Identification of the first vancomycin intermediate-resistant Staphylococcus aureus (VISA) isolate from a hospital in Portugal.

A clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) with intermediate resistance to vancomycin (minimal inhibitory concentration [MIC] of 4 mug/ml) was isolated in 2006 from a surgical wound of a patient hospitalized at the orthopedics ward of Hospital de São Marcos--Braga, in the town of Braga. A combination of molecular typing methods, including pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and staphylococcal chromosomal cassette mec typing, identified the vancomycin intermediate-resistant S. aureus VISA-BRAGA as a derivative of the epidemic MRSA (EMRSA)-15 clone, which has been isolated with increasing frequency from several Portuguese hospitals recently. Compared to another EMRSA-15 isolate with the same genetic background (including PFGE subtype) the VISA-BRAGA isolate exhibited relatively high oxacillin MIC, slow growth, loss of hemolytic activity, and increased resistance to vancomycin and to daptomycin although neither of these two antibiotics was used in therapy. The VISA-BRAGA isolate described here appears to represent the first S. aureus with decreased susceptibility to vancomycin identified in a Portuguese hospital.

Role of murF in cell wall biosynthesis: isolation and characterization of a murF conditional mutant of Staphylococcus aureus.

The Staphylococcus aureus murF gene was placed under the control of a promoter inducible by IPTG (isopropyl-beta-d-thiogalactopyranoside). It was demonstrated that murF is an essential gene; it is cotranscribed with ddlA and growth rate, level of beta-lactam antibiotic resistance, and rates of transcription of the mecA and pbpB genes paralleled the rates of transcription of murF. At suboptimal concentrations of the inducer, a UDP-linked muramyl tripeptide accumulated in the cytoplasm in parallel with the decline in the amounts of the normal pentapeptide cell wall precursor. The abnormal tripeptide component incorporated into the cell wall as a monomeric muropeptide, accompanied by a decrease in the oligomerization degree of the peptidoglycan. However, incorporation of the tripeptide into the cell wall was limited to a relatively low threshold value. Further reduction of the amounts of pentapeptide cell wall precursor caused a gradual decrease in the cellular amounts of peptidoglycan, the production of a thinner peripheral cell wall, aberrant septae, and an overall increase in the diameter of the cells. The observations suggest that the role of murF exceeds its primary function in peptidoglycan biosynthesis and may also be involved in the control of cell division.

Role of murE in the Expression of beta-lactam antibiotic resistance in Staphylococcus aureus.

It was shown earlier that Tn551 inserted into the C-terminal region of murE of parental methicillin-resistant Staphylococcus aureus strain COL causes a drastic reduction in methicillin resistance, accompanied by accumulation of UDP-MurNAc dipeptide in the cell wall precursor pool and incorporation of these abnormal muropeptides into the peptidoglycan of the mutant. Methicillin resistance was recovered in a suppressor mutant. The murE gene of the same strain was then put under the control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter P(spac). Bacteria grown in the presence of suboptimal concentrations of IPTG accumulated UDP-MurNAc dipeptide in the cell wall precursor pool. Both growth rates and methicillin resistance levels (but not resistance to other antibiotics) were a function of the IPTG concentration. Northern analysis showed a gradual increase in the transcription of murE and also in the transcription of pbpB and mecA, parallel with the increasing concentrations of IPTG in the medium. A similar increase in the transcription of pbpB and mecA, the structural genes of penicillin-binding protein 2 (PBP2) and PBP2A, was also detected in the suppressor mutant. The expression of these two proteins, which are known to play critical roles in the mechanism of staphylococcal methicillin resistance, appears to be-directly or indirectly-under the control of the murE gene. Our data suggest that the drastic reduction of the methicillin MIC seen in the murE mutant may be caused by the insufficient cellular amounts of these two PBPs.

Normally functioning murF is essential for the optimal expression of methicillin resistance in Staphylococcus aureus.

A carboxy-terminal fragment of murF was used to construct and insert a suicide plasmid into the chromosomal copy of the gene in the highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain COL by Campbell type integration. The plasmid insertion generated a mutant in which the MIC value for oxacillin was reduced from 400 microg/ml of the parental strain to 0.75 microg/ml in 90% of the cells of the mutant cultures that were heterogeneous: they contained subpopulations of bacteria with a frequency of 10(-3) that were capable of expressing resistance at nearly the parental level. The impact of the murF mutation on antibiotic resistance was selective for beta-lactam antibiotics: there was no change in the susceptibility of the mutant to D-cycloserine, fosfomycin, beta-D-chloro-alanine, moenomycin, bacitracin, or vancomycin. Analysis of the mutant peptidoglycan showed decrease in the percentage of oligomeric components in rough proportion to the accumulation of several abnormal muropeptide components, which were identified as structural variants of the disaccharide tripeptide monomer. An abnormal cell wall precursor identified as UDP MurNac tripeptide was also detected in the cytoplasmic pool of the mutant strain. A normal proportion of oligomers and a greatly reduced representation of the disaccharide tripeptide were demonstrated in the cell wall of the murF mutant's subpopulation that has retained the parental level of resistance. Northern analysis demonstrated a drastic reduction in the transcription rate of mecA in mutant F9 whereas mecA transcription increased in the subpopulation of bacteria that retained high-level resistance.

