gentamicin-sulfate and Pseudomonas-Infections

The activity of the new cephalosporin CXA-101 (CXA), previously designated FR264205, was evaluated against a collection of 236 carbapenem-resistant P. aeruginosa isolates, including 165 different clonal types, from a Spanish multicenter (127-hospital) study. The MICs of CXA were compared to the susceptibility results for antipseudomonal penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones. The MIC of CXA in combination with tazobactam (4 and 8 microg/ml) was determined for strains with high CXA MICs. The presence of acquired beta-lactamases was investigated by isoelectric focusing and PCR amplification followed by sequencing. Additional beta-lactamase genes were identified by cloning and sequencing. The CXA MIC50/MIC90 for the complete collection of carbapenem-resistant P. aeruginosa isolates was 1/4 microg/ml, with 95.3% of the isolates showing an MIC of 8 microg/ml produced a horizontally acquired beta-lactamase, including the metallo-beta-lactamase (MBL) VIM-2 (one strain), the extended-spectrum beta-lactamase (ESBL) PER-1 (one strain), several extended-spectrum OXA enzymes (OXA-101 [one strain], OXA-17 [two strains], and a newly described OXA-2 derivative [W159R] designated OXA-144 [four strains]), and a new BEL variant (BEL-3) ESBL (one strain), as identified by cloning and sequencing. Synergy with tazobactam in these 11 strains was limited, although 8 microg/ml reduced the mean CXA MIC by 2-fold. CXA is highly active against carbapenem-resistant P. aeruginosa isolates, including MDR strains. Resistance was restricted to still-uncommon strains producing an acquired MBL or ESBL.

Topics: Anti-Bacterial Agents; beta-Lactamases; Carbapenems; Cephalosporins; Drug Resistance, Multiple, Bacterial; Humans; Isoelectric Focusing; Microbial Sensitivity Tests; Polymerase Chain Reaction; Pseudomonas aeruginosa; Pseudomonas Infections

Expression of ampC, oprD, mexA, mexC, mexE, and mexX was studied in 25 Pseudomonas aeruginosa isolates from cystic fibrosis patients, including 14 isolates of the Liverpool epidemic strain. Overexpressed mexA or ampC and reduced oprD were associated with beta-lactam resistance. A specific combination of mexR, nalC, and nalD mutations occurred in 11 Liverpool strain isolates, including 7 with upregulated mexA.

Topics: Bacterial Outer Membrane Proteins; Bacterial Proteins; beta-Lactamases; Cystic Fibrosis; Drug Resistance, Bacterial; Drug Resistance, Multiple; Electrophoresis, Gel, Pulsed-Field; Humans; Membrane Transport Proteins; Mutation; Porins; Pseudomonas aeruginosa; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation

A total of 489 clinical isolates of Pseudomonas aeruginosa was investigated for metallo-beta-lactamase (MBL) production. Molecular analysis detected a blaVIM-1 gene in the chromosome of one isolate and a blaVIM-2 gene carried on the plasmid in seven isolates. Moreover, we showed that an initial screening by combined susceptibility testing of imipenem and ceftazidime followed by a confirmatory EDTA combination disk test represents a valid alternative to the molecular investigation of MBL genes, making MBL detection possible in routine diagnostic laboratories.

Topics: Anti-Bacterial Agents; beta-Lactamases; Ceftazidime; Drug Resistance, Bacterial; Germany; Hospitals, University; Humans; Imipenem; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Pseudomonas Infections

Topics: Austria; Bacterial Proteins; beta-Lactam Resistance; beta-Lactamases; beta-Lactams; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Pseudomonas aeruginosa; Pseudomonas Infections; Sequence Analysis, DNA

Topics: Anti-Bacterial Agents; beta-Lactam Resistance; beta-Lactamases; Brazil; Ceftriaxone; Clindamycin; Drug Resistance, Multiple, Bacterial; Fatal Outcome; Humans; Male; Microbial Sensitivity Tests; Middle Aged; Pseudomonas aeruginosa; Pseudomonas Infections

