SCCmec transformation requires living donor cells in mixed biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that has emerged through the horizontal acquisition of the staphylococcal cassette chromosome mec (SCCmec). Previously, we showed that SCCmec from heat-killed donors can be transferred via natural transformation in biofilms at frequencies of 10-8-10-7. Here, we show an improved transformation assay of SCCmec with frequencies up to 10-2 using co-cultured biofilms with living donor cells. The Ccr-attB system played an important role in SCCmec transfer, and the deletion of ccrAB recombinase genes reduced the frequency ∼30-fold. SCCmec could be transferred from either MRSA or methicillin-resistant coagulase-negative staphylococci to some methicillin-sensitive S. aureus recipients. In addition, the transformation of other plasmid or chromosomal genes is enhanced by using living donor cells. This study emphasizes the role of natural transformation as an evolutionary ability of S. aureus and in MRSA emergence.

Natural transformation allows transfer of SCCmec-mediated methicillin resistance in Staphylococcus aureus biofilms.

SCCmec is a large mobile genetic element that includes the mecA gene and confers resistance to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA). There is evidence that SCCmec disseminates among staphylococci, but the transfer mechanisms are unclear. Here, we show that two-component systems mediate the upregulation of natural competence genes in S. aureus under biofilm growth conditions, and this enhances the efficiency of natural transformation. We observe SCCmec transfer via natural transformation from MRSA, and from methicillin-resistant coagulase-negative staphylococci, to methicillin-sensitive S. aureus. The process requires the SCCmec recombinase genes ccrAB, and the stability of the transferred SCCmec varies depending on SCCmec types and recipients. Our results suggest that natural transformation plays a role in the transfer of SCCmec and possibly other mobile genetic elements in S. aureus biofilms.

Use of Recombinant Endolysin to Improve Accuracy of Group B Streptococcus Tests.

Group B Streptococcus (GBS) causes serious neonatal infection via vertical transmission. The prenatal GBS screening test is performed at the late stage of pregnancy to avoid risks of infection. In this test, enrichment culture is performed, followed by GBS identification. Selective medium is used for the enrichment; however, Enterococcus faecalis, which is a potential contaminant in swab samples, can interfere with the growth of GBS. Such bacterial contamination can lead to false-negative results. Endolysin, a bacteriophage-derived enzyme, degrades peptidoglycan in the bacterial cell wall; it is a promising antimicrobial agent for selectively eliminating specific bacterial genera/species. In this study, we used the recombinant endolysin EG-LYS, which is specific to E. faecalis; the endolysin potentially enriched GBS in the selective culture. First, in the false-negative model (coculture of GBS and E. faecalis, which disabled GBS detection in the subsequent GBS identification test), EG-LYS treatment at 0.1 mg/ml improved GBS detection. Next, we used 548 vaginal swabs to test the efficacy of EG-LYS treatment in improving GBS detection. EG-LYS treatment (0.1 mg/ml) increased the GBS-positive ratio to 17.9%, compared to 15.7% in the control (phosphate-buffered saline [PBS] treatment). In addition, there were an increased number of GBS colonies under EG-LYS treatment in some samples. The results were supported by the microbiota analysis of the enriched cultures. In conclusion, EG-LYS treatment of the enrichment culture potentially improves the accuracy of the prenatal GBS screening test. IMPORTANCE Endolysin is a bacteriophage-derived enzyme that degrades the peptidoglycan in the cell wall of host bacteria; it could be used as an antimicrobial agent for selectively eliminating specific bacterial genera/species. Group B Streptococcus (GBS) causes neonatal infection via vertical transmission; prenatal GBS screening test, in which enrichment culture is followed by bacterial identification, is used to detect the presence of GBS in pregnant women. However, the presence of commensal bacteria such as Enterococcus faecalis in clinical specimens can inhibit GBS growth in the selective enrichment culture, resulting in false-negative result. Here, we demonstrated that the application of originally isolated endolysin in the enrichment culture improved the test accuracy by inhibiting unwanted E. faecalis growth and therefore avoiding false-negative results, not only in experimental settings, but also in tests using vaginal swabs.

Evaluation of a rapid immunochromatographic test for the detection of Candida species from oropharyngeal samples.

