Renata Cristina Picão

Metallo-beta-lactamase detection: comparative evaluation of double-disk synergy versus combined disk tests for IMP-, GIM-, SIM-, SPM-, or VIM-producing isolates.

ABSTRACT The emergence of metallo-β-lactamase (MBL)-producing isolates is a challenge to routine microbiology laboratories, since there are no standardized methods for detecting such isolates. The aim of this study was to evaluate the accuracy of different phenotypic methods to detect MBL production among Pseudomonas spp., Acinetobacter spp., and enterobacterial isolates, including GIM, IMP, SIM, SPM, and VIM variants. A total of 46 genetically unrelated Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter sp., and enterobacterial strains producing distinct MBLs were tested. Nineteen strains were included as negative controls. The inhibition of bacterial growth and β-lactam hydrolysis caused by MBL inhibitors (IMBL) also were evaluated. The isolates were tested for MBL production by both a double-disk synergy test (DDST) and a combined disk assay (CD) using imipenem and ceftazidime as substrates in combination with distinct IMBL. One hundred percent sensitivity and specificity were achieved by DDST using 2-mercaptopropionic acid in combination with ceftazidime and imipenem for the detection of MBL production among P. aeruginosa and Acinetobacter species isolates, respectively. The CD test showed the same results for detecting MBL-producing enterobacteria by combining imipenem and EDTA, with a 5.0-mm-breakpoint increase in the size of the inhibition zone. Our results indicate that both phenotypic methods to detect MBL-producing isolates should be based on the genera to be tested, regardless of the enzyme produced by such isolates, as well as on the local prevalence of MBL producers.

Diversity of β-Lactamases Produced by Ceftazidime-Resistant Pseudomonas aeruginosa Isolates Causing Bloodstream Infections in Brazil

ABSTRACT A retrospective survey was conducted to characterize β-lactamases in a collection of 43 ceftazidime-resistant Pseudomonas aeruginosa isolates recovered from patients with bloodstream infections hospitalized at a Brazilian teaching hospital between January and December 2005. Resistance rates for carbapenems, aminoglycosides, and quinolones were over 80%, with only colistin remaining active against all isolates. Pulsed-field gel electrophoresis analysis identified seven different genotypes. AmpC overproduction was found to be the sole β-lactamase-mediated mechanism responsible for ceftazidime resistance in four isolates (9.3%). Nine isolates (20.9%) produced an extended-spectrum β-lactamase (ESBL), either GES-1 (n = 7, 16.3%) or CTX-M-2 (n = 2, 4.6%). Carbapenemase activity was detected in 30 (70%) additional isolates. Among those isolates, two isolates (4.6%) produced the ESBL GES-5, possessing the ability to hydrolyze imipenem; a single isolate (2.3%) produced the metallo-β-lactamase (MBL) IMP-1; and 27 isolates produced the MBL SPM-1 (62.8%). None of the isolates coproduced both ESBL and MBL. Insertion sequence elements ISCR4 and ISCR1 were associated with blaSPM-1 and blaCTX-M-2 genes, respectively, whereas the blaGES-1 and blaGES-5 genes were part of class 1 integron structures. This study underlines the spread of MBL- and ESBL-producing P. aeruginosa isolates as an important source of ceftazidime resistance in Brazil.

The route of antimicrobial resistance from the hospital effluent to the environment: focus on the occurrence of KPC-producing Aeromonas spp. and Enterobacteriaceae in sewage☆ , ☆☆

We investigated the antimicrobial resistance profile and the occurrence of Klebsiella pneumoniae carbapenemase (KPC)-producing Gram-negative rods in sewage samples obtained from a Brazilian teaching hospital and from the wastewater treatment plant (WWTP) that receives it for treatment. We identified multidrug-resistant bacteria as well as KPC-2-producing Aeromonas spp. and several Enterobacteriaceae species, including Kluyvera spp., in the hospital effluent and in different sites of the WWTP. Most isolates showed the blaKPC-2 gene harbored on a transposon that was carried by conjugative plasmids. The presence of KPC production among Aeromonas spp., Kluyvera spp., and other Enterobacteriaceae indicates the adaptability of such isolates to aquatic environments, not only in the hospital effluent but also throughout the WWTP. Although secondary treatment seems to decrease the amount of KPC producers in sewage, multidrug-resistant isolates are continually disposed in the urban river. Thus, sewage treatment regulations are urgently needed to decelerate the evolution of antimicrobial resistance beyond hospitals.

