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.

The emergence of metallo-␤-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 ␤-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.Since the early 1990s, new metallo-␤-lactamase (MBL)-encoding genes have been reported all over the world in clinically important pathogens, such as Pseudomonas spp., Acinetobacter spp., and members of the Enterobacteriaceae family (19,27,35,40). The emergence of MBL-encoding genes is worrisome, since they usually are carried by mobile genetic structures with great ability to spread (3,5,18,24,36). Moreover, increased mortality rates have been documented for patients infected with MBL-producing Pseudomonas aeruginosa, especially due to inadequate empirical therapy (39). Therefore, early detection of MBL-producing organisms is crucial to establish appropriate antimicrobial therapy and to prevent their inter-and intrahospital dissemination (10, 35).Several phenotypic methods based on MBL inhibition by EDTA or thiol-based compounds have been published. Although they are simple to perform and cheaper than genotypic methods, they have shown discordant results depending on the employed methodology, ␤-lactam substrates, MBL inhibitors (IMBL), and bacterial genus tested (11,14,17,21,26). In addition, SPM-, GIM-, and SIM-producing pathogens rarely have been evaluated by these studies.The high diversity and prevalence of MBL-producing P.aeruginosa, Acinetobacter spp., and Enterobacteriaceae isolates have motivated the search for an accurate MBL screening test. The aim of this study was to evaluate the accuracy of the double-disk synergy test (DDST) ...

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.

Three Klebsiella pneumoniae clinical isolates demonstrating carbapenem resistance were recovered from different patients hospitalized at two medical centers in São Paulo, Brazil. Resistance to all ␤-lactams, quinolones, and some aminoglycosides was observed for these isolates that were susceptible to polymyxin B. Carbapenem hydrolysis, which was inhibited by clavulanic acid, was observed for all K. pneumoniae isolates that belonged to the same pulsed-field gel electrophoresis (PFGE) type and a novel sequence type (ST), ST1781 (clonal complex 442 [CC442]). A 10-kb nonconjugative incompatibility group Q (IncQ) plasmid, denominated p60136, was transferred to Escherichia coli strain TOP10 cells by electroporation. The full sequencing of p60136 showed that it was composed of a mobilization system, ISKpn23, the phosphotransferase aph3A-VI, and a 941-bp open reading frame (ORF) that codified a 313-amino acid protein. This ORF was named bla BKC-1 . Brazilian Klebsiella carbapenemase-1 (BKC-1) showed a pI of 6.0 and possessed the highest identity (63%) with a ␤-lactamase of Sinorhizobium meliloti, an environmental bacterium. Hydrolysis studies demonstrated that purified BKC-1 not only hydrolyzed carbapenems but also penicillins, cephalosporins, and monobactams. However, the carbapenems were less efficiently hydrolyzed due to their very low k cat values (0.0016 to 0.031 s ؊1 ). In fact, oxacillin was the best substrate for BKC-1 (k cat /K m , 53,522.6 mM ؊1 s ؊1 ). Here, we report a new class A carbapenemase, confirming the diversity and rapid evolution of ␤-lactamases in K. pneumoniae clinical isolates.

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