PCR differentiation of seventeen genospecies of Acinetobacter

Abstract In the present study, strains of 17 reference Acinetobacter genospecies were investigated by using the polymerase chain reaction (PCR). We used primers to amplify spacer regions between the 16S and 23S genes in the prokaryotic rRNA genetic loci. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish all of the tested acinetobacters into 15 groups. The genospecies 5 ( Acinetobacter junii ), 7 ( Acinetobacter johnsonii ) and 10 produced the same characteristic PCR patterns, suggesting the identity of these three genospecies. A preliminary evaluation of the proposed scheme for PCR diagnostics was carried out. Using the proposed scheme, tested clinical strains were identified correctly to the genospecies level, and the identifications confirmed by conventional biochemical tests. On the basis of our results, PCR amplification of the 16S–23S spacer region shows significant promise as a tool for the simple identification of genospecies belonging to Acinetobacter sp. The nucleotide sequences of our primers are sufficiently highly conserved among these organisms as to permit PCR reactions to be carried out with a single set of reaction conditions and amplification parameters irrespective of species or genus.     

Presence of virulence markers in environmental Vibrio vulnificus strains

Aims

This work aims to demonstrate the presence of several genes and factors associated with virulence in strains isolated from the environment at Pueblo Viejo Lagoon, State of Veracruz, Mexico.

Methods and Results

In this study, we investigated the production of V. vulnificus virulence factors, as cytolysin (haemolysin), RTX toxin, metalloprotease, siderophores, capsular polysaccharide, adhesion structures (like type IV pili), and polar and lateral flagella, involved in swimming and swarming (or, at least, the presence of genes encoding some of them) in 40 strains of V. vulnificus isolated from water and food. The results indicate that strains of environmental origin possess potential virulence characteristics.

Conclusions

Caution should be exercised when consuming raw shellfish (especially by those more susceptible risk groups).

Significance and Impact of the Study

This is the first work focused on the evaluation of V. vulnificus virulence factors in Mexico.     

Identification of clinically relevant yeasts by PCR/RFLP

For molecular diagnosis of fungal disease using DNA amplification procedures in the routine laboratory, choice of appropriate target structures and rapid and inexpensive identification of amplification products are important prerequisites. Most diagnostic procedures described thus far are characterized by limited applicability, considerable cost for laboratory equipment or low power of discrimination between species. This study aimed at identification of a PCR target appropriate for diagnosis of clinically relevant yeasts and an affordable procedure for characterization of the PCR products to the species level. Here, we describe a PCR-based system using amplification of intergenic spacers ITS1 and ITS2 and restriction length polymorphism of PCR products after sequence-specific enzymatic cleavage. We show the evaluation of the system for clinically relevant Candida species. The simple and inexpensive procedure should be instrumental for rapid identification of medically important yeasts.     

Detection of virulence markers in clinical strains of Yersinia]

The dispersion of plasmid pYV associated virulence markers in 474 Yersinia strains isolated from people has been studied. The ability to autoagglutination, calcium dependence of growth and the specific antigens were identified in 157 strains of traditionally pathogenic Yersinia enterocolitica serovars 03, 09, Yersinia pseudotuberculosis serovar I. They were not found in 223 strains of other 12 serovars of Yersinia enterocolitica, in 40 strains of Yersinia frederiksenii, Yersinia kristensenii, Yersinia intermedia. The proportion of virulent clones in the population of Yersinia is noted to depend on the conditions of its existence in vivo or in vitro. Identification of virulence markers is acknowledged to be expedient in epidemiological and ethiological estimation of the role of isolated Yersinia strains.     

