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Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2μg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125μg/mL for both antimicrobials. The MIC90 were 4μg/mL and 2μg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil., (Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.)

Plasmid-mediated AmpC β-lactamases (PMACBLs) in Enterobacteriaceae encode resistance to third-generation cephalosporins, and these can mediate carbapenem resistance when associated with porin loss. However, no standardized phenotypic method is available for detecting these enzymes in the clinical microbiology laboratory. Limited data are available concerning the frequency of PMACBLs in Enterobacteriaceae in Brazil. This study was conducted in response to an increased cefoxitin (CFO) resistance rate of 3.7% in Escherichia coli isolates from urine samples from patients with suspected urinary tract infections during 2010. We collected 2,266 E. coli isolates prospectively during January 2012. A total of 109 (4.8%) isolates were nonsusceptible to CFO. These strains were further examined using multiplex PCR for the presence of genes encoding PMACBLs and using inhibitor assays with CFO and ceftazidime (CAZ) disks with and without phenylboronic acid. Pulsed-field gel electrophoresis was used to evaluate clonal dissemination. Genes encoding PMACBLs were detected in 1.8% of the isolates from inpatients and 0.46% of isolates from outpatients. The most prevalent gene was blaCMY-2 and blaCMY-4 was also detected. The phenotypic analysis showed 100% sensitivity and specificity for CMY-2 and CMY-4 when CFO-resistant isolates with a minimum zone diameter difference of 5 mm for CAZ or CAZ and CFO were considered positive. Although most of the isolates were nonclonal, one clonal group with two isolates was observed. Thus, the most frequent PMACBL in E. coli from São Paulo, Brazil is CMY-2, and both clonal and plasmid-mediated dissemination occur.

In Enterobacteriaceae, the blaNDM genes have been found in many different genetic contexts, and a wide diversity of plasmid scaffolds bearing those genes has been found. In August 2013, we identified NDM-1-producing Escherichia coli and Enterobacter hormaechei strains from a single rectal swab sample from a patient hospitalized in Rio de Janeiro, Brazil, who had no history of travel abroad. Complete DNA sequencing using the Illumina platform and annotation of the two plasmids harboring the blaNDM-1 gene, one from each strain, showed that they belonged to incompatibility groups IncFIIK and IncX3 and harbored a novel transposon named Tn3000. Similar genetic structures have been identified among other isolates in Brazil but also on plasmids from other continents. Our findings suggest that the blaNDM-1 gene may be transmitted by Tn3000 in different parts of the world., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We sequenced the oldest blaKPC-2-bearing plasmid isolated in Brazil and another plasmid also carried by a Klebsiella pneumoniae strain of sequence type 442 (ST442), isolated 52 months later. Both plasmids present an IncN backbone and few acquired regions. Because the 2005 plasmid presented deletions and a truncated gene within Tn4401b compared to the 2009 plasmid, we can thus infer that IncN blaKPC-2-bearing plasmids pFCF1305 and pFCF3SP had a common ancestor circulating in Brazil prior to May 2005.

An epidemic of infections after video-assisted surgery (1,051 possible cases) caused by rapidly growing mycobacteria (RGM) and involving 63 hospitals in the state of Rio de Janeiro, Brazil, occurred between August 2006 and July 2007. One hundred ninety-seven cases were confirmed by positive acid-fast staining and/or culture techniques. Thirty-eight hospitals had cases confirmed by mycobacterial culture, with a total of 148 available isolates recovered from 146 patients. Most (n = 144; 97.2%) isolates presented a PRA-hsp65 restriction pattern suggestive of Mycobacterium bolletii or Mycobacterium massiliense. Seventy-four of these isolates were further identified by hsp65 or rpoB partial sequencing, confirming the species identification as M. massiliense. Epidemic isolates showed susceptibility to amikacin (MIC at which 90% of the tested isolates are inhibited [MIC(90)], 8 microg/ml) and clarithromycin (MIC(90), 0.25 microg/ml) but resistance to ciprofloxacin (MIC(90), >or=32 microg/ml), cefoxitin (MIC(90), 128 microg/ml), and doxycycline (MIC(90), >or=64 microg/ml). Representative epidemic M. massiliense isolates that were randomly selected, including at least one isolate from each hospital where confirmed cases were detected, belonged to a single clone, as indicated by the analysis of pulsed-field gel electrophoresis (PFGE) patterns. They also had the same PFGE pattern as that previously observed in two outbreaks that occurred in other Brazilian cities; we designated this clone BRA100. All five BRA100 M. massiliense isolates tested presented consistent tolerance to 2% glutaraldehyde. This is the largest epidemic of postsurgical infections caused by RGM reported in the literature to date in Brazil.

