Carlos R.V. Kiffer

Involvement of pmrAB and phoPQ in polymyxin B adaptation and inducible resistance in non-cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

ABSTRACT During investigation of susceptibility testing methods for polymyxins, 24 multidrug-resistant clinical isolates of Pseudomonas aeruginosa were observed to have a distinct, reproducible phenotype in which skipped wells were observed during broth microdilution testing for polymyxin B. Possible mechanisms underlying this phenotype were investigated. The effects of various concentrations of polymyxin B on growth, the expression of resistance genes, and outer-membrane permeability were observed. Real-time PCR was performed to compare the expression, in response to selected concentrations of polymyxin B, of genes related to the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems in polymyxin B-susceptible isolate PAO1, polymyxin B-resistant isolate 9BR, and two isolates (19BR and 213BR) exhibiting the skipped-well phenotype. 19BR and 213BR appeared to have similar basal levels of expression compared to that of PAO1 for phoQ, arnB, and PA4773 (from the pmrAB operon), and in contrast, 9BR had 52- and 280-fold higher expression of arnB and PA4773, respectively. The expression of arnB and PA4773 increased in response to polymyxin B in a concentration-dependent manner for 9BR but not for 19BR and 213BR. For these isolates, expression was significantly increased for arnB and PA4773, as well as phoQ, only upon exposure to 2 μg/ml polymyxin B but not at a lower concentration of 0.125 μg/ml. The sequencing of the pmrAB and phoPQ operons for all three isolates revealed a number of unique mutations compared to that for PAO1. 1-N-phenylnaphthylamine (NPN) was used to study the effect of preincubation with polymyxin B on the self-promoted uptake of polymyxin B across the outer membrane. The preincubation of cells with 2 μg/ml polymyxin B affected baseline membrane permeability in 19BR and 213BR and also resulted in a reduced rate of NPN uptake in these isolates and in PAO1 but not in 9BR. The results presented here suggest that the skipped-well isolates have the ability to adapt to specific concentrations of polymyxin B, inducing known polymyxin B resistance genes involved in generating alterations in the outer membrane.

Antibiotic resistance and trend of urinary pathogens in general outpatients from a major urban city

OBJECTIVE We assessed the antimicrobial resistance patterns of pathogens responsible for urinary tract infections (UTI) in outpatients in São Paulo, Brazil, as well as the Escherichia coli antimicrobial resistance trend. MATERIALS AND METHODS Outpatients urine cultures were collected from January 2000 to December 2003. Statistical analysis considered positive results for one bacterial species with colony count >or= 100,000 CFU/mL. Stratification was done on age group and gender. Statistical tests used included chi-square and the chi-square test for trend to evaluate differences between susceptibility rates among age groups and ordering in the E. coli resistance rates per year, respectively. RESULTS There were 37,261 positive results with Enterobacteriaceae isolated in 32,530 (87.3%) and Gram-positive cocci in 2,570 (6.9%) cultures. E. coli had the highest prevalence (71.6%). Susceptibility tests were performed in 31,716 cultures. E. coli had elevated resistance rates (> 30%) to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. Significant differences between age groups and ordering among years were observed. CONCLUSIONS The use of trimethoprim-sulfamethoxazole is precluded in the population studied due to elevated resistance rates (> 30%) among most prevalent pathogens. Significant resistance rate differences among age groups and years were observed, particularly for fluoroquinolones. Fluoroquinolones should be used with caution. Nitrofurantoin should be used as empirical therapy for primary, non-complicated urinary tract infections.

Antimicrobial susceptibility of Gram-negative bacteria in Brazilian hospitals: the MYSTIC Program Brazil 2003

Establish the susceptibility pattern of Gram-negative bacteria causing infections in ICU patients, MYSTIC Program Brazil 2003. Gram-negative bacteria (n = 1,550) causing nosocomial infections were collected at 20 Brazilian centers. The central laboratory confirmed the identification and performed the susceptibility tests by Etest methodology (AB Biodisk, Solna, Sweden) for meropenem, imipenem, ciprofloxacin, ceftazidime, cefepime, cefotaxime, piperacillin/tazobactam, gentamicin, and tobramycin. Interpretation criteria used were according to National Committee for Clinical Laboratory Standards (NCCLS). Pseudomonas aeruginosa (30.3%) was the most frequent isolate, followed by E. coli (18.6%), Klebsiella pneumoniae (16.9%), Acitenobacter baumannii (8.8%), and Enterobacter cloacae (7.1%). Pseudomonas aeruginosa (n=470) isolates presented susceptibility rates of 64% to meropenem, 63.8% to piperacillin/tazobactam, 63.4% to amikacin, 58.7% to imipenem. Acitenobacter baumannii presented susceptibility rates to meropenem of 97.1%, and 73% to tobramycin. E. coli and K. pneumoniae were highly susceptible to both carbapenems. Carbapenem resistance among the Enterobacteriaceae is still rare in the region. Acitenobacter baumannii and P. aeruginosa presented elevated resistance rates to all antimicrobials. Since they play an important role in nosocomial infections in this environment, the use of empirical combination therapy to treat these pathogens may be justified.