Retrospective analysis of 189 nonredundant strains of Pseudomonas aeruginosa sequentially recovered from the sputum samples of 46 cystic fibrosis (CF) patients over a 10-year period (1998 to 2007) revealed that 53 out of 189 (28%) samples were hypersusceptible to the beta-lactam antibiotic ticarcillin (MIC < or = 4 microg/ml) (phenotype dubbed Tic(hs)). As evidenced by trans-complementation and gene inactivation experiments, the mutational upregulation of the efflux system MexXY was responsible for various degrees of resistance to aminoglycosides in a selection of 11 genotypically distinct strains (gentamicin MICs from 2 to 64 microg/ml). By demonstrating for the first time that the MexXY pump may evolve in CF strains, we found that a mutation leading to an F1018L change in the resistance-nodulation-cell division (RND) transporter MexY was able to increase pump-promoted resistance to aminoglycosides, cefepime, and fluoroquinolones twofold. The inactivation of the mexB gene (which codes for the RND transporter MexB) in the 11 selected strains showed that the Tic(hs) phenotype was due to a mutational or functional loss of function of MexAB-OprM, the multidrug efflux system known to contribute to the natural resistance of P. aeruginosa to beta-lactams (e.g., ticarcillin and aztreonam), fluoroquinolones, tetracycline, and novobiocin. Two of the selected strains synthesized abnormally low amounts of the MexB protein, and 3 of 11 strains expressed truncated MexB (n = 2) or MexA (n = 1) polypeptide as a result of mutations in the corresponding genes, while 7 of 11 strains produced wild-type though nonfunctional MexAB-OprM pumps at levels similar to or even higher than that of reference strain PAO1. Overall, our data indicate that while MexXY is necessary for P. aeruginosa to adapt to the hostile environment of the CF lung, the MexAB-OprM pump is dispensable and tends to be lost or inactivated in subpopulations of P. aeruginosa.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Bacterial Proteins; beta-Lactams; Cystic Fibrosis; Gene Expression Regulation, Bacterial; Humans; Membrane Transport Proteins; Microbial Sensitivity Tests; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Sputum

Polymyxins are often the only option to treat acquired multidrug-resistant Pseudomonas aeruginosa. Polymyxin susceptibility in P. aeruginosa PAO1 is associated with the lipopolysaccharide structure that is determined by arnBCADTEF and modulated by phoPQ and pmrAB. We examined five clonally unrelated clinical isolates of polymyxin B-resistant P. aeruginosa to investigate the molecular basis of polymyxin resistance. All isolates grew with 4 microg/ml polymyxin B (MIC, 8 microg/ml), whereas P. aeruginosa PAO1 grew with 0.25 mug/ml polymyxin B (MIC, 0.5 microg/ml). The resistant isolates were converted to susceptible ones (the MICs fell from 8 to 0.5 microg/ml) following the introduction of phoPQ (four isolates) and pmrAB (one isolate), which had been cloned from strain PAO1. DNA sequence analysis revealed that a single-nucleotide substitution in three isolates replaced a single amino acid of PhoQ, the deletion of 17 nucleotides in one isolate truncated the protein of PhoQ, and two nucleotide substitutions in one isolate replaced two amino acids of PmrB. The involvement of these amino acid substitutions or the truncated protein of PhoQ and PmrB in polymyxin B resistance was confirmed using strain PAO1 lacking phoPQ or pmrAB that was transformed by phoPQ or pmrAB containing the amino acid substitutions or the truncated protein. The resistant clinical isolates were sensitized by the inactivation of arnBCADTEF (the MICs fell from 8 to 0.5 microg/ml). These results suggest that polymyxin B resistance among clinical isolates of P. aeruginosa is associated with alterations in two-component regulatory systems of phoPQ or pmrAB.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Gene Expression Regulation, Bacterial; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Polymyxin B; Pseudomonas aeruginosa; Pseudomonas Infections; Transcription Factors

Two clonally related Pseudomonas aeruginosa isolates, recovered from two patients admitted to a pediatric intensive care unit, were found to harbor a new OXA-2 variant (Asn148Asp), designated OXA-161. The plasmid location of bla(OXA-161) was demonstrated through electroporation to PAO1, and its codification in a class I integron (together with aacA4) was demonstrated through PCR and sequencing. bla(OXA-2) and bla(OXA-161) were cloned in parallel to demonstrate the extended-spectrum beta-lactamase properties of OXA-161, conferring resistance to ceftazidime and reduced susceptibility to cefepime and aztreonam.

