ABSTRACT
OBJECTIVES: The objective of this study was to investigate whether the insertion sequence IS1294b (IS91 family) is able to mobilize the blaCMY-2 gene and its adjacent regions from one replicon to another. METHODS: Klebsiella pneumoniae Kp2735 was typed by MLST and its plasmid content was examined by S1-PFGE and PCR-based replicon typing. The genetic blaCMY-2 environment was analysed after cloning experiments and sequencing. Transposition assays were performed with an inactivation strategy based on the sacB gene, which confers sucrose-dependent lethality. RESULTS: Kp2735 (ST215) exhibited high-level resistance to ceftazidime owing to the presence of the cephalosporinase CMY-2. The blaCMY-2 gene was located on an IncI1 ST156 plasmid, p2735, of â¼95 kb. Analysis of the genetic environment revealed, upstream of blaCMY-2, the presence of ISEcp1 interrupted by IS1294b and, downstream of blaCMY-2, a region of 1395 bp belonging to the backbone of IncA/C replicons, suggesting a possible DNA transfer between the two plasmids. We showed that IS1294b is able to mobilize blaCMY-2 and its adjacent regions efficiently on the recipient plasmid with a mean frequency of 5.9%. This transfer was due to a one-ended transposition mechanism, implying the non-recognition of its terIS end. CONCLUSIONS: Our experimental data demonstrate for the first time, to our knowledge, the mobilization of a ß-lactamase gene mediated by a member of the IS91 family and highlight the important role of this mobile genetic element in the spread of antibiotic resistance genes.
Subject(s)
DNA Transposable Elements , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/genetics , Recombination, Genetic , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , Ceftazidime/pharmacology , Cephalosporinase , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Humans , Klebsiella Infections/microbiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/isolation & purification , Molecular Sequence Data , Plasmids , Replicon , Sequence Analysis, DNA , beta-Lactam ResistanceABSTRACT
A carbapenem-resistant Klebsiella pneumoniae strain, Kp5196, was responsible for an uncomplicated cystitis in a patient living at home and without history of foreign travel. This isolate produced the metallocarbapenemase NDM-1 and was resistant to all antibiotics except tetracyclines and colistin. The K. pneumoniae strain belonged to sequence type ST15, and bla(NDM-1) was carried by a nontypeable conjugative plasmid. Two months later, a similar ST15 isolate, Kp5241, was present in the patient but was additionally colistin resistant.
Subject(s)
Anti-Bacterial Agents/pharmacology , Colistin/pharmacology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Drug Resistance, Multiple, Bacterial/genetics , Humans , Plasmids/genetics , Tetracyclines/pharmacology , beta-Lactamases/geneticsABSTRACT
In a collection of 110 clinical isolates of Klebsiella pneumoniae, a single strain, Kp593, was found to exhibit a mutator phenotype with a rifampicin mutation frequency 100-fold higher than the modal value for this species. Complementation experiments with the wild-type MutL, one of the main components of the methyl-directed mismatch repair system, allowed the mutator phenotype to be reversed. Sequencing revealed substitution of the conserved residue Lys307 to Arg and site-directed mutagenesis followed by complementation experiments confirmed the critical role of this mutation. The patient infected with Kp593 relapsed a month later and the strain isolated then, Kp869, was identical to Kp593, as verified by PFGE analysis. Phenotypically, Kp869 colonies were more mucoid than those of Kp593, probably due to increased capsule synthesis as shown by electron microscopy. In addition, Kp869 exhibited a 16-fold higher amoxicillin resistance level related to a 36.4 kb tandem duplication encompassing the chromosomal bla(SHV-11) gene, which was unstable in vitro. These data suggest that the mutator phenotype found in Kp593/Kp869 is associated with beneficial mutations conferring a selective advantage, such as increased virulence factor production and antibiotic resistance. The latter was due to resistance gene duplication, an event rarely described in natural isolates. This is the first description of the in vivo occurrence of gene duplication in a mutator background.