Occurrence of carbapenemase-producing bacteria in coastal recreational waters

The spread of carbapenemase-producing Gram-negative rods is an emerging global problem. Although most infections due to carbapenemase producers are limited to healthcare institutions, reports of the occurrence of clinically relevant carbapenemase producers in sewage and polluted rivers are increasingly frequent. Polluted rivers flowing to oceans may contaminate coastal waters with multidrug-resistant bacteria, potentially threatening the safety of recreational activities in these locations. Here we assessed the occurrence of carbapenemase producers in water from touristic beaches located in Rio de Janeiro, Brazil, showing distinct pollution patterns. The presence of enterobacteria was noted, including the predominantly environmental genus Kluyvera spp., producing either Klebsiella pneumoniae carbapenemase (KPC) or Guyana extended-spectrum (GES)-type carbapenemases and often associated with quinolone resistance determinants. An Aeromonas sp. harbouring blaKPC and qnrS was also observed. These findings strengthen the role of aquatic matrices as reservoirs and vectors of clinically relevant antimicrobial-resistant bacteria, with potential to favour the spread of these resistance threats throughout the community.

Further Identification of CTX-M-2 Extended-Spectrum β-Lactamase in Pseudomonas aeruginosa

β-Lactamase production is the main mechanism for β-lactam resistance in gram-negative rods. The more threatening β-lactamases that have successfully emerged and are believed to spread only in Enterobacteriaceae are CTX-M-type extended-spectrum β-lactamases (ESBLs) (5). The acquired β-lactamases with wide activity spectra that are important in Pseudomonas aeruginosa include class B metallo-β-lactamases (mostly IMP and VIM types and especially SPM-1 in Brazil) and class A ESBLs, particularly VEB-, PER-, and GES-type enzymes (3, 8, 9). However, a single CTX-M-1-producing P. aeruginosa isolate has been reported from The Netherlands (1), as well as CTX-M-2- and CTX-M-43-positive P. aeruginosa isolates in Bolivia (2). This study reports the identification of a CTX-M-2-producing P. aeruginosa strain isolated from a Brazilian teaching hospital. During June 2005, a 63-year-old male patient with a recent hospitalization history was admitted to the intensive care unit for suspicion of pneumonia. He received ceftriaxone and clindamycin as first-line therapy. Four days later, he presented with septic shock and died. A blood culture grew P. aeruginosa (isolate P6208). Isolate P6208 was resistant to all β-lactams tested, except imipenem and ceftazidime. A double-disk synergy test was performed with ticarcillin-clavulanic acid- and cefotaxime-cefepime-containing disks. The production of an ESBL was evidenced only under unusual conditions (with a distance between the disks of 1.5 cm center to center) (Fig. ​(Fig.1).1). Isolate P6208 was also resistant to fluoroquinolones, amikacin, gentamicin, and tobramycin and was susceptible to colistin. The MICs of imipenem, ceftazidime, cefepime, and cefotaxime determined by using Etest strips (AB Biodisk, Solna, Sweden) were 1 μg/ml, 2 μg/ml, >256 μg/ml, and >32 μg/ml, respectively. FIG. 1. Double-disk synergy test with blaCTX-M-2-positive P. aeruginosa clinical isolate P6208. Arrows indicate double-disk synergy. Abbreviations: CAZ, ceftazidime; TCC, ticarcillin-clavulanic acid; CTX, cefotaxime; FEP, cefepime. HindIII-restricted total DNA from isolate P6208 as described previously (6) was used for cloning in pBK-CMV and was then transformed into Escherichia coli TOP10 and selected on agar plates containing ticarcillin (50 μg/ml) and kanamycin (30 μg/ml). The E. coli TOP10(p6208) recombinant strain displaying an ESBL phenotype was obtained. The sequencing of the 2,340-bp cloned DNA insert of recombinant plasmid p6208 identified a blaCTX-M-2 gene. It was preceded by an ISCR1 element located 498-bp upstream and followed by the qacEΔ1 gene cassette. This ISCR1-blaCTX-M-2 structure has already been identified in several enterobacterial species (7). Plasmid extraction performed by the Kieser method (4) did not evidence any plasmid. In addition, repetitive attempts to transfer the blaCTX-M-2 gene by electroporation failed, using both E. coli TOP10 and P. aeruginosa PAO1 as recipient strains. Thus, the blaCTX-M-2 gene might be likely chromosomally located in isolate P6208. This study identified a CTX-M-2-producing P. aeruginosa in Brazil. This finding is important since clinical laboratories may misidentify CTX-type enzymes in those nonfermenters, jeopardizing the choice of antimicrobial chemotherapy and the implementation of infection control measures. This report underlines that P. aeruginosa may become a hidden location for blaCTX-M genes.

