BMC Microbiology - Latest Articles

Sequence analysis for detection of first-line drug resistance in Mycobacterium tuberculosis strains from a high-incidence setting

Silke Feuerriegel — 2012-05-30T00:00:00Z

Background: Drug resistance displays a problem for the therapy of Mycobacterium tuberculosis infections. For molecular resistance testing, it is essential to have precise knowledge on genomic variations involved in resistance development. However, data from high-incidence settings are only sparely available. Therefore we performed a systematic approach and analyzed a total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone for mutations in katG, rpoB, rrs, rpsL, gidB, embB, pncA and where applicable in inhA and ahpC. Of the strains investigated 50 were either mono- or poly-resistant to isoniazid, rifampin, streptomycin, ethambutol and pyrazinamide or MDR and 47 fully susceptible strains served as controls. Results: The majority of isoniazid and rifampin resistant strains had mutations in katG315 (71.9%) and rpoB531 (50%). However, rpoB mutations in codons 511, 516 and 533 were also detected in five rifampin susceptible strains. MIC determinations revealed low-level rifampin resistance for those strains. Thus, the sensitivity and specificity of sequencing of katG for detection of drug resistance were 86.7% and 100% and for sequencing of rpoB 100% and 93.8%, respectively. Strikingly, none of the streptomycin resistant strains had mutations in rrs, but 47.5% harboured mutations in rpsL. Further changes were detected in gidB. Among ethambutol resistant strains 46.7% had mutations at embB306. Pyrazinamide resistant strains displayed a variety of mutations throughout pncA. The specificities of sequencing of rpsL, embB and pncA for resistance detection were high (96-100%), whereas sensitivities were lower (48.8%, 73.3%, 70%). Conclusions: Our study reveals a good correlation between data from molecular and phenotypic resistance testing in this high-incidence setting. However, the fact that particular mutations in rpoB are not linked to high-level resistance is challenging and demonstrates that careful interpretation of molecular resistance assays is mandatory. In addition, certain variations, especially in gidB, appear to be phylogenetically informative polymorphisms rather than markers for drug resistance.

H. pylori clinical isolates have diverse babAB genotype distributions over different topographic sites of stomach with correlation to clinical disease outcomes

Shew-Meei Sheu — 2012-05-30T00:00:00Z

Background: Intragenomic recombination between babA and babB mediates antigenic variations and may help H. pylori colonization. This study determined whether variable genotypes of babA and babB correlate to different clinical disease outcomes, and can distribute over the different gastric niches. Results: This study enrolled 92 clinical strains (45 from peptic ulcer, 27 from gastritis, and 20 from gastric cancer) to detect whether the babA and babB are at locus A or B by PCR reactions using the primers designed from the upstream and variable region of the babA and babB genes. Four genotypes of babA and babB (A B, AB B, A AB, AB AB) were found. The distribution of the 4 genotypes in 92 clinical strains was significantly different among patients with different gastric diseases (p < 0.05). The isolates from gastric cancer patients had a higher rate of AB AB genotype than those from non-cancer patients 40.0% vs. 9.7%, p< 0.05). The AB AB genotype was associated with a higher intensity of ntestinal metaplasia (p < 0.05), but did not correlate with a higher inflammation and colonization density in gastric histology (p > 0.05). Besides, the study enrolled 19 patients to verify whether variable genotypes of babAB existed in the different gastric niches. Among the patients infected with more than one babAB genotypes over antrum and corpus, there were higher rate of genotypes as A B or AB AB in isolates from antrum than in those from corpus (75.0 % vs. 16.7%, p <0.05). Conclusions: The H. pylori isolate with the AB AB genotype correlates with an increased gastric cancer risk, and colonize in an antrum predominant manner.

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains

Vanya Paralanov — 2012-05-30T00:00:00Z

Background: Ureaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars. Results: We determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.750.78 Mbp genomes and UUR serovars were 0.840.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level. Conclusions: Comparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The 4 overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that 6 ureaplasmas exist as quasispecies rather than as stable serovars in their native environment. Therefore, differentialpathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.

Gene expression profiling of Mycobacterium avium subsp. paratuberculosis in simulated multi-stress conditions and within THP-1 cells reveals a new kind of interactive intramacrophage behaviour

Andrea Cossu — 2012-05-30T00:00:00Z

Background: Recent studies have identified in Mycobacterium avium subsp. paratuberculosis (MAP), already known as a pathogen in ruminants, a potential zoonotic agent of some autoimmune diseases in humans. Therefore, considering the possible risk for public health, it is necessary a thorough understanding of MAP's gene expression during infection of human host as well as the identification of its immunogenic and/or virulence factors for the development of appropriate diagnostic and therapeutic tools. Results: In order to characterize MAP's transcriptome during macrophage infection, we analyzed for the first time the whole gene expression of a human derived strain of MAP in simulated intraphagosomal conditions and after intracellular infection of the human macrophage cell line THP-1 by using the DNA-microarray technology. Results showed that MAP shifts its transcriptome to an adaptive metabolism for an anoxic environment and nutrient starvation. It up-regulates several response factors to oxidative stress or intracellular conditions and allows, in terms of transcription, a passive surface peptidoglycan spoliation within the macrophage along with an intensification of the anabolic activity for lipidic membrane structures. Conclusions: These results indicate a possible interactive system between MAP and its host cell based on the internal mimicry unlike other intracellular pathogens, bringing new hypothesis in the virulence and pathogenicity of MAP and its importance in human health.