Evaluation of the usefulness of selected virulence markers for identification of virulent strains of Yersinia enterocolitica strains. III. Chromosome markers of virulence

It is well known that virulent strains of Y. enterocolitica bear the virulence-associated plasmid pYV. Moreover some authors consider that the pathogenic strains of these bacteria have chromosome encoded phenotypic and genotypic features such as: genes ail and yst which could be used as virulence markers. The virulent strains of Y. enterocolitica do not produce pyrasinamidase and are not able to ferment salicin and cannot hydrolyse esculin. In addition these strains produce thermostable enterotoxin called YST and protein Ail (attachment invasion locus). In contrast to phenotypic virulence makers the biological function of proteins Ail, YST and nucleotide sequence of genes ail and yst is well described. In the presented study one hundred thirty virulence plasmid bearing Y. enterocolitica strains belonging to serogroup O3 were examined for the presence of genes ail, yst, and were tested for their inability to pyrasinamidase production, salicin fermentation and esculin hydrolysis. In addition forty pYV plasmid-cured isogenic strains were included in to the study. Genes ail and yst were detected by polymerase chain reaction (PCR). The obtained results indicate that all tested 130 pYV+ Y. enterocolitica strains as well as 40 plasmid-cured isogenic strains have carried ail and yst genes. All tested strains did not produce pyrasinamidase, hydrolyse esculin and ferment salicin. This generally was in agreement with the observations done by other authors and suggest that the chromosomal virulence markers, especially well described genes ail and yst, could be useful for excluding the potential virulence of Y. enterocolitica strains, which had lost pYV plasmid and have no ail or yst genes. Therefore, in clinical studies, Y. enterocolitica strains isolated directly from patients should be primarily tested for the presence of the virulence plasmid and secondarily, the negative ones could be examined for the presence of the chromosomal virulence markers.     

Comparison of the virulence potential of Acinetobacter strains from clinical and environmental sources

Several Acinetobacter strains have utility for biotechnology applications, yet some are opportunistic pathogens. We compared strains of seven Acinetobacter species (baumannii, Ab; calcoaceticus, Ac; guillouiae, Ag; haemolyticus, Ah; lwoffii, Al; junii, Aj; and venetianus, Av-RAG-1) for their potential virulence attributes, including proliferation in mammalian cell conditions, haemolytic/cytolytic activity, ability to elicit inflammatory signals, and antibiotic susceptibility. Only Ah grew at 10(2) and 10(4) bacteria/well in mammalian cell culture medium at 37°C. However, co-culture with colonic epithelial cells (HT29) improved growth of all bacterial strains, except Av-RAG-1. Cytotoxicity of Ab and Ah toward HT29 was at least double that of other test bacteria. These effects included bacterial adherence, loss of metabolism, substrate detachment, and cytolysis. Only Ab and Ah exhibited resistance to killing by macrophage-like J774A.1 cells. Haemolytic activity of Ah and Av-RAG-1 was strong, but undetectable for other strains. When killed with an antibiotic, Ab, Ah, Aj and Av-RAG-1 induced 3 to 9-fold elevated HT29 interleukin (IL)-8 levels. However, none of the strains altered levels of J774A.1 pro-inflammatory cytokines (IL-1β, IL-6 and tumor necrosis factor-α). Antibiotic susceptibility profiling showed that Ab, Ag and Aj were viable at low concentrations of some antibiotics. All strains were positive for virulence factor genes ompA and epsA, and negative for mutations in gyrA and parC genes that convey fluoroquinolone resistance. The data demonstrate that Av-RAG-1, Ag and Al lack some potentially harmful characteristics compared to other Acinetobacter strains tested, but the biotechnology candidate Av-RAG-1 should be scrutinized further prior to widespread use.     

Ribotyping and virulence markers of Yersinia pseudotuberculosis strains isolated from animals in Brazil

