In vivo development of daptomycin resistance in vancomycin-susceptible methicillin-resistant Staphylococcus aureus severe infections previously treated with glycopeptides.

Our aim was to describe the clinical and microbiological features of four cases of severe vancomycin-susceptible methicillin-resistant Staphylococcus aureus (MRSA) infections in which the vancomycin non-susceptibility development and daptomycin resistance occurred under therapy with teicoplanin (three cases) and daptomycin switched to vancomycin (one case). Clinical data were retrospectively reviewed. On nine clinical epidemiologically unrelated daptomycin-susceptible (DAP-S) and daptomycin-resistant (DAP-R) MRSA, we performed: (i) DAP-VAN-TEC-CFX-RIF minimum inhibitory concentrations (MICs); (ii) glycopeptide resistance detection (GRD) by δ-hemolysis; (iii) glycopeptide population analysis; (iv) molecular characterization by PFGE-MLST-SCCmec-agr-typing; (v) rpoB and mprF single nucleotide polymorphisms (SNPs); (vi) dltA-mprF-atl-sceD expression by real-time quantitative polymerase chain reaction (qPCR). Three out of the four patients did not survive despite salvage treatment; two died with active MRSA infection and one died because of Stenotrophomonas maltophilia sepsis. The fourth patient, in which a reversion to a DAP-S phenotype occurred, survived with daptomycin plus trimethoprim/sulfamethoxazole and oxacillin treatment, and endovascular device removal. Daptomycin resistance development was preceded by a stable heterogeneous vancomycin-intermediate S. aureus (hVISA) or VISA phenotype acquisition, while in one case, daptomycin resistance was preceded by an unstable daptomycin heteroresistance (hDAP) behavior reverting to DAP-S during vancomycin plus rifampin therapy followed by high doses of daptomycin. All DAP-R strains showed hVISA or DAP-R traits, including mutations and/or up-regulation of genes involved in cell wall turnover and cell membrane perturbation. In our study, daptomycin resistance arose during glycopeptide therapy. The emergence of DAP-R isolates was preceded by a stable VISA or hVISA phenotype or by instability reverting to a DAP-S heteroresistant phenotype. Daptomycin, as first-line therapy for the treatment of severe MRSA infections, should be used at optimal dosage combined with other agents such as beta-lactams, to prevent daptomycin resistance occurrence.

A novel δ-hemolysis screening method for detecting heteroresistant vancomycin-intermediate Staphylococcus aureus and vancomycin-intermediate S. aureus.

We assessed a new screening method, based on δ-hemolysin production in the presence of 6 mg/liter vancomycin, to distinguish heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) from vancomycin-susceptible S. aureus (VSSA). On 37 clinical methicillin-resistant S. aureus (MRSA) isolates, hVISA and VISA displayed no δ-hemolysis whereas VSSA displayed strong δ-hemolysis, showing 91.6% sensitivity. These data, supported by real-time reverse transcription PCR (real-time RT-PCR) highlighting an hld downregulation, i.e., VSSA>hVISA>VISA, define this new assay as a valid screening method.

DNA microarray-based characterisation of Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus from Italy.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) isolates are widespread in many countries, with varying distribution and epidemiology. The aim of this study was to collect and characterise the CA-MRSA isolates circulating in Italy, since only some case reports have been published. Eighteen Panton-Valentine-positive CA-MRSA isolates were collected from different Italian hospitals during the period 2005-2009 from severe infections (skin and soft tissue infections, n = 10; necrotising pneumonia, n = 7; and sepsis, n = 1). Accessory gene regulator (agr) typing, staphylococcal cassette chromosome (SCC) mec typing, spa typing, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and DNA microarray were applied to categorise isolates into clones and to compare the relevant genetic features of each clone. Six different clones were identified, the most common (7 out of 18 isolates, 38.8%) being agrI/ST8/SCCmecIV, corresponding to the USA300 clone. Six out of the seven USA300 isolates did not harbour the arginine catabolic mobile element (ACME). Four strains (22.2%) were agrIII/ST80/SCCmecIV, corresponding to the European clone. Two of the other clones, namely, agrIII/ST88/SCCmecV and agrIII/ST772/SCCmecV, corresponded to CA-MRSA clones rarely found in other countries and probably originating from Africa or the Indian subcontinent. The results of microarray hybridisations showed that the distribution of resistance genes and other virulence factors was specific to each clone. Some characteristics could be exploited as specific markers for a clone or a group of isolates, e.g. the mer operon, recovered only in ACME-negative USA300 strains. DNA microarray contributed to a more complete description of the variety of different CA-MRSA clones circulating in Italy.