The frequency and severity of human infections associated with Corynebacterium ulcerans appear to be increasing in different countries. Here, we describe the first C. ulcerans strain producing a diphtheria-like toxin isolated from an elderly woman with a fatal pulmonary infection and a history of leg skin ulcers in the Rio de Janeiro metropolitan area.

Summary: We investigated an outbreak caused by non-tuberculous mycobacteria (NTM) related to breast implant surgery in the city of Campinas, Brazil, by means of a retrospective cohort and molecular epidemiological study. A total of 492 records of individuals having breast surgery in 12 hospitals were evaluated. Twelve isolates were analysed using four different molecular typing methods. There were 14 confirmed cases, 14 possible cases and one probable case. One probable, nine possible and 12 confirmed cases were included in a cohort study; all occurred in eight of the hospitals and the confirmed cases in five. Univariate analysis showed that patients who had had breast reconstruction surgery in hospitals A and B were more likely to have NTM infections. No risk factor was independently associated with NTM infection in the multivariate model. The isolates obtained from patients at each hospital showed different molecular patterns, excluding isolates from hospital C that repeatedly showed the same genotype for approximately one year. In conclusion, this outbreak was caused by polyclonal strains at different institutions, and in one hospital a unique genotype caused most cases. No specific risk factors were found.

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.

A cluster of cases of post-augmentation mammaplasty surgical site infections occurred between 2002 and 2004 in Campinas, in the southern region of Brazil. Rapidly growing mycobacteria were isolated from samples from 12 patients. Eleven isolates were identified as Mycobacterium fortuitum and one as Mycobacterium porcinum by PCR-restriction digestion of the hsp65 gene. These 12 isolates, plus six additional M. fortuitum isolates from non-related patients, were typed by pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: 16S-23S rRNA internal transcribed spacer (ITS) genotyping; randomly amplified polymorphic DNA (RAPD) PCR; and enterobacterial repetitive intergenic consensus (ERIC) PCR. Four novel M. fortuitum allelic variants were identified by restriction analysis of the ITS fragment. One major cluster, comprising six M. fortuitum isolates, and a second cluster of two isolates, were identified by the four methods. RAPD-PCR and ITS genotyping were less discriminative than ERIC-PCR. ERIC-PCR was comparable to PFGE as a valuable complementary tool for investigation of this type of outbreak.

Cases of human diphyllobothriasis have been reported worldwide. Only 1 case in Brazil was diagnosed by our institution from January 1998 to December 2003. By comparison, 18 cases were diagnosed from March 2004 to January 2005. All patients who became infected ate raw fish in sushi or sashimi.

Purpose: To evaluate the in vitro activity of fluoroquinolones against Mycobacterium abscessus and Mycobacterium chelonae isolated from outbreaks of infectious keratitis in Brazil., Material and Methods: Micobacterial isolates were recovered from infectious keratitis cases related outbreaks that occurred in Brazil after LASIK for myopia. Two outbreaks occurred in Rio de Janeiro in 1998 and 1999, and 3 in São Paulo between 2000 and 2003. All laboratorial analysis, including molecular identification and antibiotic susceptibility testing with determination of the minimum inhibitory concentration (MIC) levels for ciprofloxacin, ofloxacin, gatifloxacin, and moxifloxacin, were performed at Universidade Federal de São Paulo in Brazil., Results: Fifteen samples were identified as M. chelonae, and 3 were identified as M. abscessus. The outbreaks studied were designated SP-1 in 2000; SP-2 in 2000-2001; and SP-3 in 2003, R1 in 1988 and R2 in 1999. All but 1 of the M. chelonae were resistant to all fluoroquinolones with an MIC90 greater than 32 microg/mL. The only susceptible isolate had MIC levels for ciprofloxacin, ofloxacin, gatifloxacin, and moxifloxacin of 0.38 microg/mL, 0.032 microg/mL, 0.047 microg/mL, and 0.19 microg/mL, respectively. MIC levels for all 3 M. abscessus isolates tested were greater then 32 microg/mL for all fluoroquinolones tested., Conclusions: Fluoroquinolone MICs for 17 M. abscessus and M. chelonae isolates recovered from infectious keratitis cases in Brazil indicate that they are not susceptible to these drugs in vitro. Further studies to investigate the in vivo effectiveness of fluoroquinolones against mycobacteria are required because in vitro tests do not support their use in the treatment of micobacterial keratitis in this particular geographic area.