Application of molecular typing methods to characterize nosocomial coagulase-negative staphylococci collected in a Greek hospital during a three-year period (1998-2000).

A total of 143 methicillin-resistant coagulase-negative staphylococci (MR-CNS) collected between 1998 and 2000 at the University Hospital of Patras, Greece, were characterized by antibiogram and genomic typing to define the clonal types endemic in this hospital and their evolution during the 3-year period. These isolates corresponded to 93 methicillin-resistant Staphylococcus epidermidis (MRSE) and 50 other MR-CNS, which were isolated from patients in different wards, exclusively from blood and catheter tips cultures. Pulsed-field gel electrophoresis (PFGE) of SmaI macrofragments and hybridization of ClaI digests with mecA and murE DNA probes were performed. The application of these methodologies demonstrated the existence, persistence and spread of MRSE, MR-Staphylococcus haemolyticus, and MR-Staphylococcus hominis clones in this hospital, whereas the SmaI/murE hybridization pattern was shown to be a valuable tool for the MRSE identification.

Antibiotic resistance as a stress response: complete sequencing of a large number of chromosomal loci in Staphylococcus aureus strain COL that impact on the expression of resistance to methicillin.

Tn551 inactivation has identified several determinants--fem or auxiliary genes--that, in addition to the mecA gene, are also critical for the expression of high-level and homogeneous resistance to methicillin. Genetic and/or biochemical analysis has shown that of the nearly dozen aux mutations described so far most are in genes involved in cell wall synthesis (murE, pbp2, glmM, glnR, femA/B, llm, etc.) or in complex regulatory functions (sigmaB), suggesting that optimal expression of resistance may involve the cooperative functioning of a number of genes in cell wall metabolism as well as stress response. The exact mechanism of these functions is not known. In an attempt to explore this unusual aspect of methicillin resistance more fully, a Tn551 transposon library, constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL, was screened for all independent insertional mutants in which the level of methicillin resistance of the parental strain (MIC, 1,600 microg/ml) was reduced by at least 15-fold and up to 500-fold. We now describe the sequencing of 21 Tn551-inactivated genes and their vicinities in 23 new auxiliary mutants that have been studied before. Using the inverted polymerase chain reaction (IPCR), we amplified fragments corresponding to the right and left junction of the Tn551 insertions, which were then sequenced by primer walking. The two largest groups of these new auxiliary genes encoded either proteins of unknown functions (6 genes) or showed homology with genes encoding proteins involved with putative sensory/regulatory activities (7 genes: protein kinases, ABC transporters, and a catabolite control protein). Sequencing upstream and downstream allowed the identification of a number of additional open reading frames, some of which may also include functions relevant for the expression of antibiotic resistance.

Molecular cloning and DNA sequencing of the Staphylococcus aureus UDP-N-acetylmuramyl tripeptide synthetase (murE) gene, essential for the optimal expression of methicillin resistance.

The Tn551 insertion in mutant RUSA235 of a highly methicillin resistant Staphylococcus aureus strain results in drastic reduction in the level of methicillin resistance and abnormalities, both in the composition of the peptidoglycan and of the cell wall precursor pool. Cloning and sequencing of the inactivated gene indicates that it is the murE gene of Staphylococcus aureus.

Massive reduction in methicillin resistance by transposon inactivation of the normal PBP2 in a methicillin-resistant strain of Staphylococcus aureus.

Screening of a large transposon library constructed in the background of a highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain (methicillin MIC 1,600 micrograms/ml) for Tn551 mutants with reduced resistance, identified mutant RUSA130 with a methicillin MIC of 12 micrograms/ml. Cloning in E. coli followed by sequencing located the Tn551 insert omega 703 near the C-terminal of the PBP2 gene. Penicillin-binding assays with mutant RUSA130 showed the presence of normal amounts of penicillin-binding protein 2A (PBP2A) but the absence of PBP2. These observations suggest that the mecA gene product PBP2A is not the sole catalyst of peptidoglycan synthesis in MRSA growing in the presence of beta-lactam antibiotics, since an intact PBP2 is also essential for the optimal expression of methicillin resistance in MRSA.