Oropharyngeal candidiasis is the most common opportunistic fungal infectious disease. Culture methods and microscopy are used to detect the presence of Candida species in clinical specimens. We have previously developed an immunochromatographic test (ICT) to enable the simple and rapid diagnosis of candidiasis. In this study, we evaluated the performance of the ICT for the detection of Candida species from pharyngeal swabs and compared the results with those of the culture method. The isolated Candida species were identified using polymerase chain reaction-restriction fragment length polymorphism, and viable cell counts were determined using selective chromogenic agar. The detection rate of C. albicans was 63.3% and 0% among ≤102 and ≥ 106 colony-forming units (CFU)/mL of viable Candida cells from pharyngeal swabs, respectively. The detection rate of nonC. albicans Candida species, especially C. glabrata, increased commensurately from 16.7% at ≤102 CFU/mL to 75.0% at ≥106 CFU/mL. Among the 300 pharyngeal swabs analyzed, 59 cultures detected Candida species at a count of >103 CFU/mL (53 were ICT-positive). Of the remaining 241 culture-negative specimens, 219 were ICT-negative. The sensitivity, specificity, and accuracy of the ICT were 89.8%, 90.9%, and 90.7%, respectively. Taken together, the ICT evaluated can be made readily available for clinical use in detecting Candida.

Finding of Agr Phase Variants in Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant S. aureus (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative S. aureus strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress.IMPORTANCEStaphylococcus aureus is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated agr locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated agr locus with mechanisms resembling phase variation. The agr revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.

Horizontal gene transmission of the cfr gene to MRSA and Enterococcus: role of Staphylococcus epidermidis as a reservoir and alternative pathway for the spread of linezolid resistance.

OBJECTIVES: Linezolid resistance mediated by the cfr gene represents a global concern due to its dissemination among multiresistant nosocomial pathogens such as MRSA and Enterococcus. In the present work, we have evaluated the in vitro transmission of cfr pSCFS7-like plasmids from two Staphylococcus epidermidis ST2 strains (SE45 and SE50) isolated in Spanish hospitals, to clinical MRSA and Enterococcus spp. isolates obtained in Japan, a country in which cfr has not been detected yet. We have also investigated alternative mechanisms of horizontal gene transfer involved in the spread of the cfr gene. METHODS: MRSA (n = 16) and Enterococcus spp. (n = 8) clinical isolates were used as recipients in conjugative experiments. Bacteriophage-mediated transmission was tested using MR83a phage and N315, COL and Mu50 strains. A transformation assay was carried out using a natural competent strain derived from N315. RESULTS: The SE45 strain was able to transfer the cfr gene to all strains tested, while transmission from SE50 was observed only to a few strains and with less efficiency. No transmission was observed to Enterococcus spp. isolates. Even though conjugation is thought to be the main mechanism of cfr dissemination, we have demonstrated that transduction can be considered an alternative pathway for transmission of the cfr gene between MRSA strains. However, the results suggest an absence of transmission by natural transformation. CONCLUSIONS: Linezolid resistance mediated by cfr vectors, such as pSCFS7-like plasmids, can be efficiently transferred to clinical MRSA in Japanese isolates. After reaching the staphylococcal pool, the cfr gene could be spread among MRSA strains by either conjugation or transduction.

Immunochromatographic detection of the group B streptococcus antigen from enrichment cultures.

Group B Streptococcus (GBS; Streptococcus agalactiae) is a leading cause of serious neonatal infections. The Centers for Disease Control and Prevention recommends GBS screening for all pregnant women during the 35th to 37th weeks of gestation. Although GBS screening has been performed mainly by the culture-based method, it takes several days to obtain a reliable result. In this study, we developed a rapid immunochromatographic test (ICT) for the detection of GBS-specific surface immunogenic protein in 15 min using an overnight enrichment culture. The ICT was prepared using two anti-Sip monoclonal antibodies. This ICT was able to detect recombinant Sip levels of 0.5 ng/ml, or about 10(6) CFU/ml of GBS cells, in tests with 9 GBS strains of different serotypes. The cross-reactivity test using 26 species of microorganism showed no detectable false-positive result. Reactivity of the ICT with 229 GBS strains showed one false-negative result that was attributable to the production of truncated Sip. Among 260 enrichment cultures of vaginal swabs, 17 produced red to orange pigments in Granada medium, and they were all GBS and Sip positive. Among 219 pigment-negative cultures, 12 were GBS positive and 10 were Sip positive. Two Sip-negative cultures contained GBS cells below the limit of detection by the ICT. Among 207 GBS-negative cultures, only one was Sip positive, which was attributable to GBS cell debris. Thus, the sensitivity and specificity of the ICT appeared to be 93.1% and 99.6%, respectively. The newly developed ICT is readily applicable to clinical use in the detection of GBS.

