BMC Microbiology - Latest Articles

Topological analysis of a haloacid permease of a Burkholderia sp. bacterium with a PhoA-LacZ reporter

Yuk Man Tse — 2009-10-31T00:00:00Z

Background: 2-Haloacids can be found in the natural environment as degradative products of natural and synthetic halogenated compounds. They can also be generated by disinfection of water and have been shown to be mutagenic and to inhibit glyceraldehyde-3-phosphate dehydrogenase activity. We have recently identified a novel haloacid permease Deh4p from a bromoacetate-degrading bacterium Burkholderia sp. MBA4. Comparative analyses suggested that Deh4p is a member of the Major Facilitator Superfamily (MFS), which includes thousands of membrane transporter proteins. Members of the MFS usually possess twelve putative transmembrane segments (TMS). Deh4p was predicted to have twelve TMS. In this study we characterized the topology of Deh4p with a PhoA-LacZ dual reporters system. Results: Thirty-six Deh4p-reporter recombinants were constructed and expressed in E. coli. Both PhoA and LacZ activities were determined in these cells. Strength indices were calculated to determine the locations of the reporters. The results mainly agree with the predicted model. However, two of the TMS were not verified. This lack of confirmation of the TMS, using a reporter, has been reported previously. Further comparative analysis of Deh4p has assigned it to the Metabolite:H+ Symporter (MHS) 2.A.1.6 family with twelve TMS. Deh4p exhibits many common features of the MHS family proteins. Deh4p is apparently a member of the MFS but with some atypical features. Conclusions: The PhoA-LacZ reporter system is convenient for analysis of the topology of membrane proteins. However, due to the limitation of the biological system, verification of some of the TMS of the protein was not successful. The present study also makes use of bioinformatic analysis to verify that the haloacid permease Deh4p of Burkholderia sp. MBA4 is a MFS protein but with atypical features.

Evolutionary relationships among salivarius streptococci as inferred from multilocus phylogenies based on 16S rRNA-encoding, recA, secA, and secY gene sequences

Jean-Francois Pombert — 2009-10-30T00:00:00Z

Background: Streptococci are divided into six phylogenetic groups, i.e., anginosus, bovis, mitis, mutans, pyogenic, and salivarius, with the salivarius group consisting of only three distinct species. Two of these species, Streptococcus salivarius and Streptococcus vestibularis, are members of the normal human oral microflora whereas the third, Streptococcus thermophilus, is found in bovine milk. Given that S. salivarius and S. vestibularis share several physiological characteristics, in addition to inhabiting the same ecosystem, one would assume that they would be more closely related to each other than to S. thermophilus. However, the few phylogenetic trees published so far suggest that S. vestibularis is more closely related to S. thermophilus. To determine whether this phylogenetic relationship is genuine, we performed phylogenetic inferences derived from secA and secY, the general secretion housekeeping genes, recA, a gene from a separate genetic locus that encodes a major component of the homologous recombinational apparatus, and 16S rRNA-encoding gene sequences using other streptococcal species as outgroups. Results: The maximum likelihood (ML) and maximum parsimony (MP) phylogenetic inferences derived from the secA and recA gene sequences provided strong support for the S. vestibularis/S. thermophilus sister-relationship, whereas 16S rRNA-encoding and secY-based analyses could not discriminate between alternate topologies. Phylogenetic analyses derived from the concatenation of these sequences unambiguously supported the close affiliation of S. vestibularis and S. thermophilus. Conclusions: Our results corroborated the sister-relationship between S. vestibularis and S. thermophilus and the concomitant early divergence of S. salivarius at the base of the salivarius lineage.

Environmental stresses inhibit splicing in the aquatic fungus Blastocladiella emersonii

Raphaela Georg — 2009-10-29T00:00:00Z

Background: Exposure of cells to environmental stress conditions can lead to the interruption of several intracellular processes, in particular those performed by macromolecular complexes such as the spliceosome. Results: During nucleotide sequencing of cDNA libraries constructed using RNA isolated from B. emersonii cells submitted to heat shock and cadmium stress, a large number of ESTs with retained introns was observed. Among the 6,350 ESTs obtained through sequencing of stress cDNA libraries, 181 ESTs presented putative introns (2.9%), while sequencing of cDNA libraries from unstressed B. emersonii cells revealed only 0.2% of ESTs containing introns. These data indicate an enrichment of ESTs with introns in B. emersonii stress cDNA libraries. Among the 85 genes corresponding to the ESTs that retained introns, 19 showed more than one intron and three showed three introns, with intron length ranging from 55 to 333 nucleotides. Canonical splicing junctions were observed in most of these introns, junction sequences being very similar to those found in introns from genes previously characterized in B. emersonii, suggesting that inhibition of splicing during stress is apparently a random process. Confirming our observations, analyses of gpx3 and hsp70 mRNAs by Northern blot and S1 protection assays revealed a strong inhibition of intron splicing in cells submitted to cadmium stress. Conclusion: In conclusion, data indicate that environmental stresses, particularly cadmium treatment, inhibit intron processing in B. emersonii, revealing a new adaptive response to cellular exposure to this heavy metal.

Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in Korea

Moon Her — 2009-10-29T00:00:00Z

Background: A Brucella eradication program has been executed in Korea. To effectively prevent and control brucellosis, a molecular method for genetic identification and epidemiological trace-back must be established. As part of that, the MLVA typing assay was evaluated and applied to B. abortus isolates for analyzing the characteristics of the regional distribution and relationships of foreign isolates. Results: A total of 177 isolates originating from 105 cattle farms for the period 1996 to 2008 were selected as representatives for the nine provinces of South Korea. A dendrogram of strain relatedness was constructed in accordance with the number of tandem repeat units for 17 loci so that it was possible to trace back in the restricted areas. Even in a farm contaminated by one source, however, the Brucella isolates showed an increase or decrease in one TRs copy number at some loci with high DI values. Moreover, those 17 loci was confirmed in stability via in-vitro and in-vivo passage, and found to be sufficiently stable markers that can readily identify the inoculated strain even if minor changes were detected. In the parsimony analysis with foreign Brucella isolates, domestic isolates were clustered distinctively, and located near the Central and Southern American isolates. Conclusions: The MLVA assay has enough discrimination power in the Brucella species level and can be utilized as a tool for the epidemiological trace-back of the B. abortus isolates. But it is important to consider that Brucella isolates may be capable of undergoing minor changes at some loci in the course of infection or in accordance with the changes of the host.

Staphylococcus aureus biofilm formation at the physiologic glucose concentration depends on the S. aureus lineage

Sander Croes — 2009-10-28T00:00:00Z

Background: Since bacteria embedded in biofilms are far more difficult to eradicte than planktonic infections, it would be useful to know whether certain Staphylococcus aureus lineages are especially involved in strong biofilm formation. For this reason, in vitro biofilm formation of 228 clinical S. aureus isolates of distinct clonal lineages was investigated. Results: At 0.1% glucose, more than 60% of the S. aureus strains associated with multilocus sequence typing (MLST) clonal complex (CC)8 produced large amounts of biomass, compared to 0-7% for various other clonal lineages. Additionally, S. aureus bloodstream isolates associated with MLST CC8 and CC7 had similar biofilm forming capacities as their commensal counterparts. Furthermore, strong biofilm formation could not be attributed to a specific accessory gene regulator (agr) genotype, as suggested previously. The agr genotypes were strictly associated with the clonal lineages. Moreover, strong biofilm formation was not related to slime formation. Congo red agar (CRA) screening is therefore not useful as a qualitative screening method for biofilm formation. Conclusions: The adherence to polystyrene surfaces under physiologic glucose concentration (0.1%) was dependent on the clonal lineage. Strains associated with MLST CC8 were markedly more often classified as strong biofilm former at glucose concentrations of 0%, 0.1% and 0.25%.The present study reveals that the MLST CC8 associated genetic background was a predisposing factor for strong biofilm formation in vitro, under all tested glucose concentrations.

Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms

Jae-Gyu Jeon — 2009-10-28T00:00:00Z

Background: The association of specific bioactive flavonoids and terpenoids with fluoride can modulate the development of cariogenic biofilms by simultaneously affecting the synthesis of exopolysaccharides (EPS) and acid production by Streptococcus mutans, which enhanced the cariostatic effectiveness of fluoride in vivo. In the present study, we further investigated whether the biological actions of combinations of myricetin (flavonoid), tt-farnesol (terpenoid) and fluoride can influence the expression of specific genes of S. mutans within biofilms and their structural organization using real-time PCR and confocal fluorescence microscopy. Results: Twice-daily treatment (one-minute exposure) during biofilm formation affected the gene expression by S. mutans both at early (49-h) and later (97-h) stages of biofilm development. Biofilms treated with combination of agents displayed lower mRNA levels for gtfB and gtfD (associated with exopolysaccharides synthesis) and aguD (associated with S. mutans acid tolerance) than those treated with vehicle-control (p<0.05). Furthermore, treatment with combination of agents markedly affected the structure-architecture of S. mutans biofilms by reducing the biovolume (biomass) and proportions of both EPS and bacterial cells across the biofilm depth, especially in the middle and outer layers (vs. vehicle-control, p<0.05). The biofilms treated with combination of agents were also less acidogenic, and had reduced amounts of extracellular insoluble glucans and intracellular polysaccharides than vehicle-treated biofilms (p<0.05). Conclusion: The data show that the combination of naturally-occurring agents with fluoride effectively disrupted the expression of specific virulence genes, structural organization and accumulation of S. mutans biofilms, which may explain the enhanced cariostatic effect of our chemotherapeutic approach.

Response of Bacillus cereus ATCC 14579 to challenges with sublethal concentrations of enterocin AS-48

Maria Grande Burgos — 2009-10-28T00:00:00Z

Background: Enterocin AS-48 is produced by Enterococcus faecalis S48 to compete with other bacteria in their environment. Due to its activity against various Gram positive and some Gram negative bacteria it has clear potential for use as a food preservative. Here, we studied the effect of enterocin AS-48 challenges on vegetative cells of Bacillus cereus ATCC 14579 by use of transcriptome analysis. Results: Of the 5200 genes analysed, expression of 24 genes was found to change significantly after a 30 min treatment with a subinhibitory bacteriocin concentration of 0.5 ug/ml. Most of up-regulated genes encode membrane-associated or secreted proteins with putative transmembrane segments or signal sequences, respectively. One operon involved in arginine metabolism was significantly downregulated. The BC4206-BC4207 operon was found to be the most upregulated target in our experiments. BC4206 codes for a PadR type transcriptional regulator, while BC4207 codes for a hypothetical membrane protein. The operon structure and genes are conserved in B. cereus and B. thuringiensis species, but are not present in B. anthracis and B. subtilis. Using real-time qPCR, we show that these genes are upregulated when we treated the cells with AS-48, but not upon nisin treatment. Upon overexpression of BC4207 in B. cereus, we observed an increased resistance against AS-48. Expression of BC4207 in B. subtilis 168, which lacks this operon also showed increased resistance against AS-48. Conclusion: BC4207 membrane protein is directly involved in the resistance mechanism of B. cereus cells against AS-48.

Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and Titanium amplicon pyrosequencing and metagenomic approaches.

Randall Wolcott — 2009-10-27T00:00:00Z

Background: Approximately 1 out of every 100 individuals has some form of venous insufficiency, which can lead to chronic venous disease and Venous Leg Ulcer (VLU). There are known underlying pathologies which contribute to the chronic nature of VLU including biofilm phenotype infections. Results: Using pyrosequencing based approaches we evaluated VLU to characterize their microbial ecology. Results show that VLU infections are polymicrobial with no single bacterium colonizing the wounds. The most ubiquitous and predominant organisms include a previously uncharacterized bacteroidales, various anaerobes, Staphylococcus, Corynebacterium, and Serratia. Topological analysis of VLU show some notable differences in bacterial populations across the surface of the wounds highlighting the importance of sampling techniques during diagnostics. Metagenomics provide a preliminary indication that there may be protozoa, fungi and possibly an undescribed virus associated with these wounds. Conclusion: The polymicrobial nature of VLU and previous research on diabetic foot ulcers and surgical site infections suggest that the future of therapy for such wounds lies in the core of the logical and proven multiple concurrent strategy approach, which has been termed "biofilm-based wound care" and the use of individualized therapeutics rather than in a single treatment modality.

