Afonso Luis Barth

Pathogen frequency and resistance patterns in Brazilian hospitals: summary of results from three years of the SENTRY antimicrobial surveillance program

BACKGROUND Pathogen frequency and resistance patterns may vary significantly from country to country and also in different hospitals within a country. Thus, regional surveillance programs are essential to guide empirical therapy and infection control measures. METHODS Rank order of occurrence and antimicrobial susceptibility of pathogenic species causing bloodstream infections (BSI), lower respiratory tract infections (LRTI), wound or skin and soft tissue infections (WSSTI), and urinary tract infections (UTI) in hospitalized patients were determined by collecting consecutive isolates over a specified period of time, as part of the SENTRY Antimicrobial Resistance Surveillance Program (SENTRY). All isolates were tested by reference broth microdilution. RESULTS AND CONCLUSIONS A total of 3,728 bacterial strains were obtained from January, 1997, to December, 1999, from 12 Brazilian hospitals located in 4 states. The largest number of isolates were obtained from patients with BSI (2,008), followed by LRTI (822 cases), UTI (468 cases), and WSSTI (430 cases). Staphylococcus aureus was the most frequently isolated pathogen in general (22.8% - 852 isolates), followed by E. coli (13.8% - 516 cases) and Pseudomonas aeruginosa (13.3% - 496 cases). Staphylococcus aureus was also the most common species isolated from BSI (23.6%) and WSSTI (45.8%), and P. aeruginosa was the most frequent species isolated from patients with LRTI (29.4%). The main bacterial resistance problems found in this study were: imipenem resistance among P. aeruginosa (69.8% susceptibility) and Acinetobacter spp. (88.1% susceptibility); ESBL production among K. pneumoniae (48.4%) and E. coli (8.9%); resistance to third generation cephalosporins among Enterobacter spp. (68.1% susceptible to ceftazidime) and oxacillin resistance among S. aureus (34.0%) and coagulase negative staphylococci (80.1%). Only the carbapenems (88.1% to 89.3% susceptibility) showed reasonable activity against the Acinetobacter spp. isolates evaluated.

Dissemination of blaKPC-2 by the Spread of Klebsiella pneumoniae Clonal Complex 258 Clones (ST258, ST11, ST437) and Plasmids (IncFII, IncN, IncL/M) among Enterobacteriaceae Species in Brazil

ABSTRACT This article reports the spread of blaKPC-2 in the Sao Paulo and Rio de Janeiro states, facilitated by globally spread K. pneumoniae clonal complex 258 (CC258) clones (ST258, ST11, and ST437) and a diversity of plasmids (IncFII, IncN, and IncL/M, two untypeable plasmids carrying Tn4401a or Tn4401b) successfully disseminated among species of the Enterobacteriaceae (Enterobacter cloacae, Serratia marcescens, and Citrobacter freundii). It also constitutes the first description of sequence type 258 (ST258) in Brazil, which was associated with a nosocomial hospital outbreak in Ribeirao Preto city.

Pharmacokinetics of Intravenous Polymyxin B in Critically Ill Patients

BACKGROUND Although not much pharmacokinetic knowledge is available, polymyxin B is increasingly used for treatment of infections caused by gram-negative bacteria that are resistant to all other antibiotics. METHODS This study involved 8 patients who received intensive care after intravenous administration of a 60-min infusion of polymyxin B at currently recommended doses. Blood and urine samples were collected, and plasma protein binding of polymyxin B was determined. Concentrations of polymyxin B in plasma and urine samples were measured by a specific high-performance liquid chromatographic method. RESULTS Polymyxin B was well tolerated. The peak plasma concentrations at the end of the infusion varied from 2.38 to 13.9 mg/L. For 4 patients from whom it was possible to collect urine samples over a dosing interval, only 0.04%-0.86% of the dose was recovered in the urine in unchanged form. Plasma protein binding of polymyxin B was higher in samples from patients (range, 78.5%-92.4%) than in plasma samples from healthy human subjects (mean +/- standard deviation, 55.9% +/- 4.7%). Unbound plasma concentrations of polymyxin B were in the vicinity of or lower than the minimum inhibitory concentration of the pathogen. CONCLUSION To our knowledge, this is the first study to report plasma concentrations over time and urinary recovery of polymyxin B in critically ill patients after intravenous administration. Polymyxin B is eliminated mainly by nonrenal pathways, and the total body clearance appears to be relatively insensitive to renal function. Additional investigations are required to assess the appropriateness of currently recommended doses of this drug for the treatment of severe infections in critically ill persons.