A homeobox protein Phx1 regulates long-term survival and meiotic sporulation in Schizosaccharomyces pombe

Ji-Yoon Kim — 2012-05-30T00:00:00Z

Background: In the fission yeast Schizosaccharomyces pombe, the phx1+ (pombe homeobox) gene was initially isolated as a multi-copy suppressor of lysine auxotrophy caused by depletion of copper/zinc-containing superoxide dismutase (CuZn-SOD). Overproduction of Phx1 increased the synthesis of homocitrate synthase, the first enzyme in lysine biosynthetic pathway, which is labile to oxidative stress. Phx1 has a well conserved DNA-binding domain called homeodomain at the N-terminal region and is predicted to be a transcription factor in S. pombe. However, its role has not been revealed in further detail. Here we examined its expression pattern and the phenotype of its null mutant to get clues on its function. Results: Fluorescence from the Phx1-GFP expressed from a chromosomal fusion gene demonstrated that it is localized primarily in the nucleus, and is distinctly visible during the stationary phase. When we replaced the N-terminal homeobox domain of Phx1 with the DNA binding domain of Pap1, a well-characterized transcription factor, the chimeric protein caused the elevation of transcripts from Pap1-dependent genes such as ctt1+ and trr1+, suggesting that Phx1 possesses transcriptional activating activity when bound to DNA. The amount of phx1+ transcripts sharply increased as cells entered the stationary phase and was maintained at high level throughout the stationary phase. Nutrient shift down to low nitrogen or carbon sources caused phx1+ induction during the exponential phase, suggesting that cells need Phx1 for maintenance function during nutrient starvation. The delta-phx1 null mutant showed decreased viability in long-term culture, whereas overproduction of Phx1 increased viability. Decrease in long-term survival was also observed for delta-phx1 under N- or C-starved conditions. In addition, delta-phx1 mutant was more sensitive to various oxidants and heat shock. When we examined sporulation of the delta-phx1/delta-phx1 diploid strain, significant decrease in the formation of meiotic spores was observed. Conclusions: Phx1 is a transcriptional regulator whose synthesis is elevated during stationary phase and by nutrient starvation in S. pombe. It supports long-term survival and stress tolerance against oxidation and heat, and plays a key role in the formation of meiotic spores.

A fur-like protein PerR regulates two oxidative stress response related operons dpr and metQIN in streptococcus suis

Tengfei Zhang — 2012-05-30T00:00:00Z

Background: Metal ions are important micronutrients in cellular metabolism, but excess ions that cause toxic reactive oxygen species are harmful to cells. In bacteria, Fur family proteins such as Fur, Zur and PerR manage the iron and zinc uptake and oxidative stress responses,respectively. The single Fur-like protein (annotated as PerR) in Streptococcus suis has been demonstrated to be involved in zinc and iron uptake in previous studies, but the reports on oxidative stress response and gene regulation are limited. Results: In the present study, the perR gene deletion mutant [increment]perR was constructed in Streptococcus suis serotype 2 strain SC-19, and the mutant strain [increment]perR exhibited less sensitivity to H2O2 stress compared to the wild-type. The dpr and metQIN were found to be upregulated in the [increment]perR strain compared with SC-19. Electrophoretic mobility shift assays showed that the promoters of dpr and metQIN could be bound by the PerR protein. These results suggest that dpr and metQIN are members of the PerR regulon of S. suis. dpr encodes a Dps-like peroxide resistance protein, and the dpr knockout strains ([increment]dpr and [increment]dpr[increment]perR) were highly sensitive to H2O2. MetQIN is a methionine transporter, and the increased utilization of methionine in the [increment]perR strain indirectly affected the peroxide resistance. Using a promoter-EGFP genefusion reporting system, we found that the PerR regulon was induced by H2O2, and the induction was modulated by metal ions. Finally, we found that the pathogenicity of the perRmutant was attenuated and easily cleared by mice. Conclusions: These data strongly suggest that the Fur-like protein PerR directly regulates dpr and metQIN and plays a crucial role in oxidative stress response in S. suis.