Subject(s)
Gene Duplication , Klebsiella pneumoniae/genetics , beta-Lactamases/genetics , Aged, 80 and over , Cloning, Molecular , DNA, Bacterial/genetics , Drug Resistance, Bacterial , Female , Genetic Complementation Test , Humans , Klebsiella Infections/microbiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/pathogenicity , Microbial Sensitivity Tests , MutS DNA Mismatch-Binding Protein/genetics , Mutagenesis, Site-Directed , Mutation , Phenotype , Sequence Analysis, DNA , Virulence , beta-Lactams/pharmacologyABSTRACT
OBJECTIVES: To investigate the high prevalence of extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia coli (4%, 10/250 consecutive isolates) recovered during a 5 month period in the maternity ward of the University Hospital of Bordeaux, France. METHODS: beta-Lactam resistance transfer was analysed by conjugation and transformation. ESBLs were characterized by isoelectric focusing, PCR amplification and sequencing. The relatedness of the strains was examined by PFGE and phylogenetic group determination. Plasmids were characterized by incompatibility group and restriction analysis. RESULTS: Ten ESBL-producing E. coli were isolated from urinary or genital samples of eight mothers and from gastric fluids of two newborns of carrier mothers. The patients were hospitalized in five different units of the maternity ward. Transconjugants, obtained for 7 of the 10 strains, and wild-type strains exhibited various antibiotypes. Different CTX-M enzymes were characterized: CTX-M-1 (n = 4); CTX-M-14 (n = 3); CTX-M-32 (n = 2); and CTX-M-28 (n = 1). The strains recovered from two mothers and their respective babies were identical. All the other strains were epidemiologically unrelated. Furthermore, various plasmids were identified. Environmental samples from the common echographic and sampling rooms did not reveal the presence of ESBL-producing enterobacteria. CONCLUSIONS: The data argue against the occurrence of a nosocomial outbreak and support the hypothesis of an importation of community-acquired ESBL-producing strains into the hospital through colonized/infected patients. At present, not only patients transferred from other hospitals or long-term care facilities are at risk of carrying ESBL-producing enterobacteria on hospital admission, but also community patients.
Subject(s)
Escherichia coli Infections/microbiology , Escherichia coli Infections/transmission , Escherichia coli Proteins/biosynthesis , Escherichia coli/enzymology , Infectious Disease Transmission, Vertical , beta-Lactamases/biosynthesis , Adult , Anti-Bacterial Agents/pharmacology , Bacterial Typing Techniques , Community-Acquired Infections/epidemiology , Community-Acquired Infections/microbiology , Community-Acquired Infections/transmission , DNA Fingerprinting , Electrophoresis, Gel, Pulsed-Field , Environmental Microbiology , Escherichia coli/classification , Escherichia coli/drug effects , Escherichia coli/isolation & purification , Escherichia coli Infections/epidemiology , Female , France/epidemiology , Genotype , Hospitals, Maternity , Hospitals, University , Humans , Infant, Newborn , Microbial Sensitivity Tests , Plasmids/analysis , Pregnancy , beta-Lactamases/classification , beta-Lactams/pharmacologyABSTRACT
The influence of antibiotic dosages and bacterial mutator phenotypes on the emergence of linezolid-resistant mutants was evaluated in an in vitro pharmacokinetic-pharmacodynamic model. A twice-daily 0.5-h infusion of a 200-, 600-, or 800-mg dose for 48 h was simulated against four strains (MIC, 2 microg/ml): Staphylococcus aureus RN4220 and its mutator derivative MutS2, Enterococcus faecalis ATCC 29212, and a mutator clinical strain of E. faecalis, Ef1497. The peak concentrations (4.38 to 4.79, 13.4 to 14.6, and 19.2 to 19.5 microg/ml) and half-lives at beta-phase (5.01 to 6.72 h) fit human plasma linezolid pharmacokinetics. Due to its bacteriostatic property, the cumulative percentages of the dosing interval during which the drug concentration exceeded the MIC (T > MIC), 66.6 and 69.1% of the dosing interval, were not significant, except for Ef1497, with an 800-mg dose and a T > MIC of 80.9%. At the standard 600-mg dosage, resistant mutants (2- to 8-fold MIC increases) were selected only with Ef1497. A lower, 200-mg dosage did not select resistant mutants of E. faecalis ATCC 29212, but a higher, 800-mg dosage against Ef1497 did not prevent their emergence. For the most resistant mutant (MIC, 16 microg/ml), characterization of 23S rRNA genes revealed the substitution A2453G in two of the four operons, which was previously described only in in vitro mutants of archaebacteria. Nevertheless, this mutant did not yield further mutants under 600- or 200-mg treatment. In conclusion, linezolid was consistently efficient against S. aureus strains. The emergence of resistant E. faecalis mutants was probably favored by the rapid decline of linezolid concentrations against a strong mutator, a phenotype less exceptional in E. faecalis than in S. aureus.
Subject(s)
Acetamides/pharmacology , Acetamides/pharmacokinetics , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/pharmacokinetics , Enterococcus faecalis/drug effects , Enterococcus faecalis/genetics , Oxazolidinones/pharmacology , Oxazolidinones/pharmacokinetics , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Acetamides/administration & dosage , Anti-Bacterial Agents/administration & dosage , Base Sequence , DNA Primers/genetics , Dose-Response Relationship, Drug , Drug Resