Characterisation of plasmid-mediated rmtB-1 in Enterobacteriaceae clinical isolates from São Paulo, Brazil.

OBJECTIVES The emergence of 16S rRNA methyltranferases (16 RMTAses) has jeopardised the clinical use of aminoglycosides. RmtB is one of the most frequently reported in Gram-negatives worldwide. In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. METHODS All Gram-negatives classified as resistant to amikacin, gentamicin, and tobramycin by agar screening were selected for analysis. The presence of 16SRMTases encoding genes was verified by polymerase chain reaction (PCR). Antimicrobial susceptible profile was determined by broth microdilution. The genetic relationship among these isolates was accessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Selected RmtB-producing isolates were characterised by whole genome sequencing (WGS) analysis. RESULTS Twenty-two of 1,052 (2.1%) Enterobacteriaceae were detected as producers of RmtB-1 [Klebsiella pneumoniae (n = 21) and Proteus mirabilis (n = 1)]. blaKPC-2 was identified among 20 RmtB-1-producing K. pneumoniae isolates that exhibited an identical PFGE and MLST (ST258) patterns. Two K. pneumoniae isolates, the A64216 (not harboring bla KPC-2), A64477 (harboring bla KPC-2) and one P. mirabilis isolate (A64421) were selected for WGS. rmtB-1 and bla KPC-2 genes were carried by distinct plasmids. While a plasmid belonging to the IncFIIk group harbored rmtB-1 in K. pneumoniae, this gene was carried by a non-typable plasmid in P. mirabilis. In the three analysed plasmids, rmtB-1 was inserted on a transposon, downstream a Tn2. CONCLUSION Our findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China. It suggests that multiple mobilization events might have occurred in South America.

Genetic and biochemical characterization of GES-16, a new GES-type ß-lactamase with carbapenemase activity in Serratia marcescens.

We evaluated the genetic environment of blaGES-16 found in 2 carbapenem-resistant Serratia marcescens clinical isolates recovered from patients hospitalized at a tertiary hospital located in Rio de Janeiro, Brazil. We also compared the kinetics constants for GES-16 and GES-5 against several ß-lactams. Both S. marcescens isolates showed identical PFGE pattern and carried the carbapenemase-encoding gene blaGES-16 and the extended-spectrum ß-lactamase encoding gene blaOXA-10. The blaGES-16 was inserted at the first position of a defective class 1 integron, composed by a fragmented integrase gene that lacked its attI1 recombination site, followed by dfr22, aac(6')-IIc, and aadA1 genes. This integron was located on a 30-kb nonconjugative plasmid. The GES-16 showed 2 amino acid substitutions (Gln38Glu and Gly170Ser) compared to GES-1. Kinetic analysis showed that GES-16 presented hydrolytic activity against all ß-lactams tested, except for aztreonam. Imipenem was the carbapenem more efficiently hydrolyzed (highest kcat/Km) by GES-16. The kinetic parameters of GES-16 were similar to those of GES-5. In conclusion, we identified a new GES-type enzyme with carbapenemase activity in S. marcescens. The increasing diversity of such resistance determinants confirms the ongoing evolution of these ß-lactamases towards a broader spectrum of activity.

Characterisation of plasmid-mediated rmtB-1 in Enterobacteriaceae clinical isolates from São Paulo, Brazil

OBJECTIVES The emergence of 16S rRNA methyltranferases (16 RMTAses) has jeopardised the clinical use of aminoglycosides. RmtB is one of the most frequently reported in Gram-negatives worldwide. In this study, we aimed to estimate the frequency of 16S RMTAses encoding genes in Enterobacteriaceae isolated in a three-month period from a tertiary Brazilian hospital. METHODS All Gram-negatives classified as resistant to amikacin, gentamicin, and tobramycin by agar screening were selected for analysis. The presence of 16SRMTases encoding genes was verified by polymerase chain reaction (PCR). Antimicrobial susceptible profile was determined by broth microdilution. The genetic relationship among these isolates was accessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Selected RmtB-producing isolates were characterised by whole genome sequencing (WGS) analysis. RESULTS Twenty-two of 1,052 (2.1%) Enterobacteriaceae were detected as producers of RmtB-1 [Klebsiella pneumoniae (n = 21) and Proteus mirabilis (n = 1)]. blaKPC-2 was identified among 20 RmtB-1-producing K. pneumoniae isolates that exhibited an identical PFGE and MLST (ST258) patterns. Two K. pneumoniae isolates, the A64216 (not harboring bla KPC-2), A64477 (harboring bla KPC-2) and one P. mirabilis isolate (A64421) were selected for WGS. rmtB-1 and bla KPC-2 genes were carried by distinct plasmids. While a plasmid belonging to the IncFIIk group harbored rmtB-1 in K. pneumoniae, this gene was carried by a non-typable plasmid in P. mirabilis. In the three analysed plasmids, rmtB-1 was inserted on a transposon, downstream a Tn2. CONCLUSION Our findings suggested that the rmtB-1 was harbored by plasmids distinct from those previously reported in Bolivia and China. It suggests that multiple mobilization events might have occurred in South America.

Frequency of BKC-1-Producing Klebsiella Species Isolates.

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.

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.

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.

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.

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.

Influence of disk preparation on detection of metallo-beta-lactamase-producing isolates by the combined disk assay.

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

First report of plasmid-mediated qnrA1 in a ciprofloxacin-resistant Escherichia coli strain in Latin America.

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.