Antimicrobial susceptibility of gram-positive bacteria isolated in brazilian hospitals participating in the SENTRY Program (2005-2008)

We report the antimicrobial susceptibility patterns of the most frequently isolated Gram-positive bacteria in the Brazilian hospitals participating in the SENTRY Antimicrobial Surveillance Program. The strains were consecutively collected (one per patient) between January 2005 and September 2008 and susceptibility tested by reference broth microdilution methods at the JMI Laboratories (North Liberty, Iowa, USA). A total of 3,907 Gram-positive cocci were analyzed. The Gram-positive organisms most frequently isolated from bloodstream infections were Staphylococcus aureus (2,218 strains; 20.2% of total), coagulase-negative staphylococci (CoNS; 812 strains [14.7%]), and Enterococcus spp. (754 strains; 5.0%). S. aureus ranked first (28.1%) and Enterococcus faecalis ranked 7th (4.5%) among cases of skin and soft tissue infections. S. aureus was also the second most frequently isolated pathogen from patients with lower respiratory tract infections (24.9% of cases) after Pseudomonas aeruginosa (30.5%). Resistance to oxacillin was observed in 31.0% of S. aureus and the vast majority of oxacillin-resistant (MRSA) strains were also resistant to clindamycin, ciprofloxacin and levofloxacin. Vancomycin, linezolid and daptomycin were all very active against S. aureus strains tested (>99.9-100.0% susceptible), but daptomycin (MIC(50), 0.25 g/mL and MIC(90), 0.5 g/mL) was four- to eight-fold more potent than vancomycin (MIC(50) and MIC(90) of 1 g/mL) and linezolid (MIC(50), 1 g/mL and MIC(90), 2 g/mL). Vancomycin resistance increased significantly among enterococci during the study period, but it was restrict to only one medical center until 2007 and emerged in a second medical center in 2008. Daptomycin was the most active antimicrobial tested against enterococci in general (100.0% susceptible), followed by linezolid (99.9% susceptible), ampicillin (87.4%) and vancomycin (84.6%). In conclusion, daptomycin and linezolid showed excellent in vitro activity against contemporary Gram-positive organisms (3,907) collected in Brazilian hospitals monitored by the SENTRY Program, including MRSA, vancomycin-resistant enterococci (VRE) and other multi-drug-resistant organisms. Although vancomycin resistance rates in Brazil appears to be relatively low compared to those reported in the USA, VRE has emerged and rapidly disseminated in some Brazilian medical centers.

Reappraisal of Pseudomonas aeruginosa hospital-acquired pneumonia mortality in the era of metallo-β-lactamase-mediated multidrug resistance: a prospective observational study

IntroductionHospital-acquired pneumonia (HAP) due to Pseudomonas aeruginosa is associated with high mortality rates. The metallo-β-lactamases (MBLs) are emerging enzymes that hydrolyze virtually all β-lactams. We aimed to assess P. aeruginosa HAP mortality in a setting of high-rate MBL productionMethodsA prospective cohort study was performed at two tertiary-care teaching hospitals. A logistic regression model was constructed to identify risk factors for 30-day mortality.ResultsOne-hundred and fifty patients with P. aeruginosa HAP were evaluated. The 30-day mortality was 37.3% (56 of 150): 57.1% (24 of 42) and 29.6% (32 of 108) for patients with HAP by MBL-producing P. aeruginosa and by non-MBL-producing P. aeruginosa, respectively (relative risk, 1.93; 95% confidence interval (CI), 1.30–2.85). The logistic regression model identified a higher Charlson comorbidity score (odds ratio, 1.21; 95% CI, 1.04–1.41), presentation with severe sepsis or septic shock (odds ratio, 3.17; 95% CI, 1.30–7.72), ventilator-associated pneumonia (odds ratio, 2.92; 95% CI, 1.18–7.21), and appropriate therapy (odds ratio, 0.24; 95% CI, 0.10–0.61) as independent factors for 30-day mortality. MBL production was not statistically significant in the final model.ConclusionMBL-producing P. aeruginosa HAP resulted in higher mortality rates, particularly in patients with ventilator-associated pneumonia, most probably related to the less frequent institution of appropriate antimicrobial therapy. Therapeutic approaches should be reviewed at institutions with a high prevalence of MBL.

