Elsa M. Mamizuka

Cell Wall Thickening Is a Common Feature of Vancomycin Resistance in Staphylococcus aureus

ABSTRACT We have previously shown that a thickened cell wall is responsible for the vancomycin resistance of vancomycin-resistant Staphylococcus aureus (VRSA) (equivalent to vancomycin-intermediate S. aureus and glycopeptide-intermediate S. aureus) strain Mu50 (L. Cui, H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu, Antimicrob. Agents Chemother. 44:2276-2285, 2000). However, the mechanism of vancomycin resistance in other VRSA strains remained unclear. In this study, 16 clinical VRSA strains from seven countries were subjected to serial daily passage in drug-free medium. After 10 to 84 days of passage in the nonselective medium, passage-derived strains with decreased MICs of vancomycin (MIC, <4 mg/liter) were obtained. However, all of the passage-derived strains except one (15 of 16) still possessed subpopulations that were resistant to vancomycin as judged by population analysis, and vancomycin-resistant mutant strains were selected from the passage-derived strains by one-step vancomycin selection with a frequency of 4.25 × 10−6 to 1.64 × 10−3. The data indicated that vancomycin-resistant cells are frequently generated from the passage-derived strains even after vancomycin selective pressure is lifted. Cell wall thicknesses and MICs of glycopeptides (vancomycin and teicoplanin) and beta-lactams (imipenem and oxacillin) were determined for a total of 48 strains, including 15 sets of three strains: the clinical VRSA strain, the passage-derived strain, and the vancomycin-resistant mutant strain obtained from the passage-derived strain. No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (r = 0.679; P < 0.001) and between MICs of imipenem and oxacillin (r = 0.787; P < 0.001) were recognized. Moreover, all of the VRSA strains had significantly thickened cell walls, which became thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains. The data showed that cell wall thickness had high correlation with the MICs of the two glycopeptides (correlation coefficients, 0.908 for vancomycin and 0.655 for teicoplanin) but not with those of the beta-lactam antibiotics tested. These results together with coupled changes of cell wall thickness and vancomycin MICs in 16 isogenic sets of strains indicate that thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for vancomycin resistance in S. aureus.

Screening and Characterization of Mutations in Isoniazid-Resistant Mycobacterium tuberculosis Isolates Obtained in Brazil

ABSTRACT We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Paraná. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations. Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide −48, previously unreported). Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G269A substitution. Although KatG315 substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.

Molecular techniques for MRSA typing: current issues and perspectives

Staphylococcus aureus has long been recognised as an important pathogen in human disease. Serious staphylococcal infections can frequently occur in inpatients and may lead to dire consequences, especially for therapy with antimicrobial agents. The increase in the frequency of Methicillin-Resistant Staphylococcus aureus (MRSA) as the causal agent of nosocomial infection and the possibility of emergence of resistance to vancomycin demands a quick and trustworthy characterization of isolates and identification of clonal spread within hospitals. Enough information must be generated to permit the implementation of appropriate measures for control of infection, so that outbreaks can be contained. Molecular typing techniques reviewed in this manuscript include: plasmid profile analysis, analysis of chromosomal DNA after enzymatic restriction, Southern blotting, pulsed field gel electrophoresis (PFGE), techniques involving polymerase chain reaction and multilocus sequence typing (MLST). Repetitive DNA Sequence PCR (rep-PCR) may be used for screening due to its practicality, low cost and reproducibility. Because of its high discriminatory power Pulsed-Field Gel Electrophoresis (PFGE) still remains the gold standard for MRSA typing. New techniques with higher reproducibility and discriminatory power, such as Multi-Locus Sequence Typing (MLST), are appearing. These are mostly useful for global epidemiology studies. Molecular typing techniques are invaluable tools for the assessment of putative MRSA outbreaks and so should be extensively used for this purpose.

Isolation in Brazil of nosocomial Staphylococcus aureus with reduced susceptibility to vancomycin

OBJECTIVE To evaluate the possible presence of vancomycin-resistant Staphylococcus aureus (VRSA) in a Brazilian hospital. DESIGN Epidemiological and laboratory investigation of nosocomial VRSA. METHODS 140 methicillin-resistant S aureus strains isolated between November 1998 and October 1999 were screened for susceptibility to vancomycin. The screening was carried out by using brain-heart infusion agar (BHIA) supplemented with 4, 6, and 8 microg/mL of vancomycin. The minimum inhibitory concentration (MIC) determination was carried out as standardized by the National Committee for Clinical Laboratory Standards using the broth macrodilution, agar-plate dilution, and E-test methods. PATIENTS Hospitalized patients exposed to vancomycin. RESULTS 5 of the 140 isolates had a vancomycin MIC of 8 microg/mL by broth macrodilution, agar plate dilution, and E-test methods. Four VRSA strains were isolated from patients in a burn unit who had been treated with vancomycin for more than 30 days, and one from an orthopedic unit patient who had received vancomycin treatment for 7 days. Pulsed-field gel electrophoresis characterized four of the VRSA strains as belonging to the Brazilian endemic clone. All five strains were negative for vanA, vanB, and vanC genes by polymerase chain reaction. Transmission electron microscopy of the five strains revealed significantly thickened cell walls. One patient died due to infection caused by the VRSA strain. CONCLUSIONS This is the first report of isolation of VRSA in Brazil and the first report of isolation of multiple VRSA strains from one facility over a relatively short period of time. This alerts us to the possibility that VRSA may be capable of nosocomial transfer if adequate hospital infection control measures are not taken.