Ribotyping and virulence markers has been used to investigate 68 Yersinia pseudotuberculosis strains of serogroups O:1a and O:3. The strains were isolated from clinical material obtained from healthy and sick animals in the Southern region of Brazil. Ribotypes were identified by double digestion of extracted DNA with the restriction endonucleases SmaI and PstI, separation by electrophoresis and hybridization with a digoxigenin-labeled cDNA probe. The presence of the chromosomal virulence marker genes inv, irp1, irp2, psn, ybtE, ybtP-ybtQ, and ybtX-ybtS, of the IS100 insertion sequence, and of the plasmid gene lcrF was detected by polymerase chain reaction. The strains were grouped into four distinct ribotypes, all of them comprising several strains. Ribotypes 1 and 4 presented distinct profiles, with 57.3% genetic similarity, ribotypes 2 and 3 presented 52.5% genetic similarity, and genetic similarity was 45% between these two groups (1/4 and 2/3). All strains possessed the inv, irp1, and irp2 genes. Additionally, strains of serogroup O:1a carried psn, ybtE, ybtP-ybtQ, ybtX-ybtS, and IS100. As expected lcrF was only detected in strains harboring the virulence plasmid. These data demonstrate the presence of Y. pseudotuberculosis strains harboring genotypic virulence markers in the livestock from Southern Brazil and that the dissemination of these bacteria may occur between herds.     

Adhesins of Acinetobacter strains

One of the important factors contributing to the pathogenicity of bacteria is the presence of adhesins on cell surface, which facilitate colonisation in the macroorganism. The presence and type of adhesins occurring in four species of the genus Acinetobacter: A. baumannii (184), A. junii (59), A. lwoffii (65) and A. haemolyticus (22) was determined by haemagglutination test with a 3% suspension of fresh, tannic acid-treated of guinea pig, cow and human group O and AB erythrocytes, with or without the addition of one of sugar inhibitors (D-mannose, alpha-methylmannopyranoside, D-galactose-N-acetyl-D-glucosamine, L-fucose and D-ribose). In strains from all species, adhesines of the mannose-resistant (MR) type dominated. The mannose-sensitive (MS) type was present solely on the surface of one A. lwoffii strains. A. baumannii (36), A. junii (8), A. lwoffii (11) and A. haemolyticus (4) exhibited mannose-resistant hemagglutination in relation to fresh erythrocytes and that reaction was restrained by D-galactose, D-galactose and L-fucose (no other inhibitor used restrained it). The results achieved prove that cell adhesines other than those of MR type must be present on the cell surface. Additional adhesines occurred mainly in strains isolated from the respiratory and urinary tract infection simples, but were not found in isolates from blood cultures.     

The virulence markers ofSalmonella enterica serovar Typhimurium. Different phage-type strains isolated in Slovakia

Phage-typing determination of cell-surface hydrophobicity, motility, and serovar-specific virulence plasmid was performed in a collection of 154 clinical isolates of S. enterica serovar Typhimurium (SeT) isolated in Slovakia. All isolates were also examined in PCR for the presence of both stn (enterotoxin) and iroB (siderophore) genes. The DT104 was the definitive phage type most frequently identified (37.7 %), the second most frequently isolated phage type was DT41 (5.8 %); the occurrence of other phage types was not epidemiologically significant. On the basis of virulence-marker investigation, 46.1 % of isolates were hydrophobic in the assay of bacterial adherence to xylene, and 97.4 % were hydrophobic in salt-aggregation test. Motility of more than 50 mm was expressed by 20.8 % isolates. The serovar-specific 90-kb virulence plasmid was contained in 138 (89.6 %) of isolates. All SeT isolates were found (according to PCR) to carry the Salmonella-enterotoxin (stn) gene and the siderophore (iroB) gene. The increasing incidence of SeT DT104 human strains in Slovakia requires continuous attention; this can be markedly improved by surveillance efficiency and made possible by determining relationships between sporadic isolates.     

Identification of virulence factors and diagnostic markers using immunosecretome of Aspergillus fumigatus

Aspergillus fumigatus is a prime causative agent for various allergic and invasive aspergillosis. There has been a dramatic increase of such cases in last three decades yet the early diagnosis and virulence factor identification remains the challenge. In the present study secretome analysis of proteins isolated from the culture filtrate was done by 2D gel electrophoresis coupled with MS/MS and the immunosecretome analysis was carried out using immunoblotting of 2D transfer blots and probed with the sera of patients, immunized rabbit and mice. The identified proteins were analyzed further for homology with human proteins by BLAST search and for secretory signal by SignalP. A total of 65 protein spots from 2D gel resulted in identification of 24 different proteins along with their isoforms and out of which 15 proteins were identified as immunogenic in human. These findings may be helpful in the identification of virulence factors involved in aspergillosis and also useful as diagnostic markers.     

