Distribution of beta-lactamases in Acinetobacter baumannii clinical isolates and the effect of Syn 2190 (AmpC inhibitor) on the MICs of different beta-lactam antibiotics.
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The distribution of beta-lactamases in a group of 20 epidemiologically well defined Acinetobacter baumannii clinical isolates and the in vitro activity of Syn 2190, a novel beta-lactamase AmpC inhibitor, were determined. Twenty-five per cent of the strains carried and expressed a TEM-type beta-lactamase, whereas 35% had an OXA-type beta-lactamase. In nine out of 11 (82%) ceftazidime-resistant and four out of 13 (30.7%) cefepime-resistant strains, the MIC of these beta-lactam antibiotics decreased when determined in the presence of Syn 2190. Thus, our results suggest that in a high percentage of A. baumannii clinical isolates the increased production of AmpC, in combination or not with other resistance mechanisms, contributes to the resistance pattern in A. baumannii to beta-lactams. (+info)
Molecular characterization of integrons in epidemiologically unrelated clinical isolates of Acinetobacter baumannii from Italian hospitals reveals a limited diversity of gene cassette arrays.
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Integron carriage by 36 epidemiologically unrelated Acinetobacter baumannii isolates collected over an 11-year period from patients in six different Italian hospitals was investigated. Sixteen type 1 integron-positive isolates (44%) were found, 13 of which carried the same array of cassettes, i.e., aacC1, orfX, orfX', and aadA1a. As ribotype analysis of the isolates demonstrated a notable genetic diversity, horizontal transfer of the entire integron structure or ancient acquisition was hypothesized. (+info)
Genetic and phenotypic analysis of Acinetobacter baumannii insertion derivatives generated with a transposome system.
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Acinetobacter baumannii is a metabolically versatile pathogen that causes severe infections in compromised patients. However, little is known about the genes and factors involved in its basic physiology and virulence properties. Insertion mutagenesis was used to initiate the identification and characterization of some of these factors and genes in the prototype strain 19606. The utilization of the pLOFKm suicide delivery vector, which harbors a suicide mini-Tn10 derivative, proved to be unsuccessful for this purpose. The EZ::TN Tnp transposome system available from Epicentre was then used in conjunction with electroporation to generate isogenic insertional derivatives of A. baumannii 19606. Replica plating showed that 2% of the colonies that grew after electroporation on agar plates without antibiotics also grew in the presence of 40 micro g of kanamycin per ml. DNA hybridization proved that all of the kanamycin-resistant derivatives contained the EZ::TN insertion element, which was mapped to different genomic locations. Replica plating on Simmons citrate agar and microtiter plate-plastic tube assays identified growth- and biofilm-defective derivatives, respectively. The location of the insertion in several of these derivatives was determined by self-ligation of NdeI- or EcoRI-digested genomic DNA and electroporation of Escherichia coli TransforMax EC100D (pir(+)). Sequence analysis of the recovered plasmids showed that some of the A. baumannii 19606 growth-defective derivatives contain insertions within genes encoding activities required for the generation of energy and cell wall components and for the biosynthesis of amino acids and purines. A gene encoding a protein similar to the GacS sensor kinase was interrupted in four derivatives, while another had an insertion in a gene coding for a hypothetical sensor kinase. A. baumannii 19606 derivatives with defective attachment or biofilm phenotypes had insertions within genes that appear to be part of a chaperone-usher transport system described for other bacteria. DNA hybridization experiments showed that the presence of strain 19606 genes encoding regulatory and attachment or biofilm functions is widespread among other A. baumannii clinical isolates. (+info)
Comparison of a repetitive extragenic palindromic sequence-based PCR method and clinical and microbiological methods for determining strain sources in cases of nosocomial Acinetobacter baumannii bacteremia.
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Using a repetitive extragenic palindromic PCR (REP-PCR), we genotypically characterized strains causing nosocomial Acinetobacter baumannii infections and analyzed the source of bacteremia in 67 patients from an institution in which infections by this bacterium were endemic. Six different genotypes were found, including 21, 27, 3, 9, 3, and 4 strains. The probable source of bacteremia, according to clinical and/or microbiological criteria, was known in 42 patients (63%): respiratory tract (n = 19), surgical sites (n = 12), intravascular catheters (n = 5), burns (n = 3), and urinary tract (n = 3). The definite source of bacteremia, according to REP-PCR, could be established in 30 (71%) out of the 42 patients with strains from blood and other sites; in these cases clinical and microbiological criteria for the source of bacteremia were thus confirmed. In the remaining 12 patients (29%) the probable source was refuted by the REP-PCR method. The definite sources of bacteremia according to genotype were as follows: respiratory tract in 13 patients (31%), surgical sites in 8 (19%), intravascular catheters in 4 (9%), burns in 3 (7%), and urinary tract in 2 (5%). A comparison of strains from blood cultures and other sites with regard to their REP-PCR and antimicrobial resistance profiles was also made. Taking the REP-PCR as the "gold standard," the positive predictive value of antibiotype was 77% and the negative predictive value was 42%. In summary, the utility of the diagnosis of the source of nosocomial A. baumannii bacteremia using clinical and/or microbiological criteria, including antibiotyping, is limited, as demonstrated by REP-PCR. (+info)
Endemic carbapenem resistance associated with OXA-40 carbapenemase among Acinetobacter baumannii isolates from a hospital in northern Spain.
