- Volume 55, Issue 12, 2006

Protease IV is important in the pathogenesis of Pseudomonas aeruginosa-induced microbial keratitis, but little is known of its role in cystic fibrosis (CF) lung infection. In this study protease IV production was examined in 43 P. aeruginosa isolates (24 non-clonal and 19 clonal) from the lungs of chronically infected adult patients attending the Royal Prince Alfred Hospital CF Clinic, Sydney, Australia. Overall, 32/43 (74 %) isolates were positive for protease IV protein by Western blotting and 22/43 (51 %) had evidence of active protease IV on gelatin zymography. Clonal strains were 1.6 times more likely than non-clonal strains to produce protease IV [18/19 (95 %) versus 14/24 (58 %), RR=1.6, CI 1.1–2.3, P=0.007] and 3 times more likely to secrete the protein [16/19 (84 %) versus 6/24 (25 %), RR=3.4, CI 1.6–6.9, P<0.001]. Nine of the ten strains negative by both Western blotting and zymography were non-clonal, and all but one of these was positive for the protease IV gene. There was a marked strain-to-strain variation in the amount of protease IV produced. Secretion of protease IV by clonal strains may enhance their infectivity and ability to adapt to the changing CF lung environment. Overall the findings suggest that protease IV plays an important role in the pathogenesis of P. aeruginosa infection in the CF lung.

This study reports the detection of an extracellular staphylococcal product, designated secretory inhibitor of platelet microbicidal protein (SIPMP), that causes local inhibition of the bactericidal action of platelet microbicidal protein (PMP) in the fluid phase. Urethral isolates of Staphylococcus aureus (n=24) and coagulase-negative staphylococci (CNS) (n=47) from patients with or without chronic bacterial prostatitis (CBP) were tested. SIPMP production was tested by inhibition of PMP bioactivity against Bacillus subtilis and was expressed as percentage inhibition of PMP bactericidal activity. The PMP susceptibility of staphylococcal strains was determined by exposing bacterial cells to serial dilutions of PMP. Staphylococci from patients without CBP produced SIPMP at levels of 10.3±1.2 and 13.25±1.72 % for S. aureus and CNS, respectively. Strains isolated from men with CBP inhibited PMP-induced killing of B. subtilis by 23.38±4.2 % (P<0.05) and 23.69±1.87 % (P<0.01) for S. aureus and CNS, respectively. SIPMP production correlated with staphylococcal resistance to PMP (r 2=0.6082 and 0.7264 for S. aureus and CNS, respectively). SIPMP represents a hitherto unrecognized determinant of staphylococcal pathogenicity. These results suggest that SIPMP production is associated with the CBP source. Data from this study may have significant implications for the understanding of the pathogenesis of CBP.

Salmonella enterica subspecies enterica serovar Paratyphi B [O1,4,(5),12 : Hb : 1,2] can cause either an enteric fever (paratyphoid fever) or self-limiting gastroenteritis in humans. The d-tartrate non-fermenting variant S. enterica subsp. enterica serovar Paratyphi B dT− (S. Paratyphi B) is the causative agent of paratyphoid fever, and the d-tartrate fermenting variant S. enterica subsp. enterica serovar Paratyphi B dT+ (S. Paratyphi B dT+; formerly called Salmonella Java) causes gastroenteritis. S. Java is currently recognized as an emerging problem worldwide. Twelve dT+ S. Java isolates were collected in Indonesia between 2000 and 2002. One-third of them contained Salmonella genomic island 1 (SGI1), which gives the multidrug-resistant phenotype to the bacteria. In this study, a PCR-based method to detect a single nucleotide difference responsible for the inability to ferment d-tartrate, reported elsewhere, was validated. The d-tartrate fermenting phenotype of S. Java was converted to the non-fermenting phenotype by the disruption of the ORF STM 3356, and the d-tartrate non-fermenting phenotype of the ORF STM 3356-disrupted strain and the dT− reference strain was changed to the dT+ phenotype by complementing ORF STM 3356 in trans. The results show that the dT+ phenotype requires a functional product encoded by STM 3356, and support the use of the PCR-based discrimination method for S. Paratyphi B and S. Java as the standard differentiation method.

Screening for Chlamydia trachomatis infections can be performed on urine samples and genital swabs using molecular techniques. A novel approach was developed that combined an automated extraction procedure, an automated liquid-handling system and real-time PCR to detect C. trachomatis from urine or swabs. This novel real-time PCR approach was compared to the commercial Cobas Amplicor system on 628 specimens. In a retrospective analysis, 51 samples that tested positive using the Cobas assay were also positive with the real-time PCR, whereas the 49 samples negative with Cobas were also negative with the real-time PCR, for an overall agreement of 100 %. Among 528 prospective samples consecutively received at the authors' laboratory with a request for C. trachomatis PCR, five PCR reactions were inhibited when tested with Cobas. These five inhibited samples were found negative with the real-time PCR. Among the remaining 523 samples, 45 (8.6 %) were positive with both methods, 476 (91 %) were negative with both methods, and 2 (0.4 %) were positive with Cobas but negative with the real-time PCR. Thus, when considering Cobas as the gold standard, the overall agreement was 99.6 %, the sensitivity of the real-time PCR was 95.7 % and the specificity was 100 %. The two discrepant samples were retested in parallel and were found negative with both methods. When testing a batch of 25 samples, both reagent costs and laboratory technician time were reduced with the new technique (7.30 euros per sample and 134 min) compared to Cobas (11.20 euros per sample and 232 min). Moreover, due to reduced organizational constraints, the median time from sample reception to result was only 24 h using the automated platform. Overall, this novel real-time PCR approach exhibited an excellent specificity and a sensitivity similar to that of Cobas Amplicor PCR for the detection of C. trachomatis. Given its high throughput potential and low costs/laboratory technician time requirement, it may be useful for future use in large C. trachomatis screening programs.