Proteomic profiling of Rhizobium tropici PRF 81: Identification of conserved and specific responses to heat stress

Douglas Gomes — 2012-05-30T00:00:00Z

Background: Rhizobium tropici strain PRF 81 (= SEMIA 4080) has been used in commercial inoculants for application to common-bean crops in Brazil since 1998, due to its high efficiency in fixing nitrogen, competitiveness against indigenous rhizobial populations and capacity to adapt to stressful tropical conditions, representing a key alternative to application of N-fertilizers. The objective of our study was to obtain an overview of adaptive responses to heat stress of strain PRF 81, by analyzing differentially expressed proteins when the bacterium is grown at 28degreesC and 35degreesC. Results: Two-dimensional gel electrophoresis (2DE) revealed up-regulation of fifty-nine spots that were identified by MALDI-TOF/TOF-TOF. Differentially expressed proteins were associated with the functional COG categories of metabolism, cellular processes and signaling, information storage and processing. Among the up-regulated proteins, we found some related to conserved heat responses, such as molecular chaperones DnaK and GroEL, and other related proteins, such as translation factors EF-Tu, EF-G, EF-Ts and IF2. Interestingly, several oxidative stress-responsive proteins were also up-regulated, and these results reveal the diversity of adaptation mechanisms presented by this thermotolerant strain, suggesting a cross-talk between heat and oxidative stresses. Conclusions: Our data provide valuable protein-expression information relevant to the ongoing genome sequencing of strain PRF 81, and contributes to our still-poor knowledge of the molecular determinants of the thermotolerance exhibited by R. tropici species

Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests

Vicky Jespers — 2012-05-30T00:00:00Z

Background: The vaginal microbiome plays an important role in urogenital health. Quantitative real time Polymerase Chain Reaction (qPCR) assays for the most prevalent vaginal Lactobacillus species and bacterial vaginosis species G. vaginalis and A. vaginae exist, but qPCR information regarding variation over time is still very limited. We set up qPCR assays for a selection of seven species and defined the temporal variation over three menstrual cycles in a healthy Caucasian population with a normal Nugent score. We also explored differences in qPCR data between these healthy women and an 'at risk' clinic population of Caucasian, African and Asian women with and without bacterial vaginosis (BV), as defined by the Nugent score. Results: Temporal stability of the Lactobacillus species counts was high with L. crispatus counts of 10*8 copies/mL and L. vaginalis counts of 10*6 copies/mL. We identified 2 types of 'normal flora' and one 'BV type flora' with latent class analysis on the combined data of all women. The first group was particularly common in women with a normal Nugent score and was characterized by a high frequency of L. crispatus, L. iners, L. jensenii, and L. vaginalis and a correspondingly low frequency of L. gasseri and A. vaginae. The second group was characterized by the predominance of L. gasseri and L. vaginalis and was found most commonly in healthy Caucasian women. The third group was commonest in women with a high Nugent score but was also seen in a subset of African and Asian women with a low Nugent score and was characterized by the absence of Lactobacillus species (except for L. iners) but the presence of G. vaginalis and A. vaginae. Conclusions: We have shown that the quantification of specific bacteria by qPCR contributes to a better description of the non-BV vaginal microbiome, but we also demonstrated that differences in populations such as risk and ethnicity also have to be taken into account. We believe that our selection of indicator organisms represents a feasible strategy for the assessment of the vaginal microbiome and could be useful for monitoring the microbiome in safety trials of vaginal products.

Multi-locus variable number tandem repeat analysis of 7th pandemic Vibrio cholerae

Connie Lam — 2012-05-24T00:00:00Z

Background: Seven pandemics of cholera have been recorded since 1817, with the current and ongoingpandemic affecting almost every continent. Cholera remains endemic in developing countriesand is still a significant public health issue. In this study we use multilocus variable numberof tandem repeats (VNTRs) analysis (MLVA) to discriminate between isolates of the 7thpandemic clone of Vibrio cholerae. Results: MLVA of six VNTRs selected from previously published data distinguished 66 V. choleraeisolates collected between 1961-1999 into 60 unique MLVA profiles. Only 4 MLVA profilesconsisted of more than 2 isolates. The discriminatory power was 0.995. Phylogenetic analysisshowed that, except for the closely related profiles, the relationships derived from MLVAprofiles were in conflict with that inferred from Single Nucleotide Polymorphism (SNP)typing. The six SNP groups share consensus VNTR patterns and two SNP groups containedisolates which differed by only one VNTR locus. Conclusions: MLVA is highly discriminatory in differentiating 7th pandemic V. cholerae isolates andMLVA data was most useful in resolving the genetic relationships among isolates withingroups previously defined by SNPs. Thus MLVA is best used in conjunction with SNP typing in order to best determine the evolutionary relationships among the 7th pandemic V.cholerae isolates and for longer term epidemiological typing.

LNA probes substantially improve detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization.

Natarajan Priya — 2012-05-24T00:00:00Z

Background: Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci. Results: In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe. Conclusion: By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.