Patricia Marchiaro

Sensitive EDTA-based microbiological assays for detection of metallo-{beta}-lactamases in nonfermentative gram-negative bacteria.

ABSTRACT The worldwide spread of metallo-β-lactamase (MBL)-producing gram-negative bacilli represents a great concern nowadays. Sensitive assays for their specific detection are increasingly demanded to aid infection control and to prevent their dissemination. We have developed a novel microbiological assay employing crude bacterial extracts, designated EDTA-imipenem microbiological assay (EIM), to identify MBLs in nonfermentative gram-negative clinical strains. We also evaluated the ability of EIM to detect MBLs in comparison to those of other currently employed screening methods, such as the EDTA disk synergy test (EDS) with imipenem as a substrate and the Etest method. The sensitivities of EIM and Etest were similar (1 versus 0.92, respectively) and much higher than that of EDS (0.67). Moreover, both EIM and Etest displayed the maximum specificity. Modifications were introduced to EDS, including the simultaneous testing of three different β-lactams (imipenem, meropenem, and ceftazidime) and two different EDTA concentrations. This resulted in a sensitivity improvement (0.92), albeit at a cost to its specificity. A simple strategy to accurately detect MBL producers is proposed; this strategy combines (i) an initial screening of the isolates by the extended EDS assay to select the potential candidates and (ii) confirmation of the true presence of MBL activity by EIM.

A convenient microbiological assay employing cell-free extracts for the rapid characterization of Gram-negative carbapenemase producers

OBJECTIVES The dissemination of metallo and serine carbapenem-hydrolysing beta-lactamases among Gram-negative nosocomial bacteria represents an acute problem worldwide. Here, we present a rapid and sensitive assay for the characterization of carbapenemase producers to aid in infection control and prevention. METHODS The assay involves a rapid disruption of bacterial isolates with silicon dioxide microbeads, followed by the testing in cell-free extracts of hydrolytic activity towards various beta-lactams including two carbapenems (imipenem and meropenem) and a cephalosporin (ceftazidime). A parallel testing of the effects of selective beta-lactamase inhibitors such as EDTA and clavulanic acid allows differentiation of metallo carbapenemases from serine carbapenemases, and also clavulanic-acid-sensitive from -resistant enzymes among the latter. RESULTS The efficiency of bacterial disruption using silicon dioxide microbeads was identical to that of ultrasonic treatment. The subsequent microbiological assay aimed to evaluate both substrate specificity and inhibitor profile of carbapenem-hydrolysing enzymes present in the extracts and allowed an accurate differentiation of A, B and D types, as judged by the analysis of 24 well-characterized clinical strains that included metallo-beta-lactamase producers (i.e. VIM-, IMP- and SPM-type Pseudomonas producers; an L1 Stenotrophomonas maltophilia producer; and a GOB-18 Elizabethkingia meningoseptica producer) as well as serine carbapenemase producers (i.e. an SME-type Serratia marcescens producer, a GES-2 Pseudomonas aeruginosa producer, Klebsiella pneumoniae and Citrobacter freundii KPC-2 producers and OXA-type Acinetobacter baumannii producers). CONCLUSIONS We have developed a convenient microbiological assay aimed to more accurately and in a short time characterize carbapenem-hydrolysing enzymes produced by Gram-negative bacteria. The assay possesses broad applicability in the clinical setting.

Emergence of Pseudomonas aeruginosa strains producing metallo-β-lactamases of the IMP-15 and VIM-2 types in Mexico.

Eighty-six carbapenem non-susceptible Pseudomonas aeruginosa isolates collected in the National Institute of Respiratory Diseases of Mexico City were screened for the presence of metallo-beta-lactamase (MBL) activity using both E-test strips and a microbiological assay with EDTA-imipenem. Genomic comparisons and sequence analyses conducted with these isolates revealed the presence of bla(VIM-2) in two clonally related isolates, and bla(IMP-15) in a clonally unrelated isolate. Both genes were found to be carried by class 1 integrons, and bla(IMP-15) was additionally present on a broad host-range plasmid. This is the first report of co-existing P. aeruginosa strains producing different MBLs in a Mexican hospital, highlighting the necessity of appropriate surveillance to prevent dissemination of carbapenem resistance.

Biochemical Characterization of Metallo-β-Lactamase VIM-11 from a Pseudomonas aeruginosa Clinical Strain

ABSTRACT A detailed biochemical characterization of the Pseudomonas aeruginosa VIM-11 metallo-β-lactamase (MβL) is reported. The only substitution differentiating VIM-11 from VIM-2 (N165S) promoted a slightly improved catalytic efficiency of the former on 3 out of 12 substrates, notably the bulky cephalosporins. Thus, MβL-mediated resistance also may be modulated by remote mutations.

