Adriana S. Limansky

Acquisition of Resistance to Carbapenems in Multidrug-Resistant Clinical Strains of Acinetobacter baumannii: Natural Insertional Inactivation of a Gene Encoding a Member of a Novel Family of β-Barrel Outer Membrane Proteins

ABSTRACT The outer membrane proteins responsible for the influx of carbapenem β-lactam antibiotics in the nonfermentative gram-negative pathogen Acinetobacter baumannii are still poorly characterized. Resistance to both imipenem and meropenem in multidrug-resistant clinical strains of A. baumannii is associated with the loss of a heat-modifiable 29-kDa outer membrane protein, designated CarO. The chromosomal locus containing the carO gene was cloned and characterized from different clinical isolates. Only one carO copy, present in a single transcriptional unit, was found in the A. baumannii genome. The carO gene encodes a polypeptide of 247 amino acid residues with a typical N-terminal signal sequence and a predicted transmembrane β-barrel topology. Its absence from different carbapenem-resistant clinical isolates of A. baumannii resulted from the disruption of carO by distinct insertion elements. The overall data thus support the notion that CarO participates in the influx of carbapenem antibiotics in A. baumannii. Moreover, database searches identified the presence of carO homologs only in species of the genera Acinetobacter, Moraxella, and Psychrobacter, disclosing the existence of a novel family of outer membrane proteins restricted to the family Moraxellaceae of the class γ-Proteobacteria.

Loss of a 29-Kilodalton Outer Membrane Protein in Acinetobacter baumannii Is Associated with Imipenem Resistance

ABSTRACT We analyzed the possible causes of imipenem (IPM) resistance in multidrug-resistant isolates of Acinetobacter baumannii. Comparison of the outer membrane protein (OMP) profiles of two genomically related strains (Ab288 [IPM sensitive] and Ab242 [IPM resistant]) indicated the conspicuous loss of a 29-kDa polypeptide in the Ab242 strain. No carbapenemase activity was detected in any of these strains. The treatment of Ab288 with sodium salicylate resulted in IPM resistance and the loss of the 29-kDa OMP. In addition, IPM-resistant clones of Ab288 which were selected by repetitive culturing in increasing concentrations of this antibiotic also showed the absence of this 29-kDa OMP.

CarO, an Acinetobacter baumannii outer membrane protein involved in carbapenem resistance, is essential for l-ornithine uptake

We previously associated the emergence of carbapenem resistance in Acinetobacter baumannii with the loss of an outer membrane (OM) protein designated CarO. CarO was found essential for l‐ornithine uptake: CarO‐deficient strains were specifically impaired to grow only on l‐ornithine, and failed to incorporate l‐[14C] ornithine from the medium. l‐arginine, and histidine and lysine to a lower extent, could effectively compete for l‐[14C] ornithine uptake. l‐ornithine also reduced A. baumannii sensitivity to imipenem, suggesting that both compounds compete for uptake. The overall results indicate that CarO participates in the selective uptake of l‐ornithine, carbapenems, and other basic amino acids in A. baumannii.

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.

The Metallo-β-lactamase GOB is a mono-Zn(II) enzyme with a novel active site

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes able to hydrolyze and inactivate most β-lactam antibiotics. The large diversity of active site structures and metal content among MβLs from different sources has limited the design of a pan-MβL inhibitor. Here we report the biochemical and biophysical characterization of a novel MβL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MβLs. Contrasting all other related MβLs, GOB-18 is fully active against a broad range of β-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MβLs.

Acinetobacter baumannii extensively drug resistant lineages in Buenos Aires hospitals differ from the international clones I-III.