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Eighty-two carbapenem-resistant isolates of Acinetobacter baumannii from a single hospital in Bilbao were typed into two major clusters and several subclusters. Disk synergy tests and PCR indicated the production of a zinc-independent OXA-class carbapenemase. Sequencing identified this enzyme, OXA-40, as a variant of the OXA-24-OXA-25-OXA-26 cluster. (+info)
Loss of a 29-kilodalton outer membrane protein in Acinetobacter baumannii is associated with imipenem resistance.
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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. (+info)
Genetic and functional analysis of the chromosome-encoded carbapenem-hydrolyzing oxacillinase OXA-40 of Acinetobacter baumannii.
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Clinical isolate Acinetobacter baumannii CLA-1 was resistant to a series of antibiotic molecules, including carbapenems. Cloning and expression of the beta-lactamase gene content of this isolate in Escherichia coli DH10B identified a chromosome-encoded oxacillinase, OXA-40, that differed by one or two amino acid changes from OXA-24, -25, and -26 and an AmpC-type cephalosporinase. The OXA-40 beta-lactamase had a mainly narrow-spectrum hydrolytic profile, but it included ceftazidime and imipenem. Its activity was resistant to inhibition by clavulanic acid, tazobactam, sulbactam, and, like most of the other carbapenem-hydrolyzing oxacillinases, NaCl. OXA-40 had an FGN triad replacing a YGN motif at class D beta-lactamase (DBL) positions 144 to 146. Site-directed DNA mutagenesis leading to a Phe-to-Tyr change at DBL position 144 in OXA-40 gave a mutant enzyme with increased hydrolytic activity against most beta-lactams, including imipenem. Conversely, with a gene encoding the narrow-spectrum oxacillinase OXA-1 as the template, a nucleotide substitution leading to a Tyr-to-Phe change in the YGN motif of OXA-1 gave a mutant enzyme with decreased hydrolytic activity without an increase in carbapenem-hydrolyzing activity. Thus, the Phe residue in the FGN motif was not associated with carbapenem-hydrolyzing activity by itself but instead was associated with weak overall hydrolytic activity. Finally, this Phe residue in OXA-40 explained resistance to inhibition by NaCl whereas a Tyr residue in motif YGN was related to susceptibility to NaCl. (+info)
Relationship between beta-lactamase production, outer membrane protein and penicillin-binding protein profiles on the activity of carbapenems against clinical isolates of Acinetobacter baumannii.
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Twenty blood isolates of Acinetobacter baumannii were studied, representing eight pulsed-field gel electrophoresis patterns and all different antimicrobial susceptibility patterns observed during 1995-97 at the University Hospital Virgen Macarena, Seville, Spain. The MIC(90)s (mg/L) of imipenem and meropenem decreased from 16 to 0.5 and from 8 to 4, respectively, in the presence of BRL 42715 (BRL) but not clavulanic acid. Hydrolysing activity (nmol/min/mg) of bacterial supernatants against cefaloridine ranged from 8.8 to 552.3 for A. baumannii type I (imipenem MICs < or = 2), which expressed only a beta-lactamase of pI > or = 9, and from 12.3 to 1543.5 for A. baumannii type II (imipenem MICs > or = 4), which expressed a beta-lactamase of pI > or = 9 and two others of pI 6.3 and 7. The hydrolysing activities of A. baumannii type II against imipenem, meropenem and oxacillin were higher than those observed for A. baumannii type I. Ten outer membrane protein (OMP) profiles (A. baumannii types I and II) were visualized on 10% SDS-PAGE gels with 6 M urea, whereas only five OMP profiles (A. baumannii types I and II) were differentiated in 12% SDS-PAGE gels. Five A. baumannii with OMP profile type B, characterized by the absence of a 22.5 kDa OMP, were resistant to meropenem and/or imipenem. Twelve penicillin-binding protein (PBP) patterns were observed. PBP patterns of A. baumannii type II were characterized by the absence of a 73.2 kDa band (PBP 2). We concluded that production of beta-lactamases of pI 6.3 and 7.0 and reduced expression of PBP 2 are the most frequently observed mechanisms of resistance to carbapenems. In some isolates, loss of a 22.5 kDa OMP is also related to resistance to carbapenems. (+info)