Molecular characterization of methicillin-resistant coagulase-negative staphylococci from a neonatal intensive care unit

OBJECTIVE To evaluate clonal dissemination of methicillin-resistant coagulase-negative staphylococci (CNS). SETTING Neonatal intensive care unit of a 180-bed, university-affiliated general hospital. PATIENTS Neonates admitted to the neonatal intensive care unit between March 1999 and October 2000, from whom CNS were isolated as a unique pathogen. Patients from other wards from whom epidemiologically unrelated staphylococci strains were obtained served as control-patients. METHODS Conventional methods were used for phenotypic characterization of CNS. Methicillin resistance was determined by mecA polymerase chain reaction (PCR) amplification. Genotypic characterization was done by random amplification of DNA with degenerated primers (RAPD) and repetitive element sequence-based PCR (rep-PCR). RESULTS Forty methicillin-resistant CNS isolates obtained from neonates were characterized as Staphylococcus epidermidis (33), S. hominis (5), S. warneri (1), and S. auricularis (1). Both RAPD and rep-PCR indicated the presence of 4 different clones among the 33 S. epidermidis isolates. In turn, the 4 randomly selected, epidemiologically unrelated methicillin-resistant CNS strains obtained from control-patients showed 3 new profiles by RAPD and 2 by rep-PCR, which differed from the corresponding patterns mentioned earlier. Persistence of S. hominis in a neonate could be assessed by both genotypic techniques. CONCLUSIONS The molecular characterization of the methicillin-resistant CNS studied indicated dissemination of one particular methicillin-resistant CNS clone among the neonates in the ward studied. Although RAPD showed a superior power to discriminate among methicillin-resistant CNS isolates, both RAPD and rep-PCR detected intraspecific and interspecific genomic diversity.

Polyclonal outbreak of bacteremia caused by Burkholderia cepacia complex and the presumptive role of ultrasound gel

A nosocomial polyclonal outbreak associated to bacteremia caused by different Burkholderia cepacia complex (BCC) species and clones is reported. Molecular characterization identified Burkholderia stabilis, Burkholderia contaminans, and Burkholderia ambifaria among BCC isolates obtained from patients in neonatal and adult intensive care units. BCC was also isolated from an intrinsically contaminated ultrasound gel, which constituted the presumptive BCC source. Prior BCC outbreak related to contaminated ultrasound gels have been described in the setting of transrectal prostate biopsy. Outbreak caused strains and/or clones of BCC have been reported, probably because BCC are commonly found in the natural environment; most BCC species are biofilm producers, and different species may contaminate an environmental source. The finding of multiple species or clones during the analysis of nosocomial BCC cases might not be enough to reject an outbreak from a common source.

The Complete Nucleotide Sequence of the Carbapenem Resistance-Conferring Conjugative Plasmid pLD209 from a Pseudomonas putida Clinical Strain Reveals a Chimeric Design Formed by Modules Derived from Both Environmental and Clinical Bacteria

ABSTRACT The complete sequence of the carbapenem-resistance-conferring conjugative plasmid pLD209 from a Pseudomonas putida clinical strain is presented. pLD209 is formed by 3 well-defined regions: an adaptability module encompassing a Tn402-like class 1 integron of clinical origin containing blaVIM-2 and aacA4 gene cassettes, partitioning and transfer modules, and a replication module derived from plasmids of environmental bacteria. pLD209 is thus a mosaic of modules originating in both the clinical and environmental (nonclinical) microbiota.

Complete Sequence of a blaNDM-1-Harboring Plasmid in an Acinetobacter bereziniae Clinical Strain Isolated in Argentina

The New Delhi metallo-β-lactamase (NDM-1) was initially identified in clinical isolates of Escherichia coli and Klebsiella pneumoniae in Sweden from a patient previously hospitalized in India (1). Since then, blaNDM-1 has been frequently reported in Enterobacteriaceae and Acinetobacter spp. with a fast dissemination in the Indian subcontinent, the Balkan countries, China and the Middle East (2). NDM-1 producers generally associated to Enterobacteriaceae species have also been reported, albeit with much lower frequency, in Latin American countries including Guatemala, Mexico, Colombia and Brazil (3). Moreover, production of NDM-1 in this geographic region has also been noted in A. baumannii in Honduras and Brazil (3, 4), and in A. pittii in Paraguay and Brazil (5, 6). Here, we report the first case of a NDM-1-producing Acinetobacter species in Argentina, an A. bereziniae clinical isolate. We describe also the complete sequence of a blaNDM-1-containing plasmid in this strain.

Draft Genome Sequence of Acinetobacter bereziniae HPC229, a Carbapenem-Resistant Clinical Strain from Argentina Harboring blaNDM-1

ABSTRACT We report here the draft genome sequence of an NDM-1-producing Acinetobacter bereziniae clinical strain, HPC229. This strain harbors both plasmid and chromosomal resistance determinants toward different β-lactams and aminoglycosides as well as several types of multidrug efflux pumps, most likely representing an adaptation strategy for survival under different environments.

Structural diversity and similar bioactivity in synthetic bicyclononanes

ABSTRACT Simple syntheses of diverse bicyclo[3.3.1]nonanes and related compounds as the minimal substructure of bioactive natural products via Michael, aldol, and alkylation reactions from diketones are described herein. The structures of the synthesized compounds were determined by infrared spectroscopy, NMR (1H and 13C), and electrospray ionization–high-resolution mass spectrometry. We also show the in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria. The qualitative analysis has revealed that the new synthesized compounds 5, 6, 9, and 11 present antibacterial properties. GRAPHICAL ABSTRACT