Methicillin resistance and vancomycin heteroresistance in Staphylococcus aureus in cystic fibrosis patients.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are increasingly being reported among cystic fibrosis (CF) populations worldwide. In this paper, we sought to examine at the epidemiology, the molecular characterisation and the antibiotic resistance of MRSA isolates in our cohort of CF patients. All MRSA strains were collected prospectively at the University Hospital of Catania, Italy, during a two-year study between mid 2005 to mid 2007 and underwent molecular, pathotype and susceptibility characterisations. Our study demonstrates persisting infections with both hospital-associated (HA-) and community-associated (CA-)MRSA, including Panton-Valentine leukocidin (PVL)-positive strains, in our CF population with an overall prevalence of 7.8%. We demonstrated that, in these patients, persistence was sustained by either identical clones that underwent subtle changes in their toxin content or by different clones over time. The isolation of MRSA in our CF population aged 7-24 years was associated with an increased severity of the disease even if, due to the small sample of patients included and the paucity of data on the clinical outcome, these results cannot be conclusive. Furthermore, three strains were heteroresistant vancomycin-intermediate S. aureus (hVISA), questioning the use of glycopeptides in the treatment of MRSA infections in these patients.

Activity of telithromycin against multi-drug resistant Streptococcus pneumoniae and molecular characterization of macrolide and tetracycline resistance determinants.

The antistreptococcal activity of telithromycin and 11 different comparators was evaluated in 26 multi-drug resistant (MDR) Streptococcus pneumoniae strains collected during 2002-2003 as part of the ongoing PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) Italian Surveillance Program. The strains were characterized for their susceptibility to antibiotics both at the phenotypic and genotypic levels; furthermore, the association of erm(B), mef(A) class and tet(M) genes, as well as the mobile elements carrying them were determined. The strains in this study were resistant to penicillin (MIC > or = 2 mg/l) in 23.1% of cases, resistant to tetracycline in 88.4%, to cotrimoxazole in 34.6% and cefuroxime in 26.9% while only telithromycin and levofloxacin retained 100% activity against all microorganisms. Co-existence of different resistance determinants was found in 19.2% of all isolates collected in our laboratory, coming from southern Italy. Twenty-three isolates showing the MLSB phenotype of resistance possessing the erm(B) gene (88.5%), associated with tet(M), were carried on the same Tn1545-like element, while two isolates showing the M phenotype possessing the mef(A) gene alone, were carried on Tn1207.1. In only one strain were mef(E) and tet(M) together carried on Tn2009.

Presence of the ica operon in clinical isolates of Staphylococcus epidermidis and its role in biofilm production.

Staphylococcus epidermidis is an important cause of catheter-associated infections, which are attributed to its ability to form a multilayered biofilm on polymeric surfaces. This ability depends, in part, on the activity of the icaADBC locus and the icaR gene, which are involved in the production of the polysaccharide intercellular adhesin (PIA) that is functionally necessary for cell-to-cell adhesion and biofilm accumulation. The present study determined: (1) the prevalence of the icaADBC operon in S. epidermidis isolates from catheter-related and other nosocomial infections; (2) the correlation between the presence of this operon, biofilm production and resistance to antibiotics; (3) the expression of ica genes and biofilm production; and (4) the genetic relatedness of the isolates. The results showed that icaRADBC was present in 45% of the isolates included in the study, and that such isolates were significantly more resistant to the main antibiotics tested than were ica-negative isolates. The presence of the entire cluster did not always correlate with biofilm production, determined under different culture conditions, but there was evidence to suggest a correlation when at least two genes (icaAD) were co-transcribed. Eight of 18 ica-positive isolates had the entire operon in the same restriction fragment after pulsed-field gel electrophoresis, but the isolates were not clonal. Estimation of genetic relatedness indicated that ica-positive S. epidermidis isolates belonged to different lineages, distributed in only one of two major clusters, with a genetic distance of c. 0.12.

