- Volume 56, Issue 1, 2007

The OmcB protein of Chlamydia trachomatis is a cysteine-rich outer membrane polypeptide with important functional, structural and antigenic properties. The entire gene encoding the OmcB protein from C. trachomatis serovar LGV1 was cloned and expressed in Escherichia coli and the full-length protein used to raise polyclonal antibodies. Recombinant OmcB was used to show that OmcB is a surface-exposed protein that functions as a chlamydial adhesin. Infectivity inhibition assays carried out using HeLa cells with serovar LGV1 in the presence of purified anti-OmcB serum showed inhibition of infectivity, suggesting that some of the OmcB was surface exposed. Moreover, using recombinant OmcB in infectivity inhibition assays resulted in 70 % inhibition of infectivity, confirming that OmcB plays a role as an adhesin in C. trachomatis. Furthermore, recombinant OmcB protein bound to the surface of HeLa and Hec1B cells, but binding to glycosaminoglycan (GAG)-deficient cells (pgsA-745 and pgsD-677) was markedly reduced, indicating that OmcB binds to GAG-like receptors on host cells.

Previously, it has been demonstrated that the invasion of Caco-2 cells by Plesiomonas shigelloides induces apoptotic cell death. Therefore, the attachment to and colonization of eukaryotic intestinal host cells by P. shigelloides are important steps in causing pathogenicity. In this study, the participation of P. shigelloides GroEL in the attachment of P. shigelloides was examined. The groESL operon of P. shigelloides was isolated by PCR. The nucleotide sequence of the groESL operon of P. shigelloides revealed two ORFs of 294 nucleotides for groES and 1647 nucleotides for groEL. Cell fractionation and immunostaining experiments suggested that the GroEL of P. shigelloides was associated with the bacterial cell surface. The expression of the groEL gene was upregulated during the attachment and apoptosis-induction stages, and the expression of the protein was also induced during the attachment stage. Furthermore, GroEL efficiently promoted the attachment of P. shigelloides to Caco-2 cells, as measured by a FACSCalibur flow cytometer. These results demonstrated that GroEL has a positive influence on the attachment of P. shigelloides to Caco-2 cells.

Balamuthia mandrillaris is a free-living amoeba and a causative agent of fatal granulomatous encephalitis. In the transmission of B. mandrillaris into the central nervous system (CNS), haematogenous spread is thought to be the primary step, followed by blood–brain barrier penetration. The objectives of the present study were (i) to determine the effects of serum from healthy individuals on the viability of B. mandrillaris, and (ii) to determine the effects of serum on B. mandrillaris-mediated blood–brain barrier perturbations. It was determined that normal human serum exhibited limited amoebicidal effects, i.e. ∼40 % of trophozoites were killed. The residual subpopulation, although viable, remained static over longer incubations. Using human brain microvascular endothelial cells (HBMEC), which form the blood–brain barrier, it was observed that B. mandrillaris exhibited binding (>80 %) and cytotoxicity (>70 %) to HBMEC. However, normal human serum exhibited more than 60 % inhibition of B. mandrillaris binding and cytotoxicity to HBMEC. ELISAs showed that both serum and saliva samples exhibit the presence of anti-B. mandrillaris antibodies. Western blots revealed that normal human serum reacted with several B. mandrillaris antigens with approximate molecular masses of 148, 115, 82, 67, 60, 56, 44, 42, 40 and 37 kDa. Overall, the results demonstrated that normal human serum has inhibitory effects on B. mandrillaris growth and viability, as well as on their binding and subsequent cytotoxicity to HBMEC. A complete understanding of B. mandrillaris pathogenesis is crucial to develop therapeutic interventions and/or to design preventative measures.

Mannitol salt agar (MSA), CHROMagar Staph aureus (CSA) and CHROMagar MRSA (CSA-MRSA) were evaluated with nasal surveillance specimens for their ability to detect Staphylococcus aureus and meticillin-resistant S. aureus (MRSA). CSA was found to be more sensitive than MSA in detecting S. aureus (98 versus 84.3 %; P=0.03). CSA and CSA-MRSA were equivalent in the ability to detect MRSA at 24 h (89.7 versus 87.2 %) and at 48 h (94.9 versus 94.9 %). When combined with Staphaurex slide confirmation testing, both CSA and CSA-MRSA were highly specific (100 %) media for detecting MRSA from nasal swab specimens.

Nine Scottish Borrelia burgdorferi isolates were investigated in IgG Western blot tests. Sera previously found to be positive and negative when tested by routine Western blots prepared from reference strain B. burgdorferi sensu stricto antigen had different outcomes with these isolates. Two isolates, E5 (Borrelia afzelii) and G4 (B. burgdorferi sensu stricto) performed well, reproducing Western blot-positive results in 90 and 95 % of tests, respectively. When antigens from both isolates were incorporated into a single IgG Western blot, the results of a panel of sera were improved when compared to the routine reference strain IgG Western blot. All of the sera positive by the routine Western blot remained positive using the Scottish isolate antigen mix. Twenty-three of the 25 negative sera remained negative and two produced an equivocal result. Of the 15 samples that tested IgG Western blot equivocal with the B. burgdorferi sensu stricto reference strain, 11 (73 %) became weak or strong positive when tested with the B. afzelii/B. burgdorferi sensu stricto antigen mix (χ 2=14.35, Yates' correction, P<0.001). In seven of these, a clinical picture of Lyme disease was consistent with the new results. The use of Scottish strains of B. afzelii and B. burgdorferi sensu stricto to provide antigen for the IgG Western blot improves the diagnosis of Lyme disease for patients in Scotland.