Low Prevalence of blaOXA-143 in Private Hospitals in Brazil

We read with great interest C. S. Antonio et al.s letter describing the high prevalence of Acinetobacter baumannii carrying bla OXA-143 in Brazilian hospitals ([1][1]). Recently, we carried out a similar study, and although the bla OXA-143 gene was identified, its frequency was lower than that

Identification of Lactic Acid Bacteria in Fruit Pulp Processing Byproducts and Potential Probiotic Properties of Selected Lactobacillus Strains

This study aimed to identify lactic acid bacteria (LAB) in byproducts of fruit (Malpighia glabra L., Mangifera indica L., Annona muricata L., and Fragaria vesca L.) pulp processing. Fifty strains of LAB were identified using matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence (16S rRNA) analysis. Species belonging to Lactobacillus genus were the predominant LAB in all fruit pulp processing byproducts. The average congruency between the MALDI-TOF MS and 16S rRNA in LAB species identification reached 86%. Isolates of L. plantarum, L. brevis, L. pentosus, L. lactis and L. mesenteroides were identified with 100% congruency. MALDI-TOF MS and 16S rRNA analysis presented 86 and 100% efficiency of LAB species identification, respectively. Further, five selected Lactobacillus strains (L. brevis 59, L. pentosus 129, L. paracasei 108, L. plantarum 49, and L. fermentum 111) were evaluated for desirable probiotic-related properties and growth behavior on two different cultivation media. The exposure to pH 2.0 sharply decreased the counts of the different Lactobacillus strains after a 1 or 2 h incubation, while varied decreases were noted after 3 h of exposure to pH 3.0. Overall, the exposure to pH 5.0 and to bile salts (0.15, 0.30, and 1.00%) did not decrease the counts of the Lactobacillus strains. All tested Lactobacillus strains presented inhibitory activity against Staphylococcus aureus, Salmonella Typhimurium, Salmonella Enteritidis, Listeria monocytogenes and Escherichia coli, and presented variable susceptibility to different antibiotics. The selected Lactobacillus strains presented satisfactory and reproducible growth behavior. In conclusion, MALDI-TOF MS and 16S rRNA analysis revealed high efficiency and congruency for LAB species identification, and the selected Lactobacillus strains may be candidates for further investigation of novel probiotic strains.