Identification of the third type of PVL phage in ST59 methicillin-resistant Staphylococcus aureus (MRSA) strains.

The genes lukS-PV and lukF-PV for Panton-Valentine leukocidin (PVL) that confers high virulence to Staphylococcus aureus are located on the prophages (PVL phages) which have been classified into group 1 and 2 sfi21-like Siphoviridae. We report novel PVL phages lysogenized in ST59 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Japan (JCSC7247) and Taiwan (JCSC5967). The genomes of φ7247PVL and φ5967PVL showed more than 99% identity, and the regions containing the five genes located at both ends of the prophages, int (integrase), hol (holin), ami (amidase), lukS-PV, and lukF-PV, are highly homologous to extant PVL phages. The genes for the structural module are less homologous to these phages, but are highly homologous to non-PVL phages belonging to group 3 Sfi21-like Siphoviridae, for example φN315. Subsequent PCR identification and nucleotide sequencing of an additional 11 Taiwanese ST59 MRSA isolates suggested they all carry the same phage as φ5967PVL, which differed from φ7247PVL by a single base. This study adds evidence to the notion that novel PVL phages would be generated through illegitimate recombination events by acquiring the region at which hol, ami, luk, and int genes would line up upon lytic growth, and suggests that the PVL-positive MRSA clones that have emerged worldwide may carry distinct phages.

Molecular characteristics of the Taiwanese multiple drug-resistant ST59 clone of Panton-Valentine leucocidin-positive community-acquired methicillin-resistant Staphylococcus aureus from pediatric cellulitis.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), which often produces Panton-Valentine leucocidin (PVL), has emerged worldwide as a life-threatening pathogen. Herein, we describe molecular characteristics of MRSA isolated from abdominal cellulitis in a 7-year-old Japanese boy. This MRSA was PVL-positive and belonged to the Taiwanese multiple drug-resistant CA-MRSA clone with the genotype of ST59, staphylococcal cassette chromosome mec (SCCmec) VII (SCCmecV, according to recent reclassification), agr1a (a novel agr1 subtype), and SaPI (which carried seb1, a newly designated variant seb gene). This study demonstrates the first isolation of the Taiwanese PVL-positive ST59 MRSA clone in Japan. The data also demonstrate novel subtypes in agr1 and seb and suggest that a combination of agr1a, seb1, and PVL could contribute to cellulitis (and its recurrence). Recently, a variety of PVL-positive MRSA clones are accumulating in Japan.

Improved selective isolation of Bordetella pertussis by use of modified cyclodextrin solid medium.

We have developed a modified cyclodextrin solid (MCS) medium using the selective antibiotic cefdinir. MCS medium exhibited higher sensitivity (95.6%; any culture-positive sample as reference) and greater inhibition of nasopharyngeal flora than did Bordet-Gengou agar (65.2%, P = 0.009) or cyclodextrin solid medium (39.1%, P < 0.001).

Methicillin-resistant Staphylococcus saprophyticus isolates carrying staphylococcal cassette chromosome mec have emerged in urogenital tract infections.