The prevalence of CTX-M-producing members of the Enterobacteriaceae is increasing worldwide. A novel, multiplex, real-time TaqMan PCR assay to detect and type bla CTX-M genes is described which is an improvement on previously described techniques with respect to reduced assay time, elimination of the need for protracted post-PCR processing and the convenience of a single reaction vessel. Based on β-lactam antibiogram and MIC data, 478 of 1279 Enterobacteriaceae isolates from clinical blood and urine culture specimens were selected and tested for extended-spectrum β-lactamase (ESBL) production using phenotypic methods. The new TaqMan assay detected and typed bla CTX-M genes in 21 of 28 ESBL-producing isolates.

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.

Lysostaphin is a glycylglycine endopeptidase. It cleaves the pentaglycine cross-bridge structure unique to the staphylococcal cell wall and is considered to be a potential drug for Staphylococcus aureus. In the present study, the in vitro activity of recombinant lysostaphin was investigated in 257 S. aureus isolates collected from hospital patients in Beijing, China, by determination of MIC and minimum bactericidal concentration (MBC) and a time–kill curve test. An agar dilution method was used for MIC determination in all of the isolates and a macrobroth dilution method was employed to verify MIC values for a subset of the isolates. All of the S. aureus strains were sensitive to the recombinant lysostaphin with MICs ranging from 0.03 to 2 μg ml−1 in the agar dilution assay. The antibacterial activity of lysostaphin was greater than that of vancomycin and other reference agents. For most of the isolates, the MICs from the agar dilution method were higher than those from the broth dilution method. The MBCs of lysostaphin in the test isolates were between 1- and 8-fold higher than their MIC values. Bactericidal activity (>99.9 % reduction) was observed after 2 h exposure of the isolates to lysostaphin at concentrations of ⩾0.5 MIC. Lysostaphin showed a rapid bactericidal activity against the test strains of meticillin-susceptible S. aureus and meticillin-resistant S. aureus. Its activity at ⩾0.5 MIC was sustained for at least 6 h. These results will be informative for the clinical application and evaluation of lysostaphin.

Coagulase-negative Staphylococcus epidermidis has become the leading cause of foreign-body infections due to its biofilm formation on all kinds of medical-device surfaces. The biofilm development of S. epidermidis includes two steps: the initial attachment phase and the accumulative phase. In the accumulative phase, the polysaccharide intercellular adhesin (PIA), encoded by the icaADBC locus, is the major component mediating intercellular adhesion. However, recent studies have revealed the emergence of biofilm-positive/ica-negative staphylococcal clinical isolates. In this report, two ica-negative S. epidermidis clinical strains, SE1 and SE4, exhibited their heterogeneity in biofilm architecture under static and flow conditions, compared with the biofilm-positive/ica-positive RP62A strain. Strains with this type of absence of PIA from biofilms also displayed intermediate resistance to vancomycin. More importantly, the cells of both SE1 and SE4 strains were more tolerant than those of RP62A to exposure to lysostaphin and vancomycin. Based on the results, it is suggested that the biofilm-positive/ica-negative strain represents a newly emergent subpopulation of S. epidermidis clinical strains, arising from selection by antibiotics in the nosocomial milieu, which displays a survival advantage in its host environment. Recent epidemiological data support this suggestion, by showing a tendency towards an increasing proportion of this subpopulation in staphylococci-associated infections.

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially in the ability to diagnose Legionnaires' disease (LD) at an early stage of the disease in a specimen that is readily obtainable. The aim of this study was to assess the performance of PCR as a rapid diagnostic method and to compare the results of different PCR assays of serum samples from patients with LD. Samples included 151 serum samples from 68 patients with proven LD and 60 serum samples from 36 patients with respiratory tract infections other than Legionella. PCR assays were based on the 5S rRNA gene, 16S rRNA gene and the mip gene. The samples from patients with infections caused by pathogens other than Legionella all tested negative in PCR. Among the patients with proven LD 54.4 % (37/68) tested positive in 5S rRNA PCR, 52.9 % (36/68) in mip gene PCR and 30.9 % (21/68) in 16S rRNA PCR in the first available serum sample. The association between threshold cycle value in 5S PCR positive serum samples (n=49) and C-reactive protein value was determined, and showed a strong negative correlation (Pearson correlation coefficient r=−0.63, P<0.0001). In addition to existing tests for the diagnosis of LD, detection of Legionella DNA in serum could be a useful tool for early diagnosis of LD caused by any Legionella species and serogroup, and has the potential to provide a diagnosis in a time frame that could affect initial infection management.

Actinobacillus actinomycetemcomitans, a pathogen associated with oral and extra-oral infections, requires iron to grow under limiting conditions. Although incapable of producing siderophores, this pathogen could acquire iron by direct interaction with compounds such as haemin, haemoglobin, lactoferrin and transferrin. In this work the ability of different A. actinomycetemcomitans strains to bind and use different iron sources was tested. None of the strains tested used haemoglobin, lactoferrin or transferrin as sole sources of iron. However, all of them used FeCl3 and haemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, haemoglobin and haemin, but not transferrin. Insertion inactivation of hmsF, which encodes a predicted cell-envelope protein related to haemin-storage proteins produced by other pathogens, reduced haemin and Congo red binding drastically without affecting haemin utilization as an iron source under chelated conditions. Biofilm assays showed that all strains tested attached to and formed biofilms on plastic under iron-rich and iron-chelated conditions. However, scanning electron microscopy showed that smooth strains formed simpler biofilms than rough isolates. Furthermore, the incubation of rough cells in the presence of FeCl3 or haemin resulted in the formation of more aggregates and microcolonies compared with the fewer cell aggregates formed when cells were grown in the presence of the synthetic iron chelator dipyridyl. These cell responses to changes in extracellular iron concentrations may reflect those that this pathogen expresses under the conditions it encounters in the human oral cavity.