Molecular characterization of piezotolerant and stress-resistant mutants of Staphylococcus aureus.

AIMS: In the previous work, following a pressure treatment with wild-type Staphylococcus aureus, we obtained piezotolerant isolates showing altered phenotypic characteristics. This work focuses on understanding the genetic background of their altered phenotype. METHODS AND RESULTS: AK23, a representative piezotolerant isolate was subjected to DNA microarrays, corroborated by PCR product sequencing and revealed 10-gene deletion. All other piezotolerant isolates possessed the mutation encompassing the region from SAR0665 to SAR0674 genes (9351 bp) which was most likely the result of recombination between two homologous loci (ATTGCGGGTG) present in both genes. RNA microarray transcriptomic analysis showed that due to partial deletion of the low-affinity phosphate transporter pitA, the high-affinity PhoU-PstABCS operon was upregulated in AK23 which could be the reason for piezotolerance. Furthermore, AK23 showed low levels of the virulence gene regulator rnaIII resulting in the downregulation of several agr system genes explaining the impaired virulence characteristics of the mutant. CONCLUSIONS: Naturally occurring mutations can result in piezotolerance which can be of a concern for high hydrostatic pressure-treated foods. SIGNIFICANCE AND IMPACT OF THE STUDY: A locus has been identified in piezotolerant S. aureus mutants providing insight into possible mechanisms associated with phenotypic characteristics of S. aureus. Further work should study each individual gene of the locus.

Nasal carriage of methicillin-resistant and methicillin-sensitive strains of Staphylococcus sciuri in the Indonesian population.

Staphylococcus sciuri strains were unexpectedly cultured from healthy persons and patients from Indonesia during a population-based survey on nasal Staphylococcus aureus carriage. Fifty-one S. sciuri isolates were further characterized. The S. aureus mecA gene was detected by PCR in 22 isolates (43.1%), whereas S. sciuri mecA was found in 33 isolates (64.7%). The staphylococcal cassette chromosome mec (SCCmec) regions of S. aureus mecA-positive isolates contained elements of classical S. aureus SCCmec types II and/or III.

Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

BACKGROUND & AIM: The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. RESULTS: All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. CONCLUSION: Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

Influence of temperature and relative humidity on the survival of Chlamydia pneumoniae in aerosols.

The survival of Chlamydia pneumoniae in aerosols was investigated by using a chamber with a capacity of 114.5 liters. We injected 5 x 10(7) inclusion-forming units (IFU) of C. pneumoniae in aerosols with a droplet size of 3 to 5 microns. Samples were taken after 30 s and every 1 min thereafter. The survival of C. pneumoniae was measured at four temperatures (8.5, 15, 25, and 35 degrees C) and at three different relative humidities (RH) of 5, 50, and 95% for each temperature. The survival rates of Streptococcus pneumoniae, Streptococcus faecalis, Klebsiella pneumoniae, Chlamydia trachomatis LGV2, and cytomegalovirus were also determined at 25 degrees C and 95% RH and compared with that of C. pneumoniae. At the mentioned temperatures and RH, a rapid decrease of C. pneumoniae IFU was observed in the first 30 s. After this the decrease in the number of IFU was more gradual. The survival of C. pneumoniae in aerosols were optimal at 15 to 25 degrees C and 95% RH; it was good compared with those of other microorganisms. A lower death rate was observed only in S. faecalis. In C. trachomatis, the death rate during the first 30 s was higher than that in C. pneumoniae (85 and 53.3%, respectively). After the first 30 s, the death rates in the two organisms were identical. It was concluded that transmission of C. pneumoniae via aerosols was possible. There is probably a direct transmission from person to person, taking into account the relatively short survival period of C. pneumoniae in aerosols.

Evaluation of the Vitek 2 system for susceptibility testing of Streptococcus pneumoniae isolates.

Vitek 2 (bioMérieux, France) is a new commercial system that allows rapid identification and rapid determination of the minimum inhibitory concentration (MIC) of Streptococcus pneumoniae by monitoring the growth kinetics of the organisms in microwells. The accuracy of the Vitek 2 system in susceptibility testing was evaluated by determining the MICs of 50 penicillin-susceptible and 150 intermediate or penicillin-resistant Streptococcus pneumoniae isolates and comparing the results with those obtained using the agar dilution method. The essential agreement between the Vitek 2 system and the reference method was 91% for penicillin, 93% for cefotaxime and ceftriaxone, and more than 94% for amoxicillin, erythromycin, ofloxacin, co-trimoxazole, tetracycline, and imipenem. One very major error (1.1%) and one major error (0.9%) were obtained for tetracycline. The minor error rate for penicillin of 19.3% was mainly due to intermediate category isolates (n = 29) being identified as resistant and susceptible isolates (n = 6) being identified as intermediate by the commercial system. The minor error rates for amoxicillin, cefotaxime, ceftriaxone, imipenem, and ofloxacin were 25.4%, 25.4%, 29.4%, 19.2%, and 31.5%, respectively. Vancomycin, tetracycline, co-trimoxazole, and erythromycin showed minor error rates of 0-6.1%. In conclusion, Vitek 2 shows good agreement with the reference method, as demonstrated by the low numbers of major errors, but it has a tendency to overestimate MICs, resulting in minor errors.

Rapid emergence of ciprofloxacin-resistant enterobacteriaceae containing multiple gentamicin resistance-associated integrons in a Dutch hospital.

In a hematology unit in the Netherlands, the incidence of ciprofloxacin-resistant Enterobacter cloacae and Escherichia coli increased from from 1996 to 1999. Clonal spread of single genotypes of both ciprofloxacin-resistant E. coli and Enterobacter cloacae from patient to patient was documented by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. In addition, genetically heterogeneous strains were isolated regularly. Integrons associated with gentamicin resistance were detected in Enterobacter cloacae and E. coli strains. Integron-containing E. coli were detected in all hematology wards. In contrast, in Enterobacter cloacae strains two integron types were encountered only in the isolates from one ward. Although in all patients identical antibiotic regimens were used for selective decontamination, we documented clear differences with respect to the nosocomial emergence of ciprofloxacin-resistant bacterial strains and gentamicin resistance-associated integrons.

Comparison of three commercially available amplification assays, AMP CT, LCx, and COBAS AMPLICOR, for detection of Chlamydia trachomatis in first-void urine.

We compared the Gen-Probe transcription-mediated amplification assay (AMP CT), the Abbott LCx assay, and the Roche COBAS AMPLICOR assay for the detection of Chlamydia trachomatis in a mixed population in urine samples. First-void urine, urethral specimens, and cervical specimens in females were obtained from 1,000 patients (544 males and 456 females) visiting the outpatient sexually transmitted disease clinic of our hospital. The prevalence of C. trachomatis infection was 7.7% as determined by tissue culture of urethral and cervical specimens. The sensitivities of LCx, COBAS AMPLICOR, and AMP CT compared to cell culture were 79, 86, and 78%, respectively. Sensitivity and specificity were recalculated by using a new "gold standard", i.e., a sample was considered to be true positive if two or more techniques yielded positive results. Specimens positive only by cell culture or positive in only one commercial amplification technique were retested by a previously described in-house PCR. After discordance analysis the sensitivities of LCx, COBAS AMPLICOR, and AMP CT were 84, 93, and 85%, respectively. Specificity exceeded 99% for all three assays. With each method the sensitivity was lower for urine samples from females compared to urine samples from males. By application of this new gold standard, existing differences between methods are highlighted; future evaluations of new techniques should be validated against two or more amplification assays.