Objective: Investigate clonal dissemination of nosocomial multidrug-resistant Pseudomonas aeruginosa isolates within and between Brazilian intensive care units, which participated in the MYSTIC Program Brazil 2002., Methods: Thirty-six P. aeruginosa isolates resistant to meropenem or imipenem plus at least two of the following drugs: ciprofloxacin, cefepime, ceftazidime or piperacillin/tazobactam were isolated during 2002 at 4 centres in São Paulo and 1 centre in Brasília. Chromosomal restriction fragments obtained with SpeI were separated by pulsed-field gel electrophoresis (PFGE). Electrophoretic patterns were analyzed with GelCompar II v. 2.5., Results: Five major clones were identified (A, B, C, D, G). Clone A was constituted by 8 isolates with indistinguishable PFGE pattern present in 2 centres. Clone B was constituted by 4 indistinguishable isolates predominant in centre 6. Clone C had 3 indistinguishable isolates, with closely related clones (C1-3). Also, Clone D had 3 indistinguishable isolates, with closely related (D1) and possibly related (D2/D3) clones. Clones C and D were present in centre 1. Clone G was constituted by 2 indistinguishable isolates and was present in centre 7. Finally, 8 isolates were unique. Isolates from Centre 4 were unique., Conclusions: Clonal dissemination was detected within (clones A, B, C, D, and G) and between centres (clone A). These findings are important when analyzing surveillance data, since susceptibility rates may be significantly affected by the dissemination of a resistant clone.

Ralstonia pickettii and Burkholderia cepacia complex isolates are causes of healthcare-associated infection related to contamination of intravenously administered products. Based on microbiological and epidemiological data and molecular typing by pulsed-field gel electrophoresis, we report the occurrence of two outbreaks of R. pickettii and B. cepacia complex bloodstream infections. The first outbreak occurred from August 1995 to September 1996, and the second outbreak occurred from 28 March to 8 April 1998, affecting adults and neonates, respectively. Infusion of contaminated water for injection was the source of infection.

We investigated the occurrence and phenotypic and genotypic characteristics of erythromycin-resistant Streptococcus pneumoniae strains isolated in three major states in Brazil, from 1990 to 1999. Of the 931 pneumococcal strains evaluated, 40 (4.3%) were erythromycin-resistant (Ery-R). Among the 40 Ery-R strains, 90.0%, 80.0%, 27.5%, 5.0%, and 2.5% were resistant to tetracycline, trimethoprim-sulfamethoxazole, penicillin, chloramphenicol, and rifampin, respectively. None of the strains were resistant to ofloxacin or to vancomycin. Most [37 (92.5%)] of the 40 Ery-R isolates presented the MLS(B) phenotype and 3 (7.5%) strains showed the M phenotype. PCR testing indicated that all MLS(B) phenotype isolates harbored the erm(B) gene only, whereas the mef(A/E) gene was present in all isolates presenting the M phenotype. The tet(M) gene was the most frequent (86.1%) among Ery-R isolates that were also resistant to tetracycline. Pulsed-field gel electrophoresis (PFGE) analysis after SmaI digestion revealed the occurrence of clonal relationships within groups of strains belonging to serotypes 14, 19A, and 23F. All Ery-R isolates belonging to serotype 14 were susceptible to penicillin and were included in a single clonal group (named Ery(14)-A) related to the England(14-)9 internationally spread clone.