This study characterized the genetic basis of antimicrobial resistance of a number of Shigella spp. isolated from humans from 2000 to 2004 in Hiroshima prefecture, Japan. A total of 26 isolates of Shigella spp. were included in this study. Antimicrobial susceptibility tests revealed high levels of resistance, especially to ampicillin, streptomycin, trimethoprim, tetracycline, nalidixic acid and ciprofloxacin. PCR and DNA sequencing were used for screening and characterization of antibiotic-resistance determinants. PCR sequencing analysis revealed the presence of only one type of class 1 integron in one isolate of Shigella sonnei. This class 1 integron was 1904 bp and contained two gene cassettes: a probable esterase/lipase (estX) and aadA1, which confers resistance to streptomycin and spectinomycin. Two types of class 2 integron were identified in this study. One was the classic type (2158 bp) and carried the three conserved resistance gene cassettes of the class 2 integron, dfrA1, sat1 and aadA1, which confer resistance to trimethoprim, streptothricin and streptomycin/spectinomycin, respectively. This type was detected in both Shigella sonnei (14 isolates) and Shigella flexneri (five isolates). The other type was shorter (1313 bp) and carried only two gene cassettes, dfrA1 and sat1. This integron was detected in a single isolate of Shigella sonnei. PFGE patterns showed limited diversity within clusters of the same species. Furthermore, an extended-spectrum β-lactamase gene, bla OXA-30, which confers resistance to ampicillin, was characterized in all isolates of Shigella flexneri except the oldest strain, which was isolated in 2000. Southern blot hybridization and conjugation experiments showed that bla OXA-30 was located in the chromosome.

A prospective study was performed to evaluate the correlation between the proposed standard of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST) (document 7.1) and the commercial system Etest for determining the MICs of flucytosine, amphotericin B, fluconazole, itraconazole and voriconazole for a collection of 100 clinical and environmental isolates of Cryptococcus neoformans. The agreements among Etest MICs within ±2 log2 dilutions of AFST-EUCAST standard MICs were greater for flucytosine, fluconazole and voriconazole (76, 78 and 88 %, respectively) than for amphotericin B (5 %), the lowest agreement, and itraconazole (67 %). Overall, the correlation coefficients were statistically significant (P<0.05), and it is suggested that the Etest and AFST-EUCAST method are reliable alternatives and present good correlation for all drugs evaluated except amphotericin B. However, the observed differences related to MICs for susceptible, susceptible dose dependent and resistant strains between the methods suggest that it will be necessary to carry out further studies, including assessment of interlaboratory agreement and correlation of MICs by different methods with in vivo response.

A fatal case of nosocomial legionellosis in a low prevalence region (Calgary, Alberta, Canada) prompted investigation into the source of infection. Hospital water systems contaminated with Legionella pneumophila have been shown to pose a risk to compromised patients. Typing of an L. pneumophila serogroup 1 strain isolated from the patient using sequence-based typing (SBT) and amplified fragment length polymorphism (AFLP) analysis linked it to a persistent and widespread strain isolated from the hospital water system establishing a nosocomial mode of acquisition. Different SBT and AFLP patterns were determined for non-epidemiologically linked cases and isolates from different hospitals.

Previous epidemiological studies have demonstrated a potential link between the serotypes of Yersinia enterocolitica recovered from cattle, sheep and pigs and those isolated from human disease cases. Further studies utilizing amplified fragment length polymorphisms have shown a relationship at the genetic level between strains of biotypes 3 and 4 from humans and livestock, and also suggested that some biotype 1A isolates, classically defined as non-pathogenic, are closely related to biotype 3 and 4 isolates. This study sought to understand further the pathogenic potential of Y. enterocolitica isolates from livestock in Great Britain. A range of surrogate in vitro models, such as invasion of epithelial tissue cultures, survival in cultured macrophages and cytokine secretion response, was employed to assess the pathogenicity of 88 strains. The results suggested that all isolates examined were capable of adhering to and invading epithelial cells and of surviving within macrophages. However, the inflammatory response of the infected macrophages differed with the infecting Y. enterocolitica subtype, with the response to pathogenic biotype 3 and 4 isolates different to that observed with biotype 1A isolates, and with the biotype 3 O : 5,27 isolates recovered exclusively from animals. Infections of porcine tissue also suggested the possibility of host-tissue tropism within Y. enterocolitica subtypes.

An immunogenic 22 kilodalton exported Mycobacterium avium subspecies paratuberculosis (MAP) lipoprotein (P22) was previously identified, and found to belong to the LppX/LprAFG family of mycobacterial lipoproteins. N-terminal polyhistidine-tagged P22 was produced and purified from Escherichia coli. Antibody recognition of P22, and interferon-gamma (IFN-γ) responses in vitro using blood from a sheep vaccinated with Neoparasec, confirmed its immunogenicity. To evaluate the immunogenicity of P22 in vivo, five sheep were immunized with a single dose containing 0.8 mg recombinant P22 protein in adjuvant. Blood was collected at 4, 13 and 29 weeks post-immunization (p.i.) and tested for anti-P22 antibodies and P22-specific IFN-γ production. P22-specific antibodies were detected by Western blot analysis in all five Neoparasec-immunized sheep at the three time points. Three out of five P22-immunized sheep produced P22-specific antibodies for up to 13 weeks p.i., and two gave a response at 29 weeks p.i. Recombinant P22 was able to stimulate significant IFN-γ production in blood of P22-immunized sheep at 13 and 29 weeks p.i. Recombinant P22 also elicited an IFN-γ response in blood of sheep immunized with Neoparasec.