Prevalence, susceptibility profile for fluconazole and risk factors for candidemia in a tertiary care hospital in southern Brazil

Bloodstream infections caused by yeast, Candida spp, are quite important clinically and epidemiologically due to a high mortality rate and an increasing number of non-albicans species with a more resistant (differentiated susceptibility) profile. We examined species prevalence and susceptibility profile for fluconazole and the risk for nosocomial infections by Candida spp at the Hospital de Clínicas de Porto Alegre, a general tertiary care hospital in southern Brazilian, through a retrospective study, beginning with positive cultures of hospitalized patients. The distribution by species in 131 documented episodes was as follows: Candida albicans (45%), C. parapsilosis (24.4%), C. tropicalis (15.3%), C. glabrata (6.9%), C. krusei (4.6%) and 3.8% other species (C. pelicullosa, C. guilliermondii, C. lusitaniae and C. kefyr). The vast majority of samples (121- 92.4%) were susceptible to fluconazole; the resistant or dose-dependent sensitive samples included only C. krusei and C. glabrata. Blood diseases (leukemia, lymphoma), or neoplasias (solid tumors), were found in 35.0% of the candidemia episodes. We noted the previous use of antibiotics in 128 (97.7%) patients, with 79.7% using three or more antibiotics before the candidemia episode. Other risk factors included a central venous catheter in 94 (71.8%) and abdominal surgery in 32 (24.4%) patients. The overall mortality rate was 51.9%, which varied according to the underlying disease. We found that C. albicans was the most prevalent species, although the non-albicans species predominated. However, in vitro resistance to fluconazole was detected only among the species (C. glabrata and C. krusei) that tend to be resistant to the azolic compounds. Previous use of antibiotic and the use of a central venous catheter were the main risk factors among patients with candidemia.

EVALUATION OF THREE MOLECULAR TYPING TECHNIQUES FOR NONFERMENTATIVE GRAM-NEGATIVE BACILLI

OBJECTIVE To evaluate three different DNA techniques for typing nonfermentative gram-negative bacilli isolated from Latin American hospitals. DESIGN One hundred twenty-six nonfermentative gram-negative bacilli were typed. PARTICIPANTS Pseudomonas aeruginosa (n = 64) and Acinetobacter baumannii (n = 42) samples were obtained from blood cultures of patients admitted to 10 medical centers in Latin America during 1998 and Stenotrophomonas maltophilia (n = 20) samples were obtained from patients admitted to the Hospital São Paulo between 1999 and 2001. METHODS All samples were typed using automated ribotyping, PFGE, and ERIC-PCR. The discriminatory power for each technique was calculated using Hunters generalized formula. RESULTS All strains could be typed by automated ribotyping and ERIC-PCR, but two strains (1.6%) were not typeable by PFGE. All three techniques showed 100% reproducibility. The time to obtain the results was shorter for automated ribotyping and ERIC-PCR compared with PFGE. Likewise, the costs for ERIC-PCR and PFGE were lower than those for automated ribotyping. The interpretation of results was more complicated and more difficult with ERIC-PCR than with both PFGE and automated ribotyping. All techniques presented excellent discriminatory power for P. aeruginosa (0.98). PFGE presented the highest discriminatory power (0.94) for A. baumannii, and both PFGE and ERIC-PCR showed higher discriminatory power (0.90 for both) than automated ribotyping (0.82) for S. maltophilia. CONCLUSIONS PFGE showed the highest discriminatory power for typing these nonfermentative gram-negative bacilli. However, automated ribotyping and ERIC-PCR can provide results in a shorter time period with similar discriminatory power.

Carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme: Dissemination in Southern Brazil

