BMC Microbiology - Latest Articles

Proteins involved in difference of sorbitol fermentation rates of the toxigenic and nontoxigenic Vibrio cholerae El Tor strains revealed by comparative proteome analysis

Ruibai Wang — 2009-07-09T00:00:00Z

Background: The nontoxigenic V. cholerae El Tor strains ferment sorbitol faster than the toxigenic strains, hence fast-fermenting and slow-fermenting strains are defined by the sorbitol fermentation test. This test has been used for more than 40 years in cholera surveillance and strain analysis in China. Understanding the mechanisms of sorbitol metabolism in toxigenic and nontoxigenic strains may help to explore the genome and metabolism divergence in these strains. Here we used comparative proteome analysis to find the proteins which may be involved in such metabolic difference. Results: We found that the production of formate and lactic acid in the sorbitol fermentation media of the nontoxigenic strain was earlier than in the toxigenic strain. We compared the protein expression profiles of the toxigenic strain N16961 and nontoxigenic strain JS32 cultured in sorbitol fermentation media, by using fructose fermentation media as the control. Seventy-three differential protein spots were found and further identified by MALDI-MS. The difference in product of fructose-specific IIA/FPR component gene and mannitol-1-P dehydrogenase, may be involved in the difference of sorbitol transportation and dehydrogenation in the sorbitol fast- and slow-fermenting strains. The difference in the relative transcription levels of pyruvate formate-lyase to pyruvate dehydrogenase between the toxigenic and nontoxigenic strains may be also responsible for the time and ability difference of formate production between the strains. Conclusions: Multiple factors involved in different metabolism steps may affect the sorbitol fermentation in the toxigenic and nontoxigenic strains of V. cholerae El Tor.

Effect of lactoperoxidase on the antimicrobial effectiveness of the thiocyanate hydrogen peroxide combination in a quantitative suspension test

A Welk — 2009-07-09T00:00:00Z

Background,The positive antimicrobial effects of increasing concentrations of thiocyanate (SCN-) and H2O2 on the human peroxidase defence system are well known. However, little is known about the quantitative efficacy of the human peroxidase thiocyanate H2O2 system regarding Streptococcus mutans and sanguinis, as well as Candida albicans. The aim of this study was to evaluate the effect of the enzyme lactoperoxidase on the bactericidal and fungicidal effectiveness of a thiocyanate-H2O2 combination above the physiological saliva level. To evaluate the optimal effectiveness curve, the exposure times were restricted to 1, 3, 5, and 15 min. Results, The bactericidal and fungicidal effects of lactoperoxidase on Streptococcus mutans and sanguinis and Candida albicans were evaluated by using two test mixtures of a 2.0% (w/v; 0.34 M) thiocyanate and 0.4% (w/v; 0.12 M) hydrogen peroxide solution, one without and one with lactoperoxidase. Following the quantitative suspension tests (EN 1040 and EN 1275), the growth of surviving bacteria and fungi in a nutrient broth was measured. The reduction factor in the suspension test without lactoperoxidase enzyme was < 1 for all three tested organisms. Thus, the mixtures of 2.0% (w/v; 0.34 M) thiocyanate and 0.4% (w/v; 0.12 M) hydrogen peroxide had no in vitro antimicrobial effect on Streptococcus mutans and sanguinis or Candida albicans. However, the suspension test with lactoperoxidase showed a high bactericidal and fungicidal effectiveness in vitro.Conclusions,The tested thiocyanate and H2O2 mixtures showed no relevant antimicrobial effect. However, the mixtures became not only an effective bactericidal (Streptococcus mutans and sanguinis) but also a fungicidal (Candida albicans) agent by adding lactoperoxidase enzyme.

Domain swapping reveals that the N-terminal domain of the sensor kinase KdpD in Escherichia coli is important for signaling