Staphylococcus saprophyticus is a uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in female outpatients. We investigated the dissemination and antimicrobial susceptibilities of 101 S. saprophyticus isolates from the genitourinary tracts of patients in Japan. Eight of these isolates were mecA positive and showed beta-lactam resistance. Pulsed-field gel electrophoresis showed that only some isolates were isogenic, indicating that the mecA gene was apparently acquired independently by mecA-positive isolates through staphylococcal cassette chromosome mec (SCCmec). Type determination of SCCmec by multiplex PCR showed a nontypeable element in the eight mecA-positive isolates. Sequence analysis of the entire SCCmec element from a prototype S. saprophyticus strain revealed that it was nontypeable with the current SCCmec classification due to the novel composition of the class A mec gene complex (IS431-mecA-mecR1-mecI genes) and the ccrA1/ccrB3 gene complex. Intriguingly, the attachment sites of SCCmec are similar to those of type I SCCmec in S. aureus NCTC 10442. Furthermore, the genes around the mec gene complex are similar to those of type II/III SCCmec in S. aureus, while those around the ccr gene complex are similar to those of SCC15305RM found in S. saprophyticus ATCC 15305. In comparison with known SCCmec elements, this S. saprophyticus SCCmec is a novel type.

The serine 31 residue of the B subunit of Shiga toxin 2 is essential for secretion in enterohemorrhagic Escherichia coli.

Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) include Shiga toxin 1 (Stx1) as well as Shiga toxin 2 (Stx2). Stx1 is cell associated, whereas Stx2 is localized to the culture supernatant. We have analyzed the secretion of Stx2 by generating histidine-tagged StxB (StxB-H). Although neither StxB1-H nor StxB2-H was secreted in StxB-H-overexpressed EHEC, StxB2-H-overexpressed EHEC showed inhibited Stx2 secretion. On the other hand, StxB1-H-overexpressed EHEC showed no alteration of Stx2 secretion. B-subunit chimeras of Stx1 and Stx2 were used to identify the specific residue of StxB2 that the Stx2 secretory system recognizes. Alteration of the serine 31 residue to an asparagine residue (S31N) in StxB2-H enabled the recovery of Stx2 secretion. On the other hand, alteration of the asparagine 32 residue to a serine residue (N32S) in StxB1-H caused the partial secretion of a point-mutated histidine-tagged B subunit in EHEC. Based on the evidence, it appeared possible that this residue might contain secretion-related information for Stx2 secretion. To investigate this hypothesis, we constructed an isogenic mutant EHEC (Stx1B subunit, N32S) strain and an isogenic mutant EHEC (Stx2B subunit, S31N) strain. Although the mutant Stx2 was cell associated in isogenic mutant EHEC, mutant Stx1 was not extracellular. However, when we used plasmids for the expression of the mutant holotoxins, the overexpressed mutant Stx1 was found in the supernatant fraction, and the overexpressed mutant Stx2 was found in the cell-associated fraction in mutant holotoxin gene-transformed EHEC. These results indicate that the serine 31 residue of the B subunit of Stx2 contains secretion-related information.

Evaluation of a cefoxitin disk diffusion test for the detection of mecA-positive methicillin-resistant Staphylococcus saprophyticus.

In order to validate the current Clinical and Laboratory Standards Institute (CLSI) criteria for the detection of mecA-mediated resistance in Staphylococcus saprophyticus, 101 clinical isolates, including 8 mecA-positive isolates, were investigated. All the isolates were in the range of the resistant category for coagulase-negative staphylococci with the 1 microg oxacillin disk diffusion method and agar dilution method, despite 93 isolates (92%) being mecA-negative. On the other hand, the 30 microg cefoxitin disk diffusion method showed clearly distinguishable zone diameters between the mecA-positive and -negative isolates. However, four of the mecA-negative isolates that would be considered resistant were false positive, and the current interpretive criteria of the CLSI may thus require reconsideration. This study suggests that the cefoxitin disk diffusion method could be more suitable than the oxacillin disk diffusion method for detecting mecA-mediated resistance in S. saprophyticus.

Genetic characterization of the natural SigB variants found in clinical isolates of Staphylococcus aureus.

The SigB concentrations in clinical isolates of Staphylococcus aureus were measured to examine their correlation with the antibiotic resistance. The SigB concentrations in methicillin-resistant S. aureus (MRSA) were higher than in the control strain, N315, and many of methicillin-susceptible S. aureus (MSSA). Sequencing analyses of the sigB genes revealed that the strains exhibiting the high SigB concentrations have three amino acid substitutions in SigB: I11V, D141N, and Q256K. Further analysis using isogenic mutants demonstrated that D141N (or both D141N and Q256K) is essential to maintain the high SigB concentration. These substitutions affected the UV tolerance, but had no effect on the antibiotic resistance. The SigB activity was affected by these substitutions toward the stationary phase, but not during the transient heat shock response.