Topics: Amikacin; Aminoglycosides; Anti-Bacterial Agents; beta-Lactamases; Cefepime; Cephalosporins; Clavulanic Acid; Gentamicins; Humans; Imipenem; Integrons; Meropenem; Molecular Sequence Data; Penicillanic Acid; Piperacillin; Plasmids; Pseudomonas aeruginosa; Pseudomonas Infections; Spain; Tazobactam; Thienamycins

In a university hospital, time-series analysis revealed a significant relationship between antibiotic (aminoglycoside, fluoroquinolone, and cefepime) use and incidence of MexXY-OprM-overproducing Pseudomonas aeruginosa. In vitro experiments confirm that such mutants were readily selected from both PAO1 and clinical strains when grown in the presence of these antibiotics.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacterial Outer Membrane Proteins; Bacterial Proteins; Cefepime; Cephalosporins; Culture Media; Drug Resistance, Multiple; Fluoroquinolones; France; Hospitals, University; Humans; Incidence; Membrane Transport Proteins; Microbial Sensitivity Tests; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections

The in vitro activities of 10 families of antimicrobial agents alone and in combination with a synthetic polycationic polymer, polyethylenimine (PEI), against a resistant clinical isolate of Pseudomonas aeruginosa were investigated by MIC assays, checkerboard testing, and killing curve studies. At a concentration of 250 nM, PEI (10 kDa) was not directly bactericidal or bacteriostatic; but when it was used in combination with novobiocin, ceftazidime, ampicillin, ticarcillin, carbenicillin, piperacillin, cefotaxime, chloramphenicol, rifampin, or norfloxacin, it significantly reduced the MICs of these antibiotics by 1.5- to 56-fold. However, the MICs of aminoglycosides, polymyxins, and vancomycins were increased by 1.2- to 5-fold when these drugs were combined with PEI; and the MICs of tetracycline, erythromycin, ciprofloxacin, and ofloxacin were not affected when these drugs were combined with PEI. In the killing curve studies, combinations of PEI with novobiocin, ceftazidime, chloramphenicol, or rifampin resulted in 5- to 8-log(10) CFU/ml reductions in bacterial counts when 25% of the MIC of each antibiotic was used. These results indicate that infections due to resistant Pseudomonas strains could be treated by the use of a synergistic combination of PEI and antimicrobial drugs.

Topics: Aminoglycosides; Anti-Bacterial Agents; Carbenicillin; Ceftazidime; Ciprofloxacin; Drug Resistance, Bacterial; Drug Synergism; Humans; Microbial Sensitivity Tests; Novobiocin; Ofloxacin; Piperacillin; Polyethyleneimine; Pseudomonas aeruginosa; Pseudomonas Infections; Rifampin; Ticarcillin; Tobramycin

Since their initial description 2 decades ago, MexCD-OprJ-overproducing efflux mutants of Pseudomonas aeruginosa (also called nfxB mutants) have rarely been described in the clinical setting. Screening of 110 nonreplicate clinical isolates showing moderate resistance to ciprofloxacin (MIC from 0.5 microg/ml to 4 microg/ml) yielded only four mutants (3.6%) of that type harboring various alterations in the repressor gene nfxB. MexCD-OprJ upregulation correlated with an increased resistance to ciprofloxacin, cefepime, and chloramphenicol in most of the clinical strains, concomitant with a higher susceptibility to ticarcillin, aztreonam, imipenem, and aminoglycosides. Evidence was obtained that this increased susceptibility to aminoglycosides results from the impaired activity of efflux pump MexXY-OprM. Furthermore, MexCD-OprJ upregulation was found to impair bacterial growth and to have a strain-specific, variable impact on rhamnolipid, elastase, phospholipase C, and pyocyanin production. Review of patient files indicated that the four nfxB mutants were responsible for confirmed cases of infection and emerged during long-term therapy with ciprofloxacin. Taken together, these data show that, while rather infrequent among P. aeruginosa strains with low-level resistance to ciprofloxacin, MexCD-OprJ-overproducing mutants may be isolated after single therapy with fluoroquinolones and may be pathogenic.

