Search

The objective of this study was to investigate the underlying mechanism explaining reversion of clinical DAP non-susceptible (NS) MRSA isolates to DAP-susceptible (S) by analysis of genomic and cell wall characteristics of clinical DAP-NS MRSA and DAP-S MRSA isolates as well as in vitro revertant DAP-S MRSA using whole genome sequencing (WGS) and analysis of biological properties. WGS of the 4 clinical DAP-NS MRSA revealed mprF mutations resulting in amino acid substitutions or deletion. These same amino acid substitutions and deletion were also observed in the 4 in vitro revertant DAP-S strains. While WGS identified the presence of the same mprF mutations in both the DAP-NS and in vitro DAP-S revertant strains, new mutations were also detected in other genes and intergenic regions of in vitro DAP-S revertant strains. Transmission electron microscopy to assess cell-wall (CW) thickness of 4 sets strains (pre- and post-DAP therapy isolates and in vitro DAP-S revertant) showed that 3 of the 4 isolates developed increased thickness of the CW after DAP therapy. After reversion to DAP susceptibility, CW thickness was decreased to the same level as DAP-S MRSA. Our results indicate that in vitro conversion of DAP-NS MRSA to DAP-S is independent of mprF gene mutations and may be partially explained by a change in CW thickness. However, as some strains showed no change in the CW, further studies are required to elucidate the different mechanisms of resistance to DAP, and factors for conversion of DAP-NS to DAP-S.

Electroporation is a common technique necessary for genomic manipulation of Staphylococci. However, because this technique has too low efficiency to be applied to some Staphylococcal species and strains, especially to coagulase-negative Staphylococcus (CNS) isolates, basic researches on these clinically important Staphylococci are limited. Here we report on the optimization of electroporation parameters and conditions as well as on the generation of a universal protocol that can be efficiently applicable to both CNS and Coagulase-positive Staphylococci (CPS). This protocol could generate transformants of clinical Staphylococcus epidermidis isolate, with an efficiency of up to 1400 CFU/μg of plasmid DNA. Transformants of 12 other clinically important Staphylococcal species, including CNS and CPS, were also generated with this protocol. To our knowledge, this is the first report on successful electroporation in nine these Staphylococcal species.

Vancomycin-intermediate Staphylococcus aureus (VISA) and its precursor, heterogeneous VISA (hVISA), are increasingly the cause of vancomycin treatment failure. Prolonged glycopeptide treatment causes the emergence of these pathogens. However, we recently reported that hVISA can be generated by methicillin-resistant S. aureus (MRSA) exposure to imipenem (Katayama et al., Antimicrob Agents Chemother. 53:3190-6). We report here a retrospective prevalence study of VISA and hVISA on 750 MRSA clinical strains isolated from 31 Japanese national university hospitals in 1990, the year before the introduction of injectable vancomycin into clinical use in Japan in 1991. No VISA strain was identified, but population analysis identified 38 hVISA strains (5.1%) from 19 hospitals. We also determined the nucleotide sequences of vraSR, walRK, clpP, and rpoB genes whose mutations are known to be associated with vancomycin resistance. When compared with vancomycin-susceptible MRSA strain N315, six of the 38 hVISA strains possessed nonsynonymous mutations in vraSR, seven in walRK, and two in rpoB genes, Thirteen of 38 (34.2%) hVISA strains possessed at least one of these mutations. Results were consistent with our hypothesis that hVISA was present in Japanese hospitals before the clinical introduction of vancomycin.

Summary Background A new type of meticillin-resistant Staphylococcus aureus (MRSA), designated community-acquired MRSA, is becoming increasingly noticeable in the community, some strains of which cause fatal infections in otherwise healthy individuals. By contrast with hospital-acquired MRSA, community-acquired MRSA is more susceptible to non β-lactam antibiotics. We investigated the high virulence potential of certain strains of this bacterium. Methods We ascertained the whole genome sequence of MW2, a strain of community-acquired MRSA, by shotgun cloning and sequencing. MW2 caused fatal septicaemia and septic arthritis in a 16-month-old girl in North Dakota, USA, in 1998. The genome of this strain was compared with those of hospital-acquired MRSA strains, including N315 and Mu50. Findings Meticillin resistance gene ( mecA ) in MW2 was carried by a novel allelic form (type IVa) of staphylococcal cassette chromosome mec (SCC mec ), by contrast with type II in N315 and Mu50. Type IVa SCC mec did not carry any of the multiple antibiotic resistance genes reported in type II SCC mec . By contrast, 19 additional virulence genes were recorded in the MW2 genome. All but two of these virulence genes were noted in four of the seven genomic islands of MW2. Interpretation MW2 carried a range of virulence and resistance genes that was distinct from those displayed on the chromosomes of extant S aureus strains. Most genes were carried by specific allelic forms of genomic islands in the MW2 chromosome. The combination of allelic forms of genomic islands is the genetic basis that determines the pathogenicity of medically important phenotypes of S aureus , including those of community-acquired MRSA strains.

Significant advances have been made in recent years in our understanding of how methicillin resistance is acquired by Staphylococcus aureus . Integration of a staphylococcal cassette chromosome mec (SCC mec ) element into the chromosome converts drug-sensitive S . aureus into the notorious hospital pathogen methicilin-resistant S . aureus (MRSA), which is resistant to practically all β-lactam antibiotics. SCC mec is a novel class of mobile genetic element that is composed of the mec gene complex encoding methicillin resistance and the ccr gene complex that encodes recombinases responsible for its mobility. These elements also carry various resistance genes for non-β-lactam antibiotics. After acquiring an SCC mec element, MRSA undergoes several mutational events and evolves into the most difficult-to-treat pathogen in hospitals, against which all extant antibiotics including vancomycin are ineffective. Recent epidemiological data imply that MRSA has embarked on another evolutionary path as a community pathogen, as at least one novel SCC mec element seems to have been successful in converting S . aureus strains from the normal human flora into MRSA.

Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism.Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction.The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors.The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Hidehito Matsui, Juri Kimura, Masato Higashide, Yoshio Takeuchi, Kuniyuki Okue, Longzhu Cui, Taiji Nakae, Keisuke Sunakawa, Hideaki Hanaki Research Center for Infections and Antimicrobials, Kitasato Institute for Life Sciences, Kitasato University, Shirokane, Minato-Ku, Tokyo, Japan; Graduate School of Infection Control Science, Kitasato University, Shirokane, Minato-Ku, Tokyo, Japan; Kotobiken Medical Laboratories, Inc., Kamiyokoba, Ibaraki, Japan; Kohjin Bio Co., Ltd., Chiyoda, Sakado, Saitama, Japan; Kitasato University Research Organization for Infection Control Science, Shirokane, Minato-Ku, Tokyo, Japan