The pathogenicity of Aeromonas strains relative to genospecies and phenospecies identification

A high yield of Escherichia coli outer membrane proteins OmpA (about 200 mg/l) and OmpF (about 100 mg/l) was obtained in Bacillus subtilis when produced intracellularly. The yield was more than 100-fold higher than the yield of these proteins by a similar vector containing the complete signal sequence of alpha-amylase of B. amyloliquefaciens. Both proteins isolated after breakage of the B. subtilis cells by low-speed centrifugation were about 70% pure and could be solubilized by Sarkosyl, SDS and guanidine hydrochloride.     

Engineering clinically relevant volumes of vascularized bone

Vascularization remains one of the most important challenges that must be overcome for tissue engineering to be consistently implemented for reconstruction of large volume bone defects. An extensive vascular network is needed for transport of nutrients, waste and progenitor cells required for remodelling and repair. A variety of tissue engineering strategies have been investigated in an attempt to vascularize tissues, including those applying cells, soluble factor delivery strategies, novel design and optimization of bio‐active materials, vascular assembly pre‐implantation and surgical techniques. However, many of these strategies face substantial barriers that must be overcome prior to their ultimate translation into clinical application. In this review recent progress in engineering vascularized bone will be presented with an emphasis on clinical feasibility.     

Polyphosphate production by strains of Acinetobacter

Of four strains of Acinetobacter isolated from a pilot plant exhibiting enhanced biological phosphate removal from sewage, two strains (RA3116 and RA3117) accumulated more than 10 times the amount of polyphosphate accumulated by the other two strains (RA3114 and RA3123). Variants isolated from RA3116 and RA3117 showed polyphosphate levels similar to RA3114 and RA3123. No correlation was found between the polyphosphate content of the strains and levels of several enzymes that have been implicated in polyphosphate formation.     

Identification of the rhizobium strains in pea root nodules using genetic markers.

Pea plants were inoculated jointly with pairs of genetically marked strains of Rhizobium leguminosarum. Out of 297 modules examined 56 contained both inoculant strains. The ratios of the strains in the inoculum did not affect the frequencies of mixed nodules. Generally one of the strains consistently occupied the majority of the nodules and ithe mixed nodules comprised the majority of bacteria. Transfer of the P-group R factor, RP4, between certain strains of Rhizobium within mixed nodules was detected. In some cases the non-parental progeny comprised 10% of the rhizobia isolated from such nodules.     

Identification of a novel trimeric autotransporter adhesin in the cryptic genospecies of Haemophilus

Haemophilus biotype IV strains belonging to the recently recognized Haemophilus cryptic genospecies are an important cause of maternal genital tract and neonatal systemic infections and initiate infection by colonizing the genital or respiratory epithelium. To gain insight into the mechanism of Haemophilus cryptic genospecies colonization, we began by examining prototype strain 1595 and three other strains for adherence to genital and respiratory epithelial cell lines. Strain 1595 and two of the three other strains demonstrated efficient adherence to all of the cell lines tested. With a stably adherent variant of strain 1595, we generated a Mariner transposon library and identified 16 nonadherent mutants. All of these mutants lacked surface fibers and contained an insertion in the same open reading frame, which encodes a 157-kDa protein designated Cha for cryptic haemophilus adhesin. Analysis of the predicted amino acid sequence of Cha revealed the presence of an N-terminal signal peptide and a C-terminal domain bearing homology to YadA-like and Hia-like trimeric autotransporters. Examination of the C-terminal 120 amino acids of Cha demonstrated mobility as a trimer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the capacity to present the passenger domain of the Hia trimeric autotransporter on the bacterial surface. Southern analysis revealed that the gene that encodes Cha is conserved among clinical isolates of the Haemophilus cryptic genospecies and is absent from the closely related species Haemophilus influenzae. We speculate that Cha is important in the pathogenesis of disease due to the Haemophilus cryptic genospecies and is in part responsible for the apparent tissue tropism of this organism.     