Rapid method for detection of gyrA and grlA mutations in unrelated strains of Staphylococci susceptible and resistant to levofloxacin.

A panel of 150 clinical isolates of methicillin resistant and susceptible Staphylococci were investigated using a rapid and simple PCR-RFLPs technique to detect DNA nucleotide changes at the site of the most frequently reported mutations in grlA (codons 79, 80) and gyrA (codons 83, 84) genes which confer fluorquinolone resistance in Staphylococci. Convergent dual mutations in and gyrA and grlA were found in all strains exhibiting resistance to ciprofloxacin (MIC, 8 to > or =128 mg/l) and levofloxacin (MIC, 8 to > or =64 mg/l). Mutations in grlA and gyrA were also found in strains susceptible to levofloxacin and resistant to ciprofloxacin. In our sample no strains with only grlA mutations were found. Our data indicate that methicillin-resistant fluorquinolone-resistant strains are likely to have mutations in both grlA and gyrA. In contrast, methicillin-susceptible strains do not show any mutation. The genetic relatedness of a sample of representative epidemiologically unrelated MRSA strains, tested by PFGE and rep-PCR, are in agreement with the hypothesis of a clonal selection of these resistant strains.

In vitro activity of levofloxacin against coagulase-positive and -negative staphylococci.

The in vitro activity of levofloxacin compared with that of ciprofloxacin, ofloxacin and norfloxacin were examined by conventional in vitro tests against 150 clinical isolates of staphylococci, subdivided according to species and susceptibility to methicillin. Although the minimum inhibitory concentrations (MICs) of all quinolones were highest in methicillin-resistant Staphylococcus aureus strains, the activity of levofloxacin was almost complete in methicillin-resistant S. epidermidis and methicillin-resistant S. haemolyticus when compared with ciprofloxacin and ofloxacin, which showed more than 30% resistance. Methicillin-susceptible S. aureus and S. epidermidis strains were susceptible to all quinolones with few differences between the antibiotics tested. The minimal bactericidal activity of levofloxacin was within the double dilution range of MIC values for all strains tested, demonstrating its potent role against staphylococci. In time-kill studies, levofloxacin exerted bactericidal activity within 3 h against all staphylococci. These in vitro results suggest that levofloxacin is a potent fluoroquinolone against coagulase-negative staphylococci and that it is both methicillin-susceptible and resistant. Further studies are necessary to determine the role of this drug in the treatment of infections sustained by these microorganisms.

[Clonal diffusion and evolution of mecA and Tn554 polymorphisms in methicillin-resistant Staphylococcus aureus in Italy]. / Diffusione clonale ed evoluzione dei polimorfirsmi di mecA e Tn554 in Staphylococcus aureus meticillino-resistente in Italia.

The purposes of the present study were to track the geographic spread of 69 MRSA strains in Italy recovered from 7 hospitals in four towns; to detect the clonal identities among the isolates by a combination of multiple genomic typing methods and to measure temporal trends in clonal types between 1984 and 1998. Our results showed the spread of three major clones among the MRSA isolates of 1984-1995 period: the most represented MRSA clone carried the PFGE pattern A, the mecA polymorph II and had no homology with Tn554 (II::NH::A); most of these isolates were susceptible to the macrolides,being similar to the historically " archaic" MRSA strains; the clone typed I::E::A, carried the PFGE pattern A, the mecA polymorph I and Tn554E commonly defined as "Iberian clone"; unique clone, showing an uncommon PFGE pattern E. the mecA polymorph II and the Tn554 E (II::E::E) and were characterized by a uniform susceptibility to tetracycline and rifampin. During 1997-98 the representation of this clone increased instead of the classical "Iberian clone". A new multi-resistant MRSA strain, carrying the PFGE pattern B (or B'), the mecA polymorph XI and Tn554 polymorph B (XI::B::B), called "Brazilian clone", increased from being absent (1984-95) to 48%. Our molecular data show an Italian MRSA "scenario" far from the common European trends and clearly documented the spread of an archaic clonal type (II::NH::A) in 1984-95, the arrival and rapid increase of Brazilian done in 1997-98 and the chronological and geographical spread of a unique (H::E::E) called "Italian clone", instead of the widely spread Iberian MRSA clone.