Topics: Adult; Aged; Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; Ciprofloxacin; DNA Primers; DNA-Binding Proteins; DNA, Bacterial; Drug Resistance, Bacterial; Female; Genes, Bacterial; Humans; Male; Membrane Proteins; Membrane Transport Proteins; Middle Aged; Molecular Sequence Data; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Transcription Factors; Virulence

During 2003, 40 carbapenem-resistant Pseudomonas aeruginosa clinical isolates collected in a Mexican tertiary-care hospital were screened for metallo-beta-lactamase production. Thirteen isolates produced IMP-15, and 12 had a single pulsed-field gel electrophoresis pattern. The bla(IMP-15) gene cassette was inserted in a plasmid-borne integron with a unique array of gene cassettes and was named In95.

Topics: Bacterial Proteins; beta-Lactamases; Cross Infection; Electrophoresis, Gel, Pulsed-Field; Humans; Integrons; Mexico; Models, Genetic; Molecular Sequence Data; Pseudomonas aeruginosa; Pseudomonas Infections

During a survey conducted to evaluate the incidence of class B carbapenemase (metallo-beta-lactamase [MBL])-producing Pseudomonas aeruginosa strains from hospitals in Majorca, Spain, five clinical isolates showed a positive Etest MBL screening test result. In one of them, strain PA-SL2, the presence of a new bla(VIM) derivative (bla(VIM-13)) was detected by PCR amplification with bla(VIM-1)-specific primers followed by sequencing. The bla(VIM-13)-producing isolate showed resistance to all beta-lactams (except aztreonam), gentamicin, tobramycin, and ciprofloxacin. VIM-13 exhibited 93% and 88% amino acid sequence identities with VIM-1 and VIM-2, respectively. bla(VIM-13) was cloned in parallel with bla(VIM-1), and the resistance profile conferred was analyzed both in Escherichia coli and in P. aeruginosa backgrounds. Compared to VIM-1, VIM-13 conferred slightly higher levels of resistance to piperacillin and lower levels of resistance to ceftazidime and cefepime. VIM-13 and VIM-1 were purified in parallel as well, and their kinetic parameters were compared. The k(cat)/K(m) ratios for the antibiotics mentioned above were in good agreement with the MIC data. Furthermore, EDTA inhibited the activity of VIM-13 approximately 25 times less than it inhibited the activity of VIM-1. VIM-13 was harbored in a class 1 integron, along with a new variant (Ala108Thr) of the aminoglycoside-modifying enzyme encoding gene aacA4, which confers resistance to gentamicin and tobramycin. Finally, the VIM-13 integron was apparently located in the chromosome, since transformation and conjugation experiments consistently yielded negative results and the bla(VIM-13) probe hybridized only with the genomic DNA.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; beta-Lactam Resistance; beta-Lactamases; Cloning, Molecular; DNA Primers; DNA, Bacterial; Genes, Bacterial; Genetic Variation; Humans; Imipenem; Integrons; Kinetics; Meropenem; Molecular Sequence Data; Phylogeny; Pseudomonas aeruginosa; Pseudomonas Infections; Recombinant Proteins; Sequence Homology, Amino Acid; Spain; Thienamycins

The mutation frequency for carbapenem resistance in Pseudomonas aeruginosa strains that were selected with carbapenems was enhanced in the presence of subinhibitory concentrations of fluoroquinolones. The mutants showed either a loss of OprD activity or increased mexAB-oprM expression. The highest mutant isolation frequency was obtained by selection with meropenem, while doripenem inhibited mutant growth.