As a way to contribute to the assessment of Acinetobacter baumannii clinical population structure, multi-locus sequence typing (MLST) was performed in a collection of 93 isolates from Buenos Aires (1983-2012) and Rosario (2006-2009) hospitals. Sequence types (STs) were achieved by Bartual (B) and Institut Pasteur (P) schemes. PFGE typing, antimicrobial susceptibility assays, and the amplification of the OXA carbapenemase genes most prevalent in our region, were also performed. e-Burst clustered the 25 STs(B) (15 novels) into 5 clonal complexes (CC) and 5 singletons, and grouped the 18 STs(P) (12 novels) into 3 CC and 4 singletons. Bartual scheme divided the CC79(P) into two groups. CC113(B)/CC79(P) prevailed in Buenos Aires at least in 1992-2009, being responsible for epidemic and for endemic infections and acquiring the XDR (extensively drug-resistant) pattern throughout the years. While, CC119(B)/CC79(P) was apparently present before the CC113(B)/CC79(P)domain. CC103(B)/CC15(P) was the second most prevalent CC. Interestingly, CC110(B)/ST25(P) apparently increased over the last years. Conversely, CC109(B)/CC1(P) (international clone I) predominated in Rosario, although the presence of CC113(B)/CC79(P), CC103(B)/CC15(P) and CC110(B)/ST25(P) was observed. Nineteen novel STs clustered in CC79(P), CC15(P), CC113(B), CC109(B) and CC103(B), suggesting their clonal expansion during persistence. PFGE typing proved transmission of strains intra- and inter-hospitals in each city. Except for one, all the recent isolates (2007-2012) harboured the blaOXA-23-like. All isolates were susceptible to colistin. Tigecycline MIC(90) was 1mg/L and the rifampicin MIC>512mg/l was found among isolates in three hospitals. In conclusion, the international clone II (CC92(B)/CC2(P)) was not found among our isolates. CC113(B)/CC79(P), CC103(B)/CC15(P), and ST25(P), suggested also as major components in the A. baumannii population together with the international clone I, were present in Buenos Aires and Rosario with different prevalence rate. Their recent isolates showed high distribution of the blaOXA-23-like as well as the XDR pattern.

ISAba825, a Functional Insertion Sequence Modulating Genomic Plasticity and bla OXA-58 Expression in Acinetobacter baumannii

ABSTRACT ISAba825, an insertion sequence found inactivating Acinetobacter baumannii carO, was tagged with a kanamycin (Kn) resistance cassette. ISAba825::Kn effectively transposed in A. baumannii, showing preference for short, AT-enriched target sequences, generating 6- to 9-bp target duplications. Additionally, we detected the presence of ISAba825 upstream of a plasmid-borne bla OXA-58 gene, generating a hybrid promoter largely enhancing its expression and leading to carbapenem resistance. Overall, a role for ISAba825 in carbapenem resistance modulation in A. baumannii is proposed.

Differential Role of the T6SS in Acinetobacter baumannii Virulence

Gram-negative bacteria, such as Acinetobacter baumannii, are an increasing burden in hospitals worldwide with an alarming spread of multi-drug resistant (MDR) strains. Herein, we compared a type strain (ATCC17978), a non-clinical isolate (DSM30011) and MDR strains of A. baumannii implicated in hospital outbreaks (Ab242, Ab244 and Ab825), revealing distinct patterns of type VI secretion system (T6SS) functionality. The T6SS genomic locus is present and was actively transcribed in all of the above strains. However, only the A. baumannii DSM30011 strain was capable of killing Escherichia coli in a T6SS-dependent manner, unlike the clinical isolates, which failed to display an active T6SS in vitro. In addition, DSM30011 was able to outcompete ATCC17978 as well as Pseudomonas aeruginosa and Klebsiella pneumoniae, bacterial pathogens relevant in mixed nosocomial infections. Finally, we found that the T6SS of DSM30011 is required for host colonization of the model organism Galleria mellonella suggesting that this system could play an important role in A. baumannii virulence in a strain-specific manner.

In vitro susceptibility of Achromobacter spp. isolates: comparison of disk diffusion, Etest and agar dilution methods

In this study, we analysed the antimicrobial susceptibility of 92 strains of Achromobacter spp. isolated from clinical samples to 18 antimicrobial agents. The disk diffusion method and Etest were compared with the agar dilution method, and the breakpoints of susceptibility and resistance for the disk diffusion method for the antimicrobials tested were determined. The most active antibiotics were piperacillin, piperacillin/tazobactam and the carbapenems. By applying the linear least-squares regression method, breakpoints could be established for antibiotics active against this genus such as imipenem, meropenem, ertapenem and trimethoprim/sulfamethoxazole (SXT). Other active antibiotics, such as piperacillin and minocycline, could be tested by the Etest method. The less active antibiotics such as gentamicin, doxycycline and tetracycline could be tested by the disk diffusion method. For the rest of the antimicrobial agents tested, breakpoints could not be established owing to the high percentage of errors and/or the poor linear regression coefficient obtained. Therefore, these antimicrobial agents should be tested by minimal inhibitory concentration determination. In summary, we recommend the following zone diameter breakpoints for resistant and susceptible, respectively: < or = 11 mm and > or = 22 mm for imipenem; < or = 13 mm and > or = 24 mm for meropenem; < or = 17 mm and > or = 24 mm for ertapenem; < or = 15 mm and > or = 21 mm for gentamicin; < or = 27 mm and > or = 28 mm for SXT; < or = 20 mm and > or = 29 mm for tetracycline; and < or = 20 mm and > or = 24 mm for doxycycline.

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.