[Correlation between methicillin-resistance and resistance to fluoroquinolones in Staphylococcus aureus and Staphylococcus epidermidis]. / Correlazione della resistenza alla meticillina e ai fluorochinoloni in Staphylococcus aureus e Staphylococcus epidermidis.

Fluoroquinolones resistance in Staphylococci is associated to point mutations in grlA (80,84 and 116) grlB, gyrA (84,88) and gyrB genes. Almost all MRSA strains are ciprofloxacin and levofloxacin resistant while, in a lesser degree, MRCoN staphylococci show to be resistant to levofloxacin. This observation made possible to predict a different correlation between methicillin-resistance and the resistance to FQs in this two different species. In this study, we compare genomic analysis of S. aureus and S. epidermidis with the resistance to FQs. Our results show that strains of MRSA are distributed in 4 different PFGE-types while 12 MRSE strains are distributed in 9. MRSA resistant to FQs showed a unique PFGE pattern; on the contrary of FQs susceptible MRSA and MSSA. Furthermore mecA and gyrA genes are located in the same SmaI fragment in MRSA and in different in MRSE. MSSE and MRSE show more ClaI/mecA polymorphisms than MRSA. All this data confirm the clonal origin of MRSA and show that FQs resistance is linked to the presence of mec locus and both clonally spread. On the contrary in MRSE FQs-resistance is independent from MR and arise with the normal frequence of antibiotic induction.

Levofloxacin in vitro activity and time-kill evaluation of Stenotrophomonas maltophilia clinical isolates.

The in vitro activity of levofloxacin and eight other antimicrobial agents against 60 clinical isolates of Stenotrophomonas maltophilia was determined by an agar dilution method using 10(4) and 10(6) cfu/spot inocula. At the lower inoculum, 85.0% of the isolates were susceptible to levofloxacin but only 58.3% were susceptible to ofloxacin; at the higher inoculum, 78.3% were susceptible to levofloxacin and 36.7% to ofloxacin. In time-kill studies, levofloxacin exerted bactericidal activity within 4 h. With ofloxacin and ciprofloxacin bacterial regrowth was observed after 8 h. Levofloxacin may represent an alternative drug in the treatment of infections caused by S. maltophilia.

In vitro selection of glycopeptide-resistant variants of Enterococci.

In order to study the possible phenotypic and genotypic changes related to glycopeptide pressure on enterococci, a study was undertaken using stepwise in vitro exposure to achieve the following objectives: (i) to evaluate the development of resistance and cross-resistance between vancomycin and teicoplanin; (ii) to determine the stability of the acquired level of resistance; (iii) to determine the phenotypic and genotypic changes related to glycopeptide pressure; and (iv) to assess the spectrum of antibiotic-susceptibility of all strains. Our results showed that no variants resistant to glycopeptides could be selected after in vitro glycopeptide exposure experiments. However some strains showed increased MIC values: 8 mg/l to vancomycin in eight strains selected by vancomycin itself, while teicoplanin produced intermediate values to vancomycin in only three strains. The phenotypes were stable in vitro after numerous passages in antibiotic-free medium and three out of nine strains with a changed MIC level, showed 40, 42 and 43 kDa proteins in cell membrane preparations. The profile of antibiotic resistance was comparable in all isogenic strains tested with the exception of three selected strains that became susceptible to penicillin G. The pressure produced by glycopeptides, particularly vancomycin has contributed to an increased level of MIC that can influence the acquisition and/or full expression of this resistance.