Aims: To investigate phenotypic aspects including biotyping, drug susceptibility and production of extracellular enzymes and genetic diversity of Stenotrophomonas maltophilia clinical strains obtained from seven hospitals in Rio de Janeiro, Brazil., Methods and Results: Thirty-nine S. maltophilia strains were investigated by biotying, susceptibility testing, extracellular enzymes detection and by randomly amplified polymorphic DNA (RAPD)-PCR. Biotyping distinguished 13 biotypes among 39, and one of them was prevalent. The majority of the strains produced DNase, gelatinase and haemolysin. Protease, lipases and phospholipase C activities were observed in highly variable amounts. None of the strains was elastase producer. The percentage of full susceptibility, by agar dilution, was 100, 94.8, 81.6 and 26.3% for trimethoprim/sulphametoxazole, ticarcillin/clavulanate, ciprofloxacin and ceftazidime, respectively. Thirty-three RAPD-PCR profiles were obtained suggesting multiple sources of acquisition., Conclusions: The results pointed out the necessity of monitoring S. maltophilia especially in critical hospital wards, to assure effective control measures., Significance and Impact of the Study: Despite of the genetic diversity among the strains, in two situations it was observed indistinguishable RAPD-PCR profiles among strains isolated from different patients who had been hospitalized in the same hospital ward, suggesting the possibility of nosocomial transmission that until now has been rarely related.

An outbreak of extended spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBLKp) infections in a neonatal intensive care unit (NICU) prompted a prospective investigation of colonization and infection with this pathogen. From August 1, 1997 to May 30, 1999, neonates admitted to the NICU for more than 24 h were screened for ESBLKp acquisition. Neonatal gastrointestinal screening was performed by means of faecal sampling within 48 h of admission and then weekly until discharge. Isolates were typed using pulsed-field gel electrophoresis (PFGE). Time-dependent proportional hazard models were used to identify independent effects of invasive procedures and antimicrobials after controlling for duration of stay at the NICU. During the study period, 464 neonates were admitted and 383 were regularly screened. Infections occurred in 13 (3.4%) neonates and 206 (53.8%) became colonized. Independent risk factors for colonization during the first nine days in the NICU were the antimicrobial combination cephalosporin plus aminoglycoside [hazard rate (HR)=4.60; 95% CI: 1.48-14.31], and each NICU-day was associated with a 26% increase in the hazard rate for colonization (HR=1.26; 95% CI: 1.16-1.37). Previous colonization (HR=5.19; 95% CI: 1.58-17.08) and central vascular catheter use (HR=13.89; 95% CI: 2.71-71.3) were independent risk factors for infection. In an outbreak setting the proportion of neonates colonized with ESBLKp was observed to increase with the duration of stay and antimicrobial use, and once colonized, infants exposed to invasive devices may become infected.

Multidrug-resistant Pseudomonas aeruginosa nosocomial infections are increasingly recognized worldwide. The existence of metallo-beta-lactamase- and extended-spectrum beta-lactamase-producing isolates exhibiting resistance to most beta-lactam antimicrobial agents greatly complicates the clinical management of patients infected with such isolates. Since 1998, P. aeruginosa isolates resistant to all commercially available antimicrobial agents have been detected at a university-affiliated public hospital in Rio de Janeiro, Brazil. The present study was designed to characterize the antimicrobial resistance profiles and the genetic diversity of the P. aeruginosa strains isolated at this hospital and four private hospitals in Rio de Janeiro. Between April 1999 and March 2000, 200 consecutive isolates were obtained and analyzed for antimicrobial resistance. The genetic diversity of a selected number of them was evaluated by pulsed-field gel electrophoresis and PCR with the ERIC-2 primer. A predominant genotype, designated genotype A, was identified among isolates from four of the five hospitals evaluated. Eighty-four ceftazidime-resistant isolates were evaluated for metallo-beta-lactamase production, which was detected in 20 (91%) of 22 genotype A isolates and 11 (18%) of 62 isolates belonging to other genotypes (P < 0.05). Two metallo-beta-lactamase-producing genotype A isolates also produced an extended-spectrum beta-lactamase. The occurrence of multidrug-resistant P. aeruginosa strains belonging to a unique genotype in different hospitals in Rio de Janeiro underscores the importance of the contribution of a single clone to the increase in the incidence of multidrug-resistant P. aeruginosa nosocomial infections.

