes and resistance levels. In this study, a real-time PCR method for interrogating the polymorphic sites at codons 238 and 240 was developed, and this confirmed the relationship between mutant gene copy numbers and resistance levels. The bla SHV promoter region was cloned from one of the ESBL-expressing isolates, and this showed that bla SHV genes exist downstream of two different promoters within this single isolate. These promoters have both been reported previously, and they differ by virtue of the presence or absence of an IS 26 insertion. The bla SHV copy numbers in cis with the different promoters were measured, and the copy number of the IS 26 promoter was correlated with resistance levels. Cloning and analysis of PCR products showed that different bla SHV variants existed in cis with individual promoters in individual isolates but that mutant genes were more abundant downstream of the IS 26 promoter. There were no ESBL-positive isolates without this promoter. It was concluded that bla SHV in cis with the IS 26 promoter is located on an amplifiable replicon, and the presence of the IS 26 insertion may facilitate the acquisition of an ESBL-positive phenotype.

Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri , Enterococcus faecium , Streptococcus pneumoniae , Streptococcus agalactiae , and Lactobacillus plantarum . The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii . The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum , Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum , L. rhamnosus , and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes . This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.

BspA is an abundant surface protein from Lactobacillus fermentum BR11, and is required for normal cystine uptake. In previous studies, a mutant strain deficient in BspA (L. fermentum PNG201) was found to be sensitive to oxidative stress. In this study, the biochemical basis for this was explored. It was found that under aerobic batch culture conditions in de Mann–Rogosa–Sharpe medium, both L. fermentum BR11 and PNG201 entered stationary phase due to hydrogen peroxide accumulation. However, this took place at a lower optical density for PNG201 than for BR11. Measurements of hydrogen peroxide levels revealed that the BspA mutant strain overproduces this compound. Addition of 6 mM cystine to aerobic cultures was found to prevent hydrogen peroxide production by both the BR11 and PNG201 strains, but lower cystine concentrations depressed hydrogen peroxide production in BR11 more efficiently than in PNG201. Each mole of cystine was able to prevent the production of several moles of hydrogen peroxide by L. fermentum BR11, suggesting that hydrogen peroxide breakdown is dependent upon a thiol that cycles between reduced and oxidized states. It was concluded that peroxide breakdown by L. fermentum BR11 is dependent upon exogenous cystine. It is most probable that the imported l-cystine is catabolized by a cystathionine lyase and then converted into a thiol reductant for a peroxidase.

The region surrounding mecA in methicillin-resistant Staphylococcus aureus (MRSA) is highly variable. We describe an approach for the rapid genotyping of MRSA by assaying for the presence or absence of variable or mobile elements previously shown to be associated with the mecA region.

BspA is a non-covalently anchored cystine-binding protein from Lactobacillus fermentum BR11. It has previously been used to present antigens derived from infectious organisms on the L. fermentum BR11 cell surface. In this study, the capacity of BspA to present a very large polypeptide was tested. A temperature sensitive plasmid was constructed that encodes a 175-kDa chimeric protein consisting of a fusion between BspA and an N-terminally truncated derivative of the Streptococcus salivarius ATCC 25975 glucosyltransferase GtfJ. This plasmid was introduced into the L. fermentum genome. Integrants were able to incorporate 20–40 nmol sucrose derived glucose into glucan per ml culture per optical density unit. The glucosyltransferase activity was external to the cytoplasmic membrane and bound to the cell. Unlike native BspA, the BspA–GtfJ fusion could not be removed from the cell by 5 M LiCl wash.

The basic surface protein, BspA, has been used as a fusion partner to direct peptide antigens from the human immunodeficiency virus gp41 protein and the Chlamydia psittaci OmpA protein to the cell surface of Lactobacillus fermentum BR11. BspA has potential utility in the construction of live vaccines and diagnostic reagents.

