DNA methylase modifications and other linezolid resistance mutations in coagulase-negative staphylococci in Italy.

BACKGROUND: Despite 10 years of clinical use, linezolid resistance in Staphylococcus aureus and coagulase-negative staphylococci (CoNS) is still a rare phenomenon. This study reports the mechanisms of resistance and strain types seen in clusters of linezolid-resistant CoNS from two different hospitals in Italy during the period 2008-09. METHODS: Genes associated with linezolid resistance were subjected to molecular analysis and isolates were characterized by PFGE macrorestriction analysis using SmaI. RESULTS: Thirty-three linezolid-resistant isolates of methicillin-resistant CoNS comprising Staphylococcus epidermidis (24), Staphylococcus hominis (5) and Staphylococcus simulans (4) were studied. The isolates showed varying levels of linezolid resistance. Almost all isolates for which linezolid MICs were 64 mg/L possessed point mutations in domain V of 23S rRNA, while isolates for which the MICs were 256 mg/L expressed methylase activity at position A2503 mediated by the cfr gene. Overall, the isolates showed reduced susceptibility to vancomycin (MICs 1-2 mg/L) and 11 of the 33 isolates showed no susceptibility to teicoplanin. These strains were also resistant to chloramphenicol (28 of 33), lincomycin (24 of 33), erythromycin (17 of 33) and quinupristin/dalfopristin (13 of 33). S. epidermidis isolates, showing mutations or methylase modifications, belonged to different PFGE profiles and to two different sequence types (ST2 and ST23), in which the cfr gene was carried on a plasmid of ∼50 kb. CONCLUSIONS: Clinical CoNS strains with resistance to linezolid and other second-line antibiotics, as well as reduced susceptibility to glycopeptides, have emerged in Italy.

Molecular basis of resistance to macrolides, lincosamides and streptogramins in Staphylococcus hominis strains isolated from clinical specimens.

Coagulase-negative staphylococci (CoNS) are the most frequently isolated bacteria from the blood and the predominant cause of nosocomial infections. Macrolides, lincosamides and streptogramin B (MLSB) antibiotics, especially erythromycin and clindamycin, are important therapeutic agents in the treatment of methicillin-resistant staphylococci infections. Among CoNS, Staphylococcus hominis represents the third most common organism. In spite of its clinical significance, very little is known about its mechanisms of resistance to antibiotics, especially MLSB. Fifty-five S. hominis isolates from the blood and the surgical wounds of hospitalized patients were studied. The erm(C) gene was predominant in erythromycin-resistant S. hominis isolates. The methylase genes, erm(A) and erm(B), were present in 15 and 25% of clinical isolates, respectively. A combination of various erythromycin resistance methylase (erm) genes was detected in 15% S. hominis isolates. The efflux gene msr(A) was detected in 18% of isolates, alone in four isolates, and in different combinations in a further six. The lnu(A) gene, responsible for enzymatic inactivation of lincosamides was carried by 31% of the isolates. No erythromycin resistance that could not be attributed to the genes erm(A), erm(B), erm(C) and msr(A) was detected. In S. hominis, 75 and 84%, respectively, were erythromycin resistant and clindamycin susceptible. Among erythromycin-resistant S. hominis isolates, 68% of these strains showed the inducible MLSB phenotype. Four isolates harbouring the msr(A) genes alone displayed the MSB phenotype. These studies indicated that resistance to MLSB in S. hominis is mostly based on the ribosomal target modification mechanism mediated by erm genes, mainly the erm(C), and enzymatic drug inactivation mediated by lnu(A).

Genotypic and phenotypic properties of coagulase-negative staphylococci causing dialysis catheter-related sepsis.

Sixty coagulase-negative staphylococcus (CNS) isolates were recovered from the blood cultures or peritoneal dialysate effluent of 43 patients on renal dialysis. The patients had either renal dialysis catheter-related sepsis (CRS) or continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis. Isolates were characterized by biotyping, and genotyped by pulsed-field gel electrophoresis (PFGE). Phenotypic properties of the strains were also investigated. Several genotypes were identified with no one specific strain of CNS being associated with CRS. However, closely related strains were isolated from several patients within the units studied, suggesting horizontal transfer of micro-organisms. Genotypic macro-restriction profiles did not concur with phenotypic profiles or biotypes, confirming that genotyping is required for epidemiological studies. All staphylococcal strains were investigated for the production of phenotypic characteristics. Significant differences were predominantly seen in the production of lipase, esterase and elastase in strains isolated from the renal patients with CRS and CAPD-associated peritonitis, compared with a non-septic control group. These phenotypic characteristics may therefore have a role in the maintenance of CRS in renal patients.

[Molecular cloning and characterization of a N-acetylneuraminate lyase gene from Staphylococcus hominis].

A N-acetylneuraminate lyase gene (shnal) from Staphylococcus hominis was cloned into pET-28a and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant enzyme was purified and characterized. It is a homotetrameric enzyme with the optimum pH at 8.0 for the cleavage direction and the optimum pH and temperature were 7.5 and 45 degrees C for the synthetic direction. The activity of ShNAL is stable when incubated at 45 degrees C for 2 h but decreased rapidly over 50 degrees C. ShNAL showed high stability in a wide range pH from 5.0 to 10.0 with the residual activity being > 70% when the enzyme was incubated in different buffers at 4 degrees C for 24 h. Its K(m) towards N-acetylneuraminic acid, pyruvate and ManNAc were (4.0 +/- 0.2) mmol/L, (35.1 +/- 3.2) mmol/L and (131.7 +/- 12.1) mmol/L, respectively. The k(cat)/K(m) value of Neu5Ac, ManNAc, and Pyr for ShNAL were 1.9 L/(mmol x s), 0.08 L/(mmol x s) and 0.08 L/(mmol x s), respectively.

Emergence of cfr-harbouring coagulase-negative staphylococci among patients receiving linezolid therapy in two hospitals in China.

This study reports on the emergence of cfr-harbouring coagulase-negative staphylococci (CoNS) among patients who received linezolid therapy in two hospitals in Hangzhou, China. The mechanisms of resistance and transmission were analysed for these resistant isolates. Eight Staphylococcus capitis isolates, one Staphylococcus epidermidis isolate and one Staphylococcus hominis isolate, obtained from patients who had received linezolid therapy in two hospitals in Hangzhou, China, were confirmed as linezolid resistant, with MICs ranging from 8 to >256 mg l(-1). The linezolid usage data of the ten patients before isolation of the linezolid-resistant CoNS were collected. PFGE analysis showed that the eight S. capitis isolates from the two hospitals belonged to the same clone. Nine of the linezolid-resistant CoNS isolates carried the cfr gene, which was located on plasmids of a similar size. A 5.3 kb fragment containing the cfr gene, revealing 99 % identity to the sequence of the cfr-harbouring plasmid pSS-01 reported previously, was determined by PCR mapping for all cfr-positive isolates, and the cfr gene was flanked by two copies of IS256-like elements. Thus, these results document the emergence of linezolid-resistant CoNS isolates carrying the cfr gene in Hangzhou, China. Effective nosocomial infection control strategies and the judicious use of antibiotics will be required to prevent further spread of this resistance mechanism.