Comparative analysis of surface-exposed virulence factors of Acinetobacter baumannii

Background

Acinetobacter baumannii is a significant hospital pathogen, particularly due to the dissemination of highly multidrug resistant isolates. Genome data have revealed that A. baumannii is highly genetically diverse, which correlates with major variations seen at the phenotypic level. Thus far, comparative genomic studies have been aimed at identifying resistance determinants in A. baumannii. In this study, we extend and expand on these analyses to gain greater insight into the virulence factors across eight A. baumannii strains which are clonally, temporally and geographically distinct, and includes an isolate considered non-pathogenic and a community-acquired A. baumannii.

Results

We have identified a large number of genes in the A. baumannii genomes that are known to play a role in virulence in other pathogens, such as the recently studied proline-alanine-alanine-arginine (PAAR)-repeat domains of the type VI secretion systems. Not surprising, many virulence candidates appear to be part of the A. baumannii core genome of virulent isolates but were often found to be insertionally disrupted in the avirulent A. baumannii strain SDF. Our study also reveals that many known or putative virulence determinants are restricted to specific clonal lineages, which suggests that these virulence determinants may be crucial for the success of these widespread common clones. It has previously been suggested that the high level of intrinsic and adaptive resistance has enabled the widespread presence of A. baumannii in the hospital environment. This appears to have facilitated the expansion of its repertoire of virulence traits, as in general, the nosocomial strains in this study possess more virulence genes compared to the community-acquired isolate.

Conclusions

Major genetic variation in known or putative virulence factors was seen across the eight strains included in this study, suggesting that virulence mechanisms are complex and multifaceted in A. baumannii. Overall, these analyses increase our understanding of A. baumannii pathogenicity and will assist in future studies determining the significance of virulence factors within clonal lineages and/or across the species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1020) contains supplementary material, which is available to authorized users.     

Identification of unique genetic markers in Rv0927c among Mycobacterium tuberculosis W-Beijing strains

Fifty-six clinical isolates of Mycobacterium tuberculosis were analyzed by spoligotyping to determine the prevalence of W-Beijing strains. Forty-nine of the 56 isolates belonged to W-Beijing strains and 7 isolates were non-Beijing strains. Comparative two-dimensional gel electrophoresis analysis of protein patterns between the W-Beijing and non-Beijing strains identified a unique protein Rv0927c that is absent in the former but present in the latter and the reference strain M. tuberculosis H37Rv. Compared with 7 non-Beijing clinical isolates and H37Rv, all 49 W-Beijing strains had two characteristic mutations, a deletion of AGC at nucleotide position 421 of Rv0927c gene encoding a putative short dehydrogenase/reductase, causing deletion of serine codon at amino acid position 141 and a -127 G-->A mutation in Rv0927c-pstS3 intergenic region, resulting in failure to express Rv0927c. Western blot analysis indicated that polyclonal antibody raised against H37Rv Rv0927c overexpressed in Escherichia coli reacted with non-Beijing strains and H37Rv but not W-Beijing strains. Characteristic mutations of Rv0927c that are present in W-Beijing strains can be used as a novel genetic marker for rapid molecular typing of M. tuberculosis W-Beijing strains.     

Genetic virulence markers of opportunistic bacteria

The analysis of opportunistic bacteria phenotypic and genetic virulence markers indicates that pathogenicity formation is based on a structural modification of bacterial DNA which is linked with migration of interbacterial pathogenicity "islands" genetic determinants. Structural organization features of these mobile genetic elements determine high expression probability, and PCR detection of pathogenicity "islands" determinants that control adhesins, invasins, cytotoxic and cytolitic toxines synthesis may indicate etiopathogenetic significance of clinical isolates.