Enterobacter hormaechei was defined as a unique species in 1989. We describe six case patients of E. hormaechei bloodstream infection in three neonatal intensive care units in Rio de Janeiro, Brazil. E. hormaechei identification was performed on the Vitek system and confirmed by conventional testing. Strain relatedness was evaluated by pulsed field gel electrophoresis. All children recovered completely. Chart review for previous procedures revealed parenteral nutrition as the only common procedure.

Three clinical strains (Escherichia coli Rio-6, E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two bla(CTX-M) genes encoding beta-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla(CTX-M-9) gene observed in E. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated bla(CTX-M-16), observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240-->Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 microg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 microg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher k(cat) for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two bla(CTX-M-9) genes and the bla(CTX-M-16) gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M beta-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum beta-lactamases in Brazil.

Serratia marcescens Rio-5, one of 18 extended-spectrum beta-lactamase (ESBL)-producing strains isolated in several hospitals in Rio de Janeiro (Brazil) in 1996 and 1997, exhibited a high level of resistance to aztreonam (MIC, 512 microgram/ml) and a distinctly higher level of resistance to cefotaxime (MIC, 64 microgram/ml) than to ceftazidime (MIC, 8 microgram/ml). The strain produced a plasmid-encoded ESBL with a pI of 7.5 whose bla gene was not related to those of other plasmid-mediated Ambler class A ESBLs. Cloning and sequencing revealed a bla gene encoding a novel class A beta-lactamase in functional group 2be, designated BES-1 (Brazil extended-spectrum beta-lactamase). This enzyme had 51% identity with chromosomal class A penicillinase of Yersinia enterocolitica Y56, which was the most closely related enzyme and 47 to 48% identity with CTX-M-type beta-lactamases, which were the most closely related ESBLs. In common with CTX-M enzymes, BES-1 exhibited high cefotaxime-hydrolyzing activity (k(cat), 425 s(-1)). However, BES-1 differed from CTX-M enzymes by its significant ceftazidime-hydrolyzing activity (k(cat), 25 s(-1)), high affinity for aztreonam (K(i), 1 microM), and lower susceptibility to tazobactam (50% inhibitory concentration [IC(50)], 0.820 microM) than to clavulanate (IC(50), 0.045 microM). Likewise, certain characteristic structural features of CTX-M enzymes, such as Phe-160, Ser-237, and Arg-276, were observed for BES-1, which, in addition, harbored different residues (Ala-104, Ser-171, Arg-220, Gly-240) and six additional residues at the end of the sequence. BES-1, therefore, may be an interesting model for further investigations of the structure-function relationships of class A ESBLs.

To estimate the diversity of extended-spectrum beta-lactamases in Brazil, 18 strains from different species of the family Enterobacteriaceae exhibiting a positive double-disk synergy test were collected by a clinical laboratory from several hospitals in Rio de Janeiro, Brazil, in 1996 and 1997. Four strains (Proteus mirabilis, Enterobacter cloacae, Enterobacter aerogenes, and Citrobacter amalonaticus) hybridized with a 550-bp CTX-M probe. The P. mirabilis strain produced a CTX-M-2 enzyme. The E. cloacae, E. aerogenes, and C. amalonaticus isolates harbored a bla gene which was identified by cloning and sequencing as a bla(CTX-M) gene. E. coli HB101 transconjugants and the E. coli DH5alpha transformant harboring a recombinant plasmid produced a CTX-M beta-lactamase with an isoelectric point of 7.6 conferring a resistance phenotype characterized by a higher level of resistance to cefotaxime than to ceftazidime, as observed with the other CTX-M enzymes. The deduced protein sequence showed a novel Ambler class A CTX-M enzyme, named CTX-M-8, which had 83 to 88% identity with the previously described CTX-M enzymes. The phylogenic study of the CTX-M family including CTX-M-8 revealed four CTX-M types, CTX-M-8 being the first member of a new phylum of CTX-M enzymes. The evolutionary distances between the four types of CTX-M were large, suggesting that the four clusters branched off early from a distant unknown enzyme and that intermediate enzymes probably existed.