Molecular typing of epidemic and nonepidemic Vibrio cholerae isolates and differentiation of V. cholerae and V. mimicus isolates by PCR-single-strand conformation polymorphism analysis

AIMS: To examine the utility of polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis to differentiate epidemic and nonepidemic Vibrio cholerae isolates as well as to differentiate V. cholerae and Vibrio mimicus isolates. METHODS AND RESULTS: By both PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis of groEL-I on chromosome 1 and groEL-II on chromosome 2, V. cholerae isolates gave distinct profiles compared with V. mimicus isolates. In addition, PCR-SSCP analysis of groEL-I and groEL-II could differentiate between V. cholerae epidemic and nonepidemic isolates. Interestingly, the relationships among strains based on groEL-I from chromosome 1 and groEL-II from chromosome 2 were congruent with each other, highlighting the conserved evolutionary history of both chromosomes in this species. CONCLUSIONS: PCR-SSCP is a powerful typing technique, which has the ability to differentiate V. cholerae and V. mimicus isolates. The epidemic V. cholerae O1/O139 serogroup isolates represent a clonal complex distinct from non-O1/non-O139 isolates that can be identified by PCR-SSCP analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the effectiveness of using reliable molecular typing methods and in particular PCR-SSCP, to identify genetic variation among V. cholerae and V. mimicus isolates.     

Comparative typing of Pseudomonas species isolated from the aquatic environment in Greece by SDS-PAGE and RAPD analysis

AIMS: Three broadly used typing methods were employed in order to assess and compare the identification and classification of environmental Pseudomonas strains. The reproducibility, typeability and discriminatory power of the methods were also compared to evaluate their application. Finally, the potential impact on public health of the isolates is to be discussed. METHODS AND RESULTS: Pseudomonas strains (160) isolated from the aquatic environment in Greece and identified by a rapid identification commercially available system (API20NE), were subjected to whole-cell protein electrophoresis (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Randomly Amplified Polymorphic DNAs (RAPD) using two 10-mer primers. In general, the obtained results were in agreement. Twenty isolates that could not be identified by the API20NE system were classified by the other methods. CONCLUSIONS: Rapid identification systems may serve only for a first rough identification of environmental Pseudomonads. In order to acquire further information, so that conclusions about their role in the ecosystem and human health could be drawn, other phenotypic or genotypic methods have to be applied. SIGNIFICANCE AND IMPACT OF STUDY: It is important, from a public health point of view, to monitor the identities of environmental Pseudomonas isolates using specific methods due to their ubiquity, heterogeneity and their pathogenicity, either established or potential.     

MOLECULAR GENETICS OF ADAPTATION IN AN EXPERIMENTAL MODEL OF COOPERATION

The evolution of cooperation was studied in an empirical system utilizing a parasitic bacteriophage (f1) and a bacterial host. Infected cells were propagated by serial passage so that a phage could increase its representation among infected hosts only by enhancing the rate of growth of its host. Loss of infectivity was therefore without selective penalty, and phage benevolence could potentially evolve through a variety of genetic changes. The infected hosts evolved to grow faster over the course of the study, but the genetic bases of this phenotypic change were more difficult to anticipate. Two fundamentally different types of genetic changes in the phage were revealed. One involved the loss of some phage genes, resulting in a noninfectious plasmid that continued to replicate via the parental phage replicon. The second change involved integration of the phage genome into host DNA by a process that, at low frequency, could be reversed to produce infectious phage particles. Integration is a previously unknown property of wild-type f1, and in the system studied, may have resulted from the use of a phage bearing an insert containing nonfunctional DNA. The evolution of this novel function apparently depended only on the presence of a small region in the phage genome that provided some homology to the host DNA, with the host providing all necessary functions. Although f1 is one of the simplest phages known, these observations suggest that host-parasite interactions of the filamentous phages are more complicated than previously thought. More generally, the f1 system offers a useful model for many problems concerning the genetic basis of adaptation.     

A novel multiplex PCR method for Clostridium botulinum neurotoxin type A gene cluster typing

A rapid, simple and sensitive multiplex PCR method for boNT/A gene cluster typing was developed by combining the results of BoNT/A subtype (boNT/A1 or /A2) gene detection with ha33 and/or p47 gene detection. Ten isolates associated with infant botulism in Japan were examined and divided into boNT/A gene cluster types 2 and 3 by origin (honey feeding or not) and period (1986–1987 or 1999–2007). It is suggested that this multiplex PCR method will be be useful for epidemiological studies of botulism.     

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.     