Fractionation of the culture medium showed that Streptococcus salivarius ATCC 25975 secreted a glucosyltransferase (Gtf) that was primer independent. On the basis of this observation, a gene library of S. salivarius chromosomal DNA cloned into lambda L47.1 was screened for a gene(s) coding for such an activity. As a result of this screening process, two new gtf genes, gtfL and gtfM, both of which coded for primer-independent Gtf activities, were isolated. GtfL produced an insoluble glucan that was refractory to digestion by the endo-(1-->6)-alpha-D-glucanase. of Chaetonium gracile, while GtfM produced a soluble glucan that was readily degraded by the glucanase. Comparison of the deduced amino acid sequences of gtfL and gtfM with 10 other available Gtf sequences allowed the relatedness of the conserved catalytic regions to be assessed. This analysis showed that the 12 enzymes did not form clusters based on their primer dependencies or on their product solubilities. Further analysis of the YG repeats in the C-terminal glucan-binding domains of GtfJ, GtfK, GtfL, and GtfM from S. salivarius showed that there was strong homology between a block of contiguous triplet YG repeats present in the four alleles. These blocks of YG repeats were coded for by a region of each gene that appeared to have arisen as a result of a recent duplication event(s).

Summary: Many strains of oral streptococci secrete glucosyltransferases (GTFs) that polymerize sucrose into glucans that form an integral part of the plaque matrix on the tooth surface. Recently, we reported the cloning of two closely linked GTF-encoding genes (gtfJ and gtfK) from Streptococcus salivarius ATCC 25975 as well as the sequence of gtfJ, which encodes a primer-dependent GTF that synthesizes an insoluble product (a GTF-I). In this communication we report the sequence of gtfK, which encodes a primer-dependent GTF that synthesizes a soluble product (a GTF-S), as well as the sequence of a small downstream open reading frame of unknown function. The deduced sequence of GtfK was compared with those of seven other streptococcal Gtfs and an unrooted phylogenetic tree constructed. This analysis suggested that Gtfs with similar product specificities do not form phylogenetic clusters and was consistent with currently accepted phylogenetic schemes. The tree was tested by constructing a series of “sub-trees” from different blocks of the alignment. Evidence was obtained for recombination events involving gtfB and gtfC from S. mutans GS-5, gtfJ and gtfK from S. salivarius, as well as the gtfI genes from S. downei and S. sobrinus. The recombination events between gtfB and gtfC, and between the two gtfI genes, were confirmed by examining divergences at silent sites.

The ftf gene, coding for the cell-bound beta-D-fructosyltransferase (FTF) of Streptococcus salivarius ATCC 25975, has been analyzed, and its deduced amino acid sequence has been compared with that of the secreted FTF of Streptococcus mutans and the levansucrases (SacBs) of Bacillus species. A unique proline-rich region detected at the C terminus of the FTF of S. salivarius preceded a hydrophobic terminal domain. This proline-rich region was shown to possess strong homology to the product of the prgC gene from pCF10 in Enterococcus faecalis, which encodes a pheromone-responsive protein of unknown function, as well as homology to the human proline-rich salivary protein PRP-4. A series of 3'-OH deletions of the S. salivarius ftf gene expressed in Streptococcus gordonii Challis LGR2 showed that the C terminus was required for cell surface attachment in this heterologous organism, as only the complete gene product was cell bound. This cell-bound activity was released in the presence of sucrose, suggesting that the mode of attachment and release of the S. salivarius FTF in S. gordonii was similar to that in its native host.

The oral micro-organism Streptococcus salivarius ATCC 25975 synthesizes extracellular glucosyltransferases (GTFs) which polymerize the glucose moiety of sucrose into glucan polymers. Two separate genes encoding the activities of a GTF-I (a GTF that synthesizes an insoluble product) and a GTF-S (a GTF that synthesizes soluble product) were cloned into bacteriophage lambda L47.1. The inserts in the lambda-clones were characterized by restriction mapping and Southern hybridization and were found to overlap, implying that the two genes lay very close to one another on the S. salivarius chromosome. Both genes were subcloned into phagemid vector pIBI30 where they were expressed at a high level. The GTF-I-encoding gene was named gtfJ and the GTF-S-encoding gene, gtfK. Nucleotide sequencing showed that gtfJ and most probably gtfK were closely related to the gtf genes of the mutans streptococci. Sequence alignment also indicated that gtfK lay very close to and downstream from gtfJ, and that both were transcribed in the same direction.

