Adriana G. Nicoletti

Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results

OBJECTIVES The aim of this study was to evaluate the presence of carbapenemases in a Klebsiella pneumoniae collection and the performance of the modified Hodge test (MHT) to correctly identify this phenotype. METHODS Twenty-eight K. pneumoniae clinical isolates with reduced susceptibility to carbapenems were evaluated. Antimicrobial susceptibility and molecular typing were performed by agar dilution and PFGE, respectively. The MHT was performed using both standard and high inoculum of test organisms. Imipenem hydrolysis was investigated by spectrophotometric assays and carbapenemase-encoding genes were identified by PCR and amplicon sequencing. Porin loss was investigated by both PCR and SDS-PAGE. RESULTS Susceptibility rates for imipenem, meropenem and ertapenem were 93%, 57% and 11%, respectively. The PFGE analysis showed seven unrelated genotypes. By testing standard inoculum and ertapenem or meropenem discs, 25% (n = 7) and 21% (n = 6) of the isolates were classified as carbapenemase producers, respectively. When a higher inoculum was employed, these rates increased to 54% (n = 15) and 43% (n = 12), respectively. No imipenem hydrolysis was detected. PCRs identified bla(CTX-M) in 27 (96%) isolates, of which 2 isolates also carried bla(GES-1.) SDS-PAGE and PCR assays revealed that all isolates had lost at least one outer membrane protein, except for a single isolate that was found to express both OmpK35 and OmpK36. CONCLUSIONS False detection of carbapenemase production was observed by the MHT possibly as a result of extended-spectrum beta-lactamase (ESBL) production coupled with porin loss as reported before. Clinical laboratories must be aware of this fact, especially in geographical areas where ESBL-producing isolates are highly prevalent.

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.

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.

First Report of Plasmid-Mediated qnrA1 in a Ciprofloxacin-Resistant Escherichia coli Strain in Latin America

ABSTRACT Among 144 ciprofloxacin-resistant Escherichia coli isolated in Brazil, one (0.69%) QnrA1-producing isolate was detected. The qnrA1 gene was associated with ISCR1. The QnrA1 determinant was carried on a 41-kb conjugative plasmid, which also carried a FOX-type cephalosporinase encoding gene and a class 1 integron with the aadB and catB3 cassettes. This is the first report of a qnrA-carrying isolate in a Latin American country.

Detection of GES-5-producing Klebsiella pneumoniae in Brazil

Sir, The production of carbapenemases or extended-spectrum b-lactamases (ESBLs) and/or AmpC b-lactamases coupled with porin loss are the most common mechanisms of carbapenem resistance in Klebsiella spp. Amino acid substitutions in the active site of GES-type ESBLs may enhance their spectrum of activity against carbapenems. The variants GES-2, -4, -5, -6 and -11 are able to hydrolyse imipenem; GES-5 hydrolyses this agent most efficiently. To date, GES-5-producing Klebsiella pneumoniae has only been identified in Korea. In this report we investigated the mechanisms responsible for carbapenem resistance in a K. pneumoniae subsp. ozaenae from Brazil. KOZ-Ban2 was isolated from a rectal surveillance swab of an elderly patient admitted to a private hospital in São Paulo, Brazil, in 2008. The patient was admitted with the diagnosis of community-acquired pneumonia and was successfully treated with piperacillin/tazobactam and moxifloxacin. Two weeks later, the patient presented a new episode of pneumonia that was empirically treated with meropenem and teicoplanin, with good clinical outcome. After 5 weeks of hospitalization, the patient was admitted to the intensive care unit with septic shock and respiratory failure. Candida sp. was isolated from the bloodstream at the same time that KOZ-Ban2 was recovered from the rectal swab. The patient died 24 h later. KOZ-Ban2 identification was performed by the BD Phoenix Automated Microbiology System. Susceptibility testing was performed by CLSI agar dilution, and showed that KOZ-Ban2 was resistant to all b-lactams including carbapenems, amikacin, gentamicin, nalidixic acid and ciprofloxacin (Table 1). PCR and amplicon sequencing, using previously reported conditions and primers specific for KPC-, MBLand ESBL-encoding genes and the 50 and 30 conserved sequences of integrons, identified the blaGES-5 gene. This gene was harboured in the first position of a class 1 integron, followed by the aacA4, blaOXA-17, dfrA21 and catB3 cassettes, encoding an aminoglycoside acetyltransferase, an extended-spectrum oxacillinase capable of hydrolysing cefotaxime and cefepime better than ceftazidime, a dihydrofolate reductase and a chloramphenicol acetyltransferase, respectively. This new integron, named In91, had a weak Pc version with the sequence 235 TGGACA and 210 TAAGCT and an inactive P2 (GQ139471). The search for plasmid-mediated quinolone resistance determinants yielded negative results. PCR and sequencing of ompK35 and ompK36 genes revealed that the size of the ompK36 amplicon was 650 bp lower than expected. Sequencing showed that 674 bp was missing in KOZ-Ban2 ompK36 (GQ139473). In addition, although the ompK35 amplicon was the expected size, its sequencing revealed one change at position 689, where the codon TGG changed to the stop codon TAG, probably resulting in a deficient porin that lacked the last 130 amino acids (GQ139472). SDS–PAGE of outer membrane protein (OMP) profiles was performed as previously reported, using a carbapenem-susceptible K. pneumoniae for comparison. The carbapenem-susceptible isolate showed three bands near the 36 kDa band of the molecular weight marker, whereas KOZ-Ban2 had a single band, probably corresponding to OmpA (data not shown). These findings suggest that neither OmpK35 nor OmpK36 was expressed in KOZ-Ban2. Mating-out assays were performed using streptomycin-resistant Escherichia coli K12 as the recipient strain, and selection was