Pyrosequencing-based analysis reveals a novel capsular gene cluster in a KPC-producing Klebsiella pneumoniae clinical isolate identified in Brazil

BackgroundAn important virulence factor of Klebsiella pneumoniae is the production of capsular polysaccharide (CPS), a thick mucus layer that allows for evasion of the hosts defense and creates a barrier against antibacterial peptides. CPS production is driven mostly by the expression of genes located in a locus called cps, and the resulting structure is used to distinguish between different serotypes (K types). In this study, we report the unique genetic organization of the cps cluster from K. pneumoniae Kp13, a clinical isolate recovered during a large outbreak of nosocomial infections that occurred in a Brazilian teaching hospital.ResultsA pyrosequencing-based approach showed that the cps region of Kp13 (cpsKp13) is 26.4 kbp in length and contains genes common, although not universal, to other strains, such as the rmlBADC operon that codes for L-rhamnose synthesis. cpsKp13 also presents some unique features, like the inversion of the wzy gene and a unique repertoire of glycosyltransferases. In silico comparison of cpsKp13 RFLP pattern with 102 previously published cps PCR-RFLP patterns showed that cpsKp13 is distinct from the C patterns of all other K serotypes. Furthermore, in vitro serotyping showed only a weak reaction with capsular types K9 and K34. We confirm that K9 cps shares common genes with cpsKp13 such as the rmlBADC operon, but lacks features like uge and Kp13-specific glycosyltransferases, while K34 capsules contain three of the five sugars that potentially form the Kp13 CPS.ConclusionsWe report the first description of a cps cluster from a Brazilian clinical isolate of a KPC-producing K. pneumoniae. The gathered data including K-serotyping support that Kp13’s K-antigen belongs to a novel capsular serotype. The CPS of Kp13 probably includes L-rhamnose and D-galacturonate in its structure, among other residues. Because genes involved in L-rhamnose biosynthesis are absent in humans, this pathway may represent potential targets for the development of antimicrobial agents. Studying the capsular serotypes of clinical isolates is of great importance for further development of vaccines and/or novel therapeutic agents. The distribution of K-types among multidrug-resistant isolates is unknown, but our findings may encourage scientists to perform K-antigen typing of KPC-producing strains worldwide.

Outbreak of OXY-2-Producing Klebsiella oxytoca in a Renal Transplant Unit

ABSTRACT We describe a Klebsiella oxytoca infection outbreak in a renal transplant unit that involved seven patients. All strains belonged to a single pulsed-field gel electrophoresis pattern and were resistant to amoxicillin-clavulanate, cefuroxime, piperacillin-tazobactam, and aztreonam but susceptible to ceftriaxone, ceftazidime, cefepime, and imipenem. Chromosomal β-lactamase hyperproduction was caused by a point mutation in the blaOXY-2 gene promoter region.

Widespread distribution of CTX-M and plasmid-mediated AmpC β-lactamases in Escherichia coli from Brazilian chicken meat

The dissemination of plasmid-mediated antimicrobial resistance genes may pose a substantial public health risk. In the present work, the occurrences of blaCTX-M and plasmid-mediated ampC and qnr genes were investigated in Escherichia coli from 16 chicken carcasses produced by four commercial brands in Brazil. Of the brands tested, three were exporters, including one of organic chicken. Our study assessed 136 E. coli isolates that were grouped into 77 distinct biotypes defined by their origin, resistance profiling, the presence of β-lactamase and plasmid-mediated quinolone resistance genes and enterobacterial repetitive intergenic consensus-polimerase chain reaction typing. The blaCTX-M-15, blaCTX-M-2 and blaCTX-M-8 genes were detected in one, 17 and eight different biotypes, respectively (45 isolates). Twenty-one biotypes (46 isolates) harboured blaCMY-2. Additionally, blaCMY-2 was identified in isolates that also carried either blaCTX-M-2 or blaCTX-M-8. The qnrB and/or qnrS genes occurred in isolates carrying each of the four types of β-lactamase determinants detected and also in oxyimino-cephalosporin-susceptible strains. Plasmid-mediated extended-spectrum β-lactamase (ESBL) and AmpC determinants were identified in carcasses from the four brands tested. Notably, this is the first description of blaCTX-M-15 genes in meat or food-producing animals from South America. The blaCTX-M-8, blaCTX-M-15 and blaCMY-2 genes were transferable in conjugation experiments. The findings of the present study indicate that plasmid-mediated ESBL and AmpC-encoding genes are widely distributed in Brazilian chicken meat.