First Isolation of Metallo-β-Lactamase-Producing Multiresistant Klebsiella pneumoniae from a Patient in Brazil

ABSTRACT A multiresistant Klebsiella pneumoniae isolate was taken from the blood of a 75-year-old patient with nosocomial pneumonia who developed septic shock and failed therapy with imipenem. The isolate presented an MIC of imipenem of 128 μg/ml, and the production of a metallo-β-lactamase was confirmed by phenotypic and genotypic techniques. We here report, for the first time, the detection of a metalloenzyme (IMP-1)-producing K. pneumoniae clinical strain in Latin America. The gene responsible for this phenotype was found to be blaIMP-1, carried in a class 1 integron.

Prevalence of SCCmec Type IV in Nosocomial Bloodstream Isolates of Methicillin-Resistant Staphylococcus aureus

ABSTRACT Over a period of 7 months, 151 consecutive methicillin-resistant Staphylococcus aureus blood isolates were evaluated. None was community acquired. Twenty (13%) were susceptible to four or more antimicrobials, and 95% of these isolates were identified as SCCmec type IV. Molecular typing demonstrated four patterns, with one predominant pattern. Although usually community acquired, SCCmec type IV in our setting is clearly nosocomial.

Characterization of the Brazilian endemic clone of methicillin-resistant Staphylococcus aureus (MRSA) from hospitals throughout Brazil

The objective of this study was to characterize patterns of the Brazilian endemic clone of methicillin-resistant Staphylococcus aureus (MRSA) from hospitals throughout Brazil. We studied 83 MRSA strains isolated from patients hospitalized in 27 public and private hospitals in 19 cities located in 14 Brazilian states from September, 1995, to June, 1997. The MRSA strains were typed using antibiograms, bacteriophage typing and pulsed field gel electrophoresis (PFGE). The analysis of genomic DNA by PFGE showed that 65 isolates presented the same PFGE pattern. This pattern was present in all of the hospitals studied indicating the presence of an endemic MRSA clone widely disseminated throughout Brazilian hospitals (BEC). All isolates belonging to the BEC proved to be resistant to ciprofloxacin, erythromycin, lincomycin, trimethoprim-sulphamethoxazole, and tetracycline. Variable susceptibility to these drugs was found only in isolates belonging to clones other than the BEC. The results show that, among MRSA, the BEC is common in Brazil. The best method for mapping changes in the frequency of this clone among MRSA is pulsed field gel electrophoresis. Use of molecular mapping is an important tool for monitoring the spread of potentially dangerous microbes.

First Report of the Globally Disseminated IncX4 Plasmid Carrying the mcr-1 Gene in a Colistin-Resistant Escherichia coli Sequence Type 101 Isolate from a Human Infection in Brazil

ABSTRACT A colistin-resistant Escherichia coli strain was recovered from a patient with a diabetic foot infection in Brazil. Whole-genome analysis revealed that the E. coli isolate belonged to the widespread sequence type (ST) 101 and harbored the mcr-1 gene on an IncX4 plasmid that was highly similar to mcr-1-bearing IncX4 plasmids that were recently identified in Enterobacteriaceae from food, animal, and human samples recovered on different continents. These results suggest that self-transmissible IncX4-type plasmids may represent promiscuous plasmids contributing to the intercontinental spread of the mcr-1 gene.

Early Dissemination of KPC-2-Producing Klebsiella pneumoniae Strains in Brazil

A recent publication by Monteiro et al. reported the first detection of KPC-2 β-lactamase in carbapenem-resistant Klebsiella pneumoniae strains in Brazil, which occurred in 2006 ([5][1]). The authors reported that, between September and November of 2006, four carbapenem-resistant K. pneumoniae

Antimicrobial Particles from Cationic Lipid and Polyelectrolytes

Hybrid nanoparticles from cationic lipid and polymers were prepared and characterized regarding physical properties and antimicrobial activity. Carboxymethylcellulose (CMC) and polydiallyldimethylammonium chloride (PDDA) were sequentially added to cationic bilayer fragments (BF) prepared from ultrasonic dispersion in water of the synthetic and cationic lipid dioctadecyldimethylammonium bromide (DODAB). Particles thus obtained were characterized by dynamic light-scattering for determination of z-average diameter (Dz) and zeta-potential (zeta). Antimicrobial activity of the DODAB BF/CMC/PDDA particles against Pseudomonas aeruginosa or Staphylococcus aureus was determined by plating and CFU counting over a range of particle compositions. DODAB BF/CMC/PDDA particles exhibited sizes and zeta-potentials strictly dependent on DODAB, CMC, and PDDA concentrations. At 0.1 mM DODAB, 0.1 mg/mL CMC, and 0.1 mg/mL PDDA, small cationic particles with Dz = 100 nm and zeta = 30 mV were obtained. At 0.5 mM DODAB, 0.5 mg/mL CMC and 0.5 mg/mL PDDA, large cationic particles with Dz = 470 nm and zeta = 50 mV were obtained. Both particulates were highly reproducible regarding physical properties and yielded 0% of P. aeruginosa viability (10(7) CFU/mL) at 1 or 2 microg/mL PDDA dissolved in solution or in form of particles, respectively. 99% of S. aureus cells died at 10 microg/mL PDDA alone or in small or large DODAB BF/CMC/PDDA particles. The antimicrobial effect was dependent on the amount of positive charge on particles and independent of particle size. A high microbicide potency for PDDA over a range of nanomolar concentrations was disclosed. P. aeruginosa was more sensitive to all cationic assemblies than S. aureus.