Antimicrobial susceptibility in intensive care units: MYSTIC Program Brazil 2002

OBJECTIVE Establish the susceptibility pattern of Gram-negative bacteria causing infections in ICU patients, MYSTIC Program Brazil 2002. MATERIAL AND METHODS Gram-negative bacteria (n = 503) causing nosocomial infections were collected at seven Brazilian centers. The central laboratory confirmed the identification and performed the susceptibility tests by E-test methodology (AB Biodisk, Solna, Sweden) for meropenem, imipenem, ciprofloxacin, ceftazidime, cefepime, cefotaxime, piperacillin/tazobactam, gentamicin, and tobramycin. Interpretation criteria used were according to National Committee for Clinical Laboratory Standards (NCCLS). RESULTS Pseudomonas aeruginosa (33%) was the most frequently isolated, followed by A. baumannii (17.1%), K. pneumoniae (12.1%), E. coli (10.5%), and E. cloacae (7.9%). Pseudomonas aeruginosa isolates had susceptibility rates of 67.5% to piperacillin/tazobactam, 59.8% to meropenem, 57.3% to imipenem. A. baumannii presented susceptibility rates to meropenem of 89.5%, 88.4% to imipenem, and 74.4% to tobramycin. E. coli and K. pneumoniae were fully susceptible to both carbapenems. CONCLUSIONS Carbapenem resistance among Enterobacteriaceae is still rare in this region. A. baumannii and P. aeruginosa presented elevated resistance rates to all antimicrobials. Since these two bacterial species play an important role in nosocomial infections, the use of empirical combination therapy to treat these pathogens may be justified.

Klebsiella pneumoniae with multiple antimicrobial resistance

A Klebsiella pneumoniae strain was isolated from the urine of a patient at one of the centers participating in the 2001 edition of the MYSTIC program in Brazil. The initial phenotypic findings of the isolated K. pneumoniae presented an unusual MIC of 8 microg/mL to meropenem, 2 microg/mL to imipenem, elevated MICs to broad spectrum cephalosporins (ceftazidime/cefotaxime/cefepime MIC > 256 microg/mL), aminoglycosides (gentamycin > 256 microg/mL and tobramycin = 48 microg/mL), piperacillin/tazobactam (MIC > 256 microg/mL) and susceptibility to ciprofloxacin (MIC = 0.25 microg/mL). The strain also tested positive for ESBL production with double-disk and E-test methodologies. More detailed investigation revealed that the strain produced a SHV-4 type enzyme and also lacked a 36 kDa outer membrane porin.

Estimated prevalence of immunity to poliomyelitis in the city of Säo Paulo, Brazil: a population-based survey

Objectives. Estimate the prevalence of immunity to poliomyelitis (anti-polio antibodies) in the city of São Paulo/Brazil through a population-based survey. Methods. A quantitative and inductive method was used to draw a representative sample of the population. Randomization and stratification (based on sex, age and residence region) was done, and 1,059 individuals were studied on a home-visit basis (structured questionnaires and blood samples). A microneutralization test was performed to detect anti-polio antibodies against serotypes 1, 2 and 3. Results. The estimated prevalence of immunity to poliomyelitis was high, with 94.6% prevalence of anti-polio 1 antibodies, 98.8% anti-polio 2 and 91.9% anti-polio 3. Despite this high prevalence, there were significantly lower prevalence levels in some groups, specially among age and residence region groups. Discussion. Routine child immunization and NIDs with OPV have provided excellent levels of serological immunity to poliomyelitis in the population of the city of São Paulo, Brazil. However, there may be specific groups with a lower prevalence of immunity. Estimations of the prevalence of immunity to poliomyelitis were made in a population-based survey, which could be used as an auxiliary tool for supporting the polio eradication program.

A pharmacodynamic strategy to optimize empirical antibiotic therapy for gram-negative bacteria in a Brazilian Intensive Care Unit

Pharmacodynamic analyses were proposed to determine optimal empirical antibiotic therapy against Gram-negative bacteria isolated in a Brazilian ICU. Due to high resistance rates, standard regimens of cefepime, ciprofloxacin, meropenem, and piperacillin/tazobactam were not able to attain significant bactericidal CFR. Prolonged infusion of meropenem achieved 88% CFR, making it a possible empirical regimen in this ICU until susceptibilities become available. Still, even through administration of high dose prolonged infusions, 12.0% of simulated subjects did not achieve bactericidal exposure, suggesting that combination therapy would frequently be required in this setting. In conclusion, we recommend that in the presence of identified resistance problems among Gram-negative bacteria in a unit or hospital, MIC testing of formulary agents should be conducted along with pharmacodynamic simulation to assist in choosing an optimal antibiotic and dosage regimen for empirical use of severe infections until cultures and susceptibilities become available.

Treatment of nosocomial pneumonia: an experience with meropenem

This study aimed at evaluating the efficacy and safety of meropenem as first choice treatment for nosocomial pneumonia (NP) in intensive care units (ICU) in Hospital das Clínicas (HC) - University of São Paulo; a hospital with high incidence of antimicrobial resistance. Prospective, open, and non-comparative trial with meropenem were done in patients with ventilator-associated or aspiration NP in 2 ICUs at HC - University of São Paulo. Etiologic investigation was done through bronchoalveolar lavage and blood cultures prior to study entry. Twenty-five (25) critically ill patients with NP were enrolled (mean age 40 years). Ventilator-acquired pneumonia was responsible for 76% of cases and aspiration NP for 24%. Specific etiologic agents were identified and considered to be clinically and temporally responsible for NP in 11 (44%) patients. A. baumanii was responsible for 6 cases (55%), P. aeruginosa for 3 (27%), and S. aureus for 2 (18%). At completion of treatment, 19 patients (76%) showed either cure (48%) or improvement (28%) after use of meropenem therapy. Mortality was 12% at the end of therapy (8% after excluding 1 non-evaluable patient). After 4 to 6 weeks of follow-up, 12 (48%) patients had improved or been totally cured, and overall mortality was 24%. Clinical complications were observed in 11 patients (44%), with none of them definitely related to the study drug. Meropenem as monotherapy was effective and well-tolerated in most NP patients in our ICU. The low mortality rate in this study might have been due to first choice use of this drug. Controlled, drug comparative clinical trials are needed to support this preliminary observation.