Characterization of isolates of Salmonella enterica serovar typhimurium displaying high-level fluoroquinolone resistance in Japan.

Strains of the multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium isolated in Japan were examined for high-level fluoroquinolone resistance. Since the first isolation in 2000 (described in reference 13), we have identified 12 human and 5 nonhuman isolates with high-level fluoroquinolone-resistance (ciprofloxacin MIC of 24 microg/ml or more). Most of these isolates shared some features including definitive phage type (DT 12/193), resistance type (ACSSuTNCp; resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, nalidixic acid, and ciprofloxacin), and genotype on pulsed-field gel electrophoresis that were different from those of the MDR S. enterica Typhimurium DT 104. Mutations in quinolone resistance-determining regions of gyrA and parC were also conserved in almost all of the isolates despite the absence of any apparent epidemiological relationships among cases. This suggests that a specific clonal group of the serovar Typhimurium with high levels of fluoroquinolone resistance is disseminating among animals and humans in Japan.

Whole genome sequence of Staphylococcus saprophyticus reveals the pathogenesis of uncomplicated urinary tract infection.

Staphylococcus saprophyticus is a uropathogenic Staphylococcus frequently isolated from young female outpatients presenting with uncomplicated urinary tract infections. We sequenced the whole genome of S. saprophyticus type strain ATCC 15305, which harbors a circular chromosome of 2,516,575 bp with 2,446 ORFs and two plasmids. Comparative genomic analyses with the strains of two other species, Staphylococcus aureus and Staphylococcus epidermidis, as well as experimental data, revealed the following characteristics of the S. saprophyticus genome. S. saprophyticus does not possess any virulence factors found in S. aureus, such as coagulase, enterotoxins, exoenzymes, and extracellular matrix-binding proteins, although it does have a remarkable paralog expansion of transport systems related to highly variable ion contents in the urinary environment. A further unique feature is that only a single ORF is predictable as a cell wall-anchored protein, and it shows positive hemagglutination and adherence to human bladder cell associated with initial colonization in the urinary tract. It also shows significantly high urease activity in S. saprophyticus. The uropathogenicity of S. saprophyticus can be attributed to its genome that is needed for its survival in the human urinary tract by means of novel cell wall-anchored adhesin and redundant uro-adaptive transport systems, together with urease.

Identification of CTX-M-14 {beta}-lactamase in a Salmonella enterica serovar enteritidis isolate from Japan.

We analyzed the resistance to cefotaxime of a Salmonella enterica serovar Enteritidis isolate from a stool culture of a 4-year-old boy. It produced a beta-lactamase CTX-M-14, encoded by two related R plasmids. The region surrounding the blaCTX-M-14 gene had an original mosaic structure containing insertion sequences (IS26 and IS903D).

[Susceptibility testing and molecular epidemiology of clinical strains of Bordetella pertussis isolated in Japan from 2001 to 2002].

We determined antimicrobial susceptibilities and analyzed molecular epidemiology of 26 strains of Bordetella pertussis clinically isolated and then performed pulsed-field gel electrophoresis (PFGE) in Japan (Japanese Pertussis Surveillance Group Participants), from 2001 to 2002. The MICs of erythromycin, clindamycin, tetracyclines, fluoroquinorones, trimethoprim-sulfamethoxazole and rifampicin of all isolates against these showed 1 microgram/ml or less. Sparfloxacin is the most potent agent, of which the MICs showed 0.008-0.016 microgram/ml. Results of DNA fingerprinting by pulsed-field gel electrophoresis (PFGE) differentiated three types (Type I; 11 strains (42%), type II; 14 strains (54%) and type III; 1 strains (4%)). However, no relation between regions and identical PFGE patterns was found in this study. Further, surveillance of the antimicrobial susceptibilities and molecular epidemiology of B. pertussis will be required.