The phs mutation of Escherichia coli has been suggested to affect the Na+/H+ antiport (D. Zilberstein, E. Padan, and S. Schuldiner, FEBS Lett. 168:327-330, 1980). We have recently shown that the mutation affects the rpoA gene and thus affects transcription. The extent of the pleiotropy of the phs mutation was investigated. In addition to the previously reported growth defect on L-glutamate and melibiose, the mutation also affects at least two other metabolic systems. The transport and metabolism of arabinose is impaired and the transport of sulfate is reduced. The extent to which the effects of the phs mutation on metabolism are due to a defect in the Na+/H+ antiport was investigated, and no causal role for this transport system in the metabolic defects was found.

Extraction of Lactobacillus fermentum BR11 cells with 5 M LiCl yielded a preparation containing a single predominant polypeptide with an apparent molecular mass of 32 kDa. A clone encoding an immunoreactive 32-kDa polypeptide was isolated from a pUC18 library of L. fermentum BR11 DNA by screening with an antiserum raised against whole cells of L. fermentum BR11. Sequence determination of the insert in the clone revealed a complete 795-bp open reading frame (ORF) that defines a 28,625-Da polypeptide (BspA). N-terminal sequencing of the LiCl-extracted polypeptide from L. fermentum BR11 confirmed that it is the same as the cloned BspA. BspA was found to have a sequence similar to those of family III of the bacterial solute-binding proteins. The sequences of two ORFs upstream of bspA are consistent with bspA being located in an operon encoding an ATP-binding cassette-type uptake system. Unusually, BspA contains no lipoprotein cleavage and attachment motif (LXXC), despite its origin in a gram-positive bacterium. Biotin labelling and trypsin digestion of whole cells indicated that this polypeptide is exposed on the cell surface. The isoelectric point as predicted from the putative mature sequence is 10.59. It was consequently hypothesized that the positively charged BspA is anchored by electrostatic interaction with acidic groups on the cell surface. It was shown that BspA could be selectively removed from the surface by extraction with an acidic buffer, thus supporting this hypothesis.

Chlamydial infection in koalas causes conjunctivitis, rhinitis, urinary tract disease and female infertility. The chlamydial strains responsible have previously been designated as Chlamydia psittaci and have been subdivided into two types, I and II. In the present study we determined the DNA sequence of a 530 bp segment of the omp 2 gene from seven koala chlamydial strains, the type strain of C. pecorum, and the horse strain N16, and compared these with previously reported omp 2 gene sequences from 10 other chlamydial strains. Omp 2 sequence data clearly separated the seven koala strains into two genetic groups. Koala omp 2 group A corresponds to the previous type I designation and includes a conjunctival strain and a systemic disease-causing strain. The koala group A omp 2 sequence is 99% similar to the human C. pneumoniae IOL-207 strain and also 99% similar to the horse strain, N16. We propose that these koala strains be referred to as koala C. pneumoniae in future. Koala omp 2 group B corresponds to the previous koala type II designation and includes three urogenital tract strains plus two ocular strains. The koala group B omp 2 sequence is 99% similar to the C. pecorum type strain, VR628, while being only 77% similar to the EAE C. psittaci strain and only 71% similar to the koala group A strains. We propose that these koala strains be referred to as koala C. pecorum in future. The fact that two different species of Chlamydia infect koalas reinforces the observation that chlamydial species are not host restricted and makes us rethink the epidemiology of chlamydial disease in this marsupial.