Topics: Base Sequence; beta-Lactam Resistance; Carbapenems; DNA Primers; DNA, Bacterial; Doripenem; Fluoroquinolones; Genes, Bacterial; Humans; In Vitro Techniques; Meropenem; Mutation; Pseudomonas aeruginosa; Pseudomonas Infections; Thienamycins

Intubated patients frequently become colonized by Pseudomonas aeruginosa, which is subsequently responsible for ventilator-associated pneumonia. This pathogen readily acquires resistance against available antimicrobials. Depending on the resistance mechanism selected for, resistance might either be lost or persist after removal of the selective pressure. We investigated the rapidity of selection, as well as the persistence, of antimicrobial resistance and determined the underlying mechanisms. We selected 109 prospectively collected P. aeruginosa tracheal isolates from two patients based on their prolonged intubation and colonization periods, during which they had received carbapenem, fluoroquinolone (FQ), or combined beta-lactam-aminoglycoside therapies. We determined antimicrobial resistance phenotypes by susceptibility testing and used quantitative real-time PCR to measure the expression of resistance determinants. Within 10 days after the initiation of therapy, all treatment regimens selected resistant isolates. Resistance to beta-lactam and FQ was correlated with ampC and mexC gene expression levels, respectively, whereas imipenem resistance was attributable to decreased oprD expression. Combined beta-lactam-aminoglycoside resistance was associated with the appearance of small-colony variants. Imipenem and FQ resistance persisted for prolonged times once the selecting antimicrobial treatment had been discontinued. In contrast, resistance to beta-lactams disappeared rapidly after removal of the selective pressure, to reappear promptly upon renewed exposure. Our results suggest that resistant P. aeruginosa is selected in less than 10 days independently of the antimicrobial class. Different resistance mechanisms lead to the loss or persistence of resistance after the removal of the selecting agent. Even if resistant isolates are not evident upon culture, they may persist in the lung and can be rapidly reselected.

Topics: Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Drug Resistance, Bacterial; Humans; Longitudinal Studies; Pneumonia, Ventilator-Associated; Polymerase Chain Reaction; Pseudomonas aeruginosa; Pseudomonas Infections

In this study, we systematically investigated the resistance mechanisms to beta-lactams, aminoglycosides, and fluoroquinolones of 120 bacteremic strains of Pseudomonas aeruginosa. Pulsed-field gel electrophoresis genotyping showed that 97 of these strains were represented by a single isolate, 10 by 2 and 1 by 3 clonally related isolates, respectively. Seventy-five percent (90 out of 120) of the bacteremic P. aeruginosa strains displayed a significant resistance to one or more of the tested antimicrobials (up to 11 for 1 strain). These strains were found to harbor a great diversity of resistance mechanisms (up to 7 in 1 strain), leading to various levels of drug resistance. Interestingly, 11 and 36% of the isolates appeared to overproduce the MexAB-OprM and MexXY-OprM efflux systems, respectively. Altogether, our results show that P. aeruginosa may accumulate intrinsic (overproduction of cephalosporinase AmpC, increased drug efflux, fluoroquinolone target mutations, and deficient production of porin OprD) and exogenous (production of secondary beta-lactamases and aminoglycoside-modifying enzymes) resistance mechanisms without losing its ability to generate severe bloodstream infections. Consequently, clinicians should be aware that multidrug-resistant P. aeruginosa may remain fully pathogenic.

Topics: Aminoglycosides; Anti-Bacterial Agents; Bacteremia; beta-Lactams; DNA, Bacterial; Drug Resistance, Bacterial; Electrophoresis, Gel, Pulsed-Field; Fluoroquinolones; Genotype; Humans; Microbial Sensitivity Tests; Pseudomonas aeruginosa; Pseudomonas Infections; Reverse Transcriptase Polymerase Chain Reaction