Isolation of Cronobacter spp. (formerly Enterobacter sakazakii) from infant food, herbs and environmental samples and the subsequent identification and confirmation of the isolates using biochemical, chromogenic assays, PCR and 16S rRNA sequencing

Ziad Jaradat — 2009-10-27T00:00:00Z

Background: Cronobacter spp. (formerly Enterobacter sakazakii), are a group of Gram-negative pathogens that have been implicated as causative agents of meningitis and necrotizing enterocolitis in infants. The pathogens are linked to infant formula; however, they have also been isolated from a wide range of foods and environmental samples. Results: In this study, 233 samples of food, infant formula and environment were screened for the presence of Cronobacter spp. in an attempt to find its source. Twenty nine strains were isolated from samples of spices, herbs, infant foods, and dust obtained from household vacuum cleaners. Among the 76 samples of infant food, infant formula, milk powder and non-milk dairy products tested, only one sample of infant food contained Cronobacter spp. (1.4%). The other Cronobacter spp. isolates recovered include two from household vacuum dust, and 26 from 67 samples of herbs and spices. Among the food categories analyzed, herbs and spices harbored the highest number of isolates, indicating plants as a possible reservoir of this pathogen. Initial screening with API 20E test strips yielded 42 presumptive isolates. Further characterization using 3 chromogenic media (alpha-MUG, DFI and EsPM) and 8 sets of PCR primers detecting ITS (internal transcribed spacer sequences), 16S rRNA, zpx, gluA, gluB, OmpA genes followed by nucleotide sequencing of some PCR amplicons did not confirm the identity of all the isolates as none of the methods proved to be free of both false positives or false negatives. The final confirmation step was done by 16S rRNA sequence analysis identifying only 29 of the 42 isolates as Cronobacter spp. Conclusion: Our studies showed that Cronobacter spp. are highly diverse and share many phenotypic traits with other Enterobacteriaceae members highlighting the need to use several methods to confirm the identity of this pathogen. None of the biochemical, chromogenic or PCR primers proved to be a reliable method for confirmation of the identity of the isolates as all of them gave either false positives or false negatives or both. It is therefore concluded that 16S rRNA sequencing is pivotal to confirm the identity of the isolates.

Classification of Myoviridae bacteriophages using protein sequence similarity

Rob Lavigne — 2009-10-26T00:00:00Z

Background: We advocate unifying classical and genomic classification of bacteriophages by integration of proteomic data and physicochemical parameters. Our previous application of this approach to the entirely sequenced members of the Podoviridae fully supported the current phage classification of the International Committee on Taxonomy of Viruses (ICTV). It appears that horizontal gene transfer generally does not totally obliterate evolutionary relationships between phages. Results: CoreGenes/CoreExtractor proteome comparison techniques applied to 102 Myoviridae suggest the establishment of three subfamilies (Peduovirinae, Teequatrovirinae, the Spounavirinae) and eight new independent genera (Bcep781, BcepMu, FelixO1, HAP1, Bzx1, PB1, phiCD119, and phiKZ-like viruses). The Peduovirinae subfamily, derived from the P2-related phages, is composed of two distinct genera: the "P2-like viruses", and the "HP1-like viruses". At present, the more complex Teequatrovirinae subfamily has two genera, the "T4-like" and "KVP40-like viruses". In the genus "T4-like viruses" proper, four groups sharing >70% proteins are distinguished: T4-type, 44RR-type, RB43-type, and RB49-type viruses. The Spounavirinae contain the "SPO1-"and "Twort-like viruses." Conclusion: The hierarchical clustering of these groupings provide biologically significant subdivisions, which are consistent with our previous analysis of the Podoviridae.