Influence of Disk Preparation on Detection of Metallo-β-Lactamase-Producing Isolates by the Combined Disk Assay

ABSTRACT The combined disk assay has been used for detection of metallo-β-lactamase-producing isolates. We have observed that the size of inhibition zones produced by many β-lactam/metallo-β-lactamase inhibitor (IMBL) combinations may differ depending on the way that the combined disks were prepared. Among the 10 β-lactam/IMBL combinations tested, only the imipenem/EDTA combination produced similar results.

Detection of PER-2-Producing Enterobacter cloacae in a Brazilian Liver Transplantation Unit

High rates of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates have been documented in many Brazilian hospitals, with CTX-M-2 being the most frequent ESBL reported ([1][1]). Resistance to broad-spectrum cephalosporins among Enterobacter cloacae isolates is usually due

Coproduction of KPC-2 and QnrB19 in Klebsiella pneumoniae ST340 isolate in Brazil

Few reports described the presence of bla(KPC) and qnr genes in the same isolate. This study reports the combination of bla(KPC-2) and qnrB19 genes in Klebsiella pneumoniae ST340 isolate in Brazil. These findings draw attention to this combination in ST340 isolate, which is part of the CC258, disseminated in Latin America.

Comparison of phenotypic tests for detecting BKC-1-producing Enterobacteriaceae isolates

Carbapenemase-producing Enterobacteriaceae may exhibit in vitro susceptibility to carbapenems, especially those producing weak carbapenemases. Routine clinical laboratories have employed phenotypic tests for screening such isolates. BKC-1 is a recently reported carbapenemase that shows weak carbapenemase activity. In this study, we aimed to evaluate the behavior of distinct phenotypic methods against BKC-1-producing Enterobacteriaceae.

Frequency of BKC-1-Producing Klebsiella Species Isolates.

ABSTRACT BKC-1 is a new class A serine carbapenemase that was recently identified in Klebsiella pneumoniae clinical isolates. The principal objective of this study was to evaluate the frequency of blaBKC-1 by testing a collection of Klebsiella isolates. Only 2 of 635 Klebsiella isolates (0.3%) carried blaBKC-1. The two BKC-1-producing isolates belonged to clonal complex 442 and possessed identical pulsed-field gel electrophoresis patterns. The blaBKC-1 gene was inserted into a 10-kb plasmid that was identical to the previously reported plasmid, p60136. The BKC-producing K. pneumoniae isolates presented also possessed other mechanisms for beta-lactam resistance, such as genes encoding extended-spectrum beta-lactamases and mutations in the genes ompK35 and ompK36, encoding the major porins.