Ralf Heermann — 2009-07-09T00:00:00Z

Background: The KdpD/KdpE two-component system of Escherichia coli regulates expression of the kdpFABC operon encoding the high affinity K+ transport system KdpFABC. The input domain of KdpD comprises a domain that belongs to the family of universal stress proteins (Usp). It has been previously demonstrated that UspC binds to this domain, resulting in KdpD/KdpE scaffolding under salt stress. However the mechanistic significance of this domain for signaling remains unclear. Here, we employed a "domain swapping" approach to replace the KdpD-Usp domain with four homologous domains or with the six soluble Usp proteins of E. coli. Results: Full response to salt stress was only achieved with a chimera that contains UspC, probably due to unaffected scaffolding of the KdpD/KdpE signaling cascade by soluble UspC. Unexpectedly, chimeras containing either UspF or UspG not only prevented kdpFABC expression under salt stress but also under K+ limiting conditions, although these hybrid proteins exhibited kinase and phosphotransferase activities in vitro. These are the first KdpD derivatives that do not respond to K+ limitation due to alterations in the N-terminal domain. Analysis of the KdpD-Usp tertiary structure revealed that this domain has a net positively charged surface, while UspF and UspG are characterized by net negative surface charges. Conclusions: The Usp domain within KdpD not only functions as a binding surface for the scaffold UspC, but it is also important for KdpD signaling. We propose that KdpD sensing/signaling involves alterations of electrostatic interactions between the large N- and C-terminal cytoplasmic domains.

Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: detection of clonal groups B2 ST95 and D ST59 with different host distribution

Azucena Mora — 2009-07-07T00:00:00Z

Background: Extraintestinal pathogenic Escherichia coli (ExPEC) strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties. Results: A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia) originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST), Pulsed Field Gel Electrophoresis (PFGE) and genotyping based on several genes known for their association with ExPEC or avian pathogenic Escherichia coli (APEC) virulence.APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, P=0.001), whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, P=0.0000). Seven virulence genes showed significant differences, being fimAvMT78 and sat genes linked to human isolates, while papGII, tsh, iron, cvaC and iss were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs) ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006), were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries. Conclusions: Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides evidence that some APEC isolates may act as potential pathogens for humans and, consequently, poultry as a food-borne source, suggesting no host specificity for this type of isolates. A novel and important finding has been the detection of the clonal group D O1:K1:H7/NM ST59 almost exclusively in humans, carrying pathogenic genes linked to the phylogenetic group D. This finding would suggest D O1:K1:H7/NM ST59 as a host specific pathotype for humans.

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains

Magdalena Wiesner — 2009-07-03T00:00:00Z

Background: Bacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome. Results: We found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes(STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population. Conclusions: Despite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal backgrounds allowed us to discovergenetic subgroups within the population. This study provides information about the importance of the accessory genome in generating genetic variability within a bacterial population

Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant's clinical samples using multiplex PCR

Mustapha Berri — 2009-07-01T00:00:00Z

Background: Chlamydiosis and Q fever, two zoonosis, are important causes of ruminants' abortion around the world. They are caused respectively by strictly intracellular and Gram negative bacterium Chlamydophila abortus (Cp. abortus) and Coxiella burnetii (C. burnetii). Chlamydophila pecorum (Cp. pecorum) is commonly isolated from the digestive tract of clinically inconspicuous ruminants but the abortive and zoonotic impact of this bacterium is still unknown because Cp. pecorum is rarely suspected in abortion cases of small ruminants. We have developed a multiplex PCR (m-PCR) for rapid simultaneous differential detection of Cp. abortus, Cp. pecorum and C. burnetii in clinical samples taken from infected animals. Results: Specific PCR primers were designed and a sensitive and specific m-PCR was developed to detect simultaneously, in one tube reaction, three specific fragments of 821, 526 and 687-bp long for Cp. abortus, Cp. pecorum and C. burnetii respectively. This m-PCR assay was performed on 253 clinical samples taken from infected ruminant's flocks that have showed problems of abortion diseases. Thus, 67 samples were infected by either one of the three pathogens: 16 (13 vaginal swabs and 3 placentas) were positive for Cp. abortus, 2 were positive for Cp. pecorum (1 vaginal swab and 1 placenta) and 49 samples (33 vaginal swabs, 11 raw milks, 4 faeces and 1 placenta) were positive for C. burnetii. Two vaginal swabs were m-PCR positive of both Cp. abortus and C. burnetii and none of the tested samples was shown to be infected simultaneously with the three pathogens. Conclusions: We have successfully developed a rapid multiplex PCR that can detect and differentiate Cp. abortus, Cp. pecorum and C. burnetii; with a good sensitivity and specificity. The diagnosis of chlamydiosis and Q fever may be greatly simplified and performed at low cost. In addition, the improvement in diagnostic techniques will enhance our knowledge regarding the prevalence and the pathogenetic significance of Q fever and chlamydiosis

Discovery of novel inhibitors of Streptococcus pneumoniae based on the virtual screening with the homology-modeled structure of histidine kinase (VicK)