Acinetobacter baumannii has emerged as an important nosocomial pathogen due to its ability of long-term survival in the hospital environment, which facilitate spreading, causing institutional outbreaks, and due to its acquired multiple mechanisms of antimicrobial resistance [1]. A number of factors such as immunosuppression, use of invasive devices, and the use of antimicrobial agents have been reported as increasing the risk of infection or colonization by this opportunistic pathogen [2]. The carbapenems used to be an effective therapeutic option for the treatment of Acinetobacter spp. infections. However, in the last few years, carbepenem-resistant Acinetobacter species have been recovered worldwide. Loss of porins, alterations in the penicillin-binding protein (PBP) affinity, and different class B (metalloenzymes) and D (OXA enzymes) b-lactamases have been associated with resistance to carbapenems in A. baumannii [3]. The OXA carbapenemases of Acinetobacter are divided into four phylogenetic subgroups: OXA-23, OXA24 and OXA-58 (acquired enzymes), and OXA-51 that is intrinsic to A. baumannii [4]. In Brazil, the first report of acquired oxacillinase has been described in Curitiba [5] in 2003, but carbapenem resistance seems to constitute an emerging problem in other regions of the country and in Latin America [6]. In Porto Alegre, a city with 1.4 million inhabitants and 7,700 beds situated in 25 hospitals, the first isolate of carbapenem-resistant A. baumannii (CRAB) was identified only in 2004, but, further, CRAB has been reported to the local health department in an unprecedented outbreak involving 16 hospitals and more than 500 detected cases from 2004 to 2008 [7]. The goal of this study was to investigate the mechanisms of emerging resistance to carbapenems in multiresistant A. baumannii isolates and to characterize the molecular type using pulsed-field gel electrophoresis (PFGE). A total of 53 CRAB isolates were obtained from patients attending two university tertiary teaching hospitals in Porto Alegre from July to December 2007 (one isolate per patient). Three isolates were obtained from the environment in hospital 2 through surveillance culture during an outbreak investigation. Isolates were identified using the GN32 card using the API 20NE system (bioMérieux, Basingstoke, United Kingdom). Antimicrobial susceptibility testing was performed by the disk diffusion method, as described by the Clinical Laboratory Standards Institute (CLSI) [8]. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were established using the E-test. The presence of genes coding for blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaOXA-51-like were detected by multiplex polymerase chain reaction (PCR) as described previously with minor modifications [9]. Positive controls included Acinetobacter spp. strains known to produce OXA-23-like, OXA-24-like, and OXA-58-like enzymes [5], and the type strain of A. baumannii ATCC 19606 (negative control) for these genes and positive for OXA-51-like (intrinsic to A. baumannii). All three isolates from the hospital environment, as well as a set of 13 clinical isolates of CRAB, randomly selected from all clinical isolates, which were stored at –80 C, were selected for molecular typing. Macrorestriction analysis of chromosomal DNA with SmaI (Invitrogen, Paisley,

Dissemination of Pseudomonas aeruginosa Producing SPM-1-like and IMP-1-like Metallo-β-lactamases in Hospitals from Southern Brazil

Background:Metallo-β-lactamase (MBL) is an emerging resistance mechanism among Pseudomonas aeruginosa. The prevalence of this mechanism is particularly high in Latin America. We aimed to describe the prevalence and molecular characteristics of SPM-1-like, IMP-1-like and VIM type MBLs among ceftazidime and/or imipenem-resistant nosocomial P. aeruginosa isolates.Methods:Pseudomonas aeruginosa isolates resistant to ceftazidime and/or imipenem recovered from hospitalized patients from two teaching hospitals from Porto Alegre, Brazil, were prospectively selected. Isolates were tested for MBL production using two phenotypic screening tests. Those isolates with positive results were further tested for the presence of MBL genes (SPM-1-like, IMP-1-like and VIM type) and submitted to molecular typing.Results:A total of 92 isolates were analyzed and 33 (35.9%) were presumptively MBL producers by phenotypic tests. The SPM-1-like gene was found in 18 isolates and IMP-1-like in 5 isolates. In ten isolates the MBL type could not be identified. Three IMP-1-like isolates were susceptible to imipenem. SPM-1-like isolates comprised a single clone, and IMP-1-like isolates another single clone.Conclusion:The prevalence of MBL production among ceftazidime-resistant P. aeruginosa isolates is relatively high in both hospitals. Infection control measures have been challenged and further improvements in such measures are required to prevent dissemination of these isolates among hospitals. This is the first report of IMP-1-like MBLs in P. aeruginosa in southern Brazil.

In Vitro Antioxidant, Anticoagulant and Antimicrobial Activity and in Inhibition of Cancer Cell Proliferation by Xylan Extracted from Corn Cobs

Xylan is one of most abundant polymer after cellulose. However, its potential has yet to be completely recognized. Corn cobs contain a considerable reservoir of xylan. The aim of this work was to study some of the biological activities of xylan obtained from corn cobs after alkaline extraction enhanced by ultrasonication. Physical chemistry and infrared analyses showed 130 kDa heteroxylan containing mainly xylose:arabinose: galactose:glucose (5.0:1.5:2.0:1.2). Xylan obtained exhibited total antioxidant activity corresponding to 48.5 mg of ascorbic acid equivalent/g of xylan. Furthermore, xylan displayed high ferric chelating activity (70%) at 2 mg/mL. Xylan also showed anticoagulant activity in aPTT test. In antimicrobial assay, the polysaccharide significantly inhibited bacterial growth of Klebsiella pneumoniae. In a test with normal and tumor human cells, after 72 h, only HeLa tumor cell proliferation was inhibited (p < 0.05) in a dose-dependent manner by xylan, reaching saturation at around 2 mg/mL, whereas 3T3 normal cell proliferation was not affected. The results suggest that it has potential clinical applications as antioxidant, anticoagulant, antimicrobial and antiproliferative compounds.