Molecular characterization of Enterococcus faecium isolated from hospitalized patients in Korea

AIMS: Multilocus sequence typing (MLST) was performed for vancomycin-resistant Enterococcus faecium (VREF) from diverse geographical areas in Korea to obtain insights into the genetic relationships with other molecular profiles. To understand the diversity of lineages, vancomycin-susceptible E. faecium (VSEF) were included. METHODS AND RESULTS: A total of 60 E. faecium isolates were analysed by MLST and esp profile. Molecular typing of Tn1546 of 30 VREF strains was evaluated by overlapping PCR of Tn1546 and DNA sequencing. Seven sequence types (ST) were found among 30 VSEF isolates, and four STs were found among 30 VREF isolates. The types most frequently encountered were ST 78 (26 isolates) and ST 203 (16 isolates). Of the 60 E. faecium isolates, 35 isolates were positive for the esp gene. On molecular typing of Tn1546, all VREF isolates were divided into four main types. Strains with the same ST showed divergence in Tn1546 types and strains with the same Tn1546 type represented different STs. CONCLUSIONS: An association between Tn1546 typing and MLST was not found. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the horizontal spread of Tn1546 between strains plays a major role in the dissemination of vancomycin resistance in Korea.     

The use of (GTG)5 oligonucleotide as an RAPD primer to type Campylobacter concisus

AIM: DNA fingerprinting using (GTG)(5) oligonucleotide as a primer in a random amplified polymorphic DNA (RAPD) assay was assessed by typing isolates of Campylobacter concisus strains, collected over a period of 8 years. METHODS AND RESULTS: RAPD analysis using the (GTG)(5) oligonucleotide as a primer was used to type 100 isolates of C. concisus comprising mostly isolates from children with diarrhoea. Using this method, 86% of the isolates were found to be genotypically diverse. Of these heterogeneous isolates, 25 of the strains were also shown to be genetically distinct in a previous study using pulsed field gel electrophoresis. The remaining isolates (14) could be classified into five profile groups based on the DNA fingerprinting patterns. The assay successfully identified epidemiologically linked strains from the unrelated genetically diverse pool of strains. CONCLUSIONS: Laboratory RADP typing using the (GTG)(5) primer proved to be useful in distinguishing related strains of C. concisus from a large pool of unrelated strains of this organism. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD typing using (GTG)(5) is a simple method that could be used to investigate the epidemiology of C. concisus. The results suggest that homologous lineages of C. concisus may exist within an otherwise heterogeneous species complex. However, these data need to be confirmed using a more robust typing method.     

Automation of MLST using third-generation liquid-handling technology

The molecular characterization of bacterial pathogens of clinical significance is increasingly important. Methods, such as multilocus sequence typing (MLST), allow bacterial strains to be characterized during case clusters, for antibiotic-resistant strains to be monitored, and for the impact of new vaccines to be assessed. Our laboratory performs MLST on Neisseria meningitidis, Streptococcus pneumonaie, Haemophilus influenzae, and Staphylococcus aureus. We have developed high-throughput automated methods to allow MLST to be performed in a time scale useful in a clinical setting. Here we describe the automation of MLST on a third-generation liquid-handling robot.     

Crystal Structures of a CTXφ pIII Domain Unbound and in Complex with a Vibrio cholerae TolA Domain Reveal Novel Interaction Interfaces

Vibrio cholerae colonize the small intestine where they secrete cholera toxin, an ADP-ribosylating enzyme that is responsible for the voluminous diarrhea characteristic of cholera disease. The genes encoding cholera toxin are located on the genome of the filamentous bacteriophage, CTXφ, that integrates as a prophage into the V. cholerae chromosome. CTXφ infection of V. cholerae requires the toxin-coregulated pilus and the periplasmic protein TolA. This infection process parallels that of Escherichia coli infection by the Ff family of filamentous coliphage. Here we demonstrate a direct interaction between the N-terminal domain of the CTXφ minor coat protein pIII (pIII-N1) and the C-terminal domain of TolA (TolA-C) and present x-ray crystal structures of pIII-N1 alone and in complex with TolA-C. The structures of CTXφ pIII-N1 and V. cholerae TolA-C are similar to coliphage pIII-N1 and E. coli TolA-C, respectively, yet these proteins bind via a distinct interface that in E. coli TolA corresponds to a colicin binding site. Our data suggest that the TolA binding site on pIII-N1 of CTXφ is accessible in the native pIII protein. This contrasts with the Ff family phage, where the TolA binding site on pIII is blocked and requires a pilus-induced unfolding event to become exposed. We propose that CTXφ pIII accesses the periplasmic TolA through retraction of toxin-coregulated pilus, which brings the phage through the outer membrane pilus secretin channel. These data help to explain the process by which CTXφ converts a harmless marine microbe into a deadly human pathogen.