Nan Li — 2009-06-27T00:00:00Z

Background: Due to the widespread abusage of antibiotics, antibiotic-resistance in Streptococcus pneumoniae (S.pneumoniae) has been increasing quickly in recent years, and it is obviously urgent to develop new types of antibiotics. Two-component systems (TCSs) are the major signal transduction pathways in bacteria and have emerged as potential targets for antibacterial drugs. Among the 13 pairs of TCSs proteins presenting in S.pneumoniae, VicR/K is the unique one essential for bacterium growth, and block agents to which, if can be found, may be developed as effective antibiotics against S.pneumoniae infection. Results: Using a structure-based virtual screening (SBVS) method, 105 compounds were computationally identified as potential inhibitors of the histidine kinase (HK) VicK protein from the compound library SPECS. Six of them were then validated in vitro to be active in inhibiting the growth of S.pneumoniae without obvious cytotoxicity to Vero cell. In mouse sepsis models, these compounds are still able to decrease the mortality of the mice infected by S.pneumoniae and one compound even has significant therapeutic effect. Conclusion: To our knowledge, these compounds are the first reported inhibitors of HK with antibacterial activity in vitro and in vivo, and are novel lead structures for developing new drugs to combat pneumococcal infection.

Characterization of the Zur regulon and direct Zur targets in Yersinia pestis

Yingli Li — 2009-06-25T00:00:00Z

Background: The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. Results: We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis upon exposure to zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in γ-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. Conclusion: Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in γ-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.

Carbon and arsenic metabolism in Thiomonas strains: differences revealed diverse adaptation processes

Christopher Bryan — 2009-06-23T00:00:00Z

Background: Thiomonas strains are ubiquitous in arsenic-contaminated environments. Differences between Thiomonas strains in the way they have adapted and respond to arsenic have never been studied in detail. For this purpose, five Thiomonas strains, that are interesting in terms of arsenic metabolism were selected: Tm. arsenivorans, Thiomonas sp. WJ68 and 3As are able to oxidise As(III), while Ynys1 and Tm. perometabolis are not. Moreover, Tm. arsenivorans and 3As present interesting physiological traits, in particular that these strains are able to use As(III) as an electron donor. Results: The metabolism of carbon and arsenic was compared in the five Thiomonas strains belonging to two distinct phylogenetic groups. Greater physiological differences were found between these strains than might have been suggested by 16S rRNA/rpoA gene phylogeny, especially regarding arsenic metabolism. Physiologically, Tm. perometabolis and Ynys1 were unable to oxidise As(III) and were less arsenic-resistant than the other strains. Genetically, they appeared to lack the aox arsenic-oxidising genes and carried only a single ars arsenic resistance operon. Thiomonas arsenivorans belonged to a distinct phylogenetic group and increased its autotrophic metabolism when arsenic concentration increased. Differential proteomic analysis revealed that in Tm. arsenivorans, the rbc/cbb genes involved in the assimilation of inorganic carbon were induced in the presence of arsenic, whereas these genes were repressed in Thiomonas sp. 3As. Conclusions: Taken together, these results show that these closely related bacteria differ substantially in their response to arsenic, amongst other factors, and suggest different relationships between carbon assimilation and arsenic metabolism.

Population structure of Helicobacter pylori among ethnic groups in Malaysia: recent acquisition of the bacterium by the Malay population

Chin Yen Tay — 2009-06-19T00:00:00Z

Background: Helicobacter pylori is a major gastric bacterial pathogen. This pathogen has been shown to follow the routes of human migration by their geographical origin and currently the global H. pylori population has been divided into six ancestral populations, three from Africa, two from Asia and one from Europe. Malaysia is made up of three major ethnic populations, Malay, Chinese and Indian, providing a good population for studying recent H. pylori migration and admixture. Results: Seventy eight H. pylori isolates, including 27 Chinese, 35 Indian and 16 Malay isolates from Malaysia were analysed by multilocus sequence typing (MLST) of seven housekeeping genes and compared with the global MLST data. STRUCTURE analysis assigned the isolates to previously identified H. pylori ancestral populations, hpEastAsia, hpAsia2 and hpEurope, and revealed a new subpopulation, hspIndia, within hpAsia2. Statistical analysis allowed us to identify population segregation sites that divide the H. pylori populations and the subpopulations. The majority of Malay isolates were found to be grouped together with Indian isolates. Conclusion: The majority of the Malay and Indian H. pylori isolates share the same origin while the Malaysian Chinese H. pylori is distinctive. The Malay population, known to have a low infection rate of H. pylori, was likely to be initially H. pylori free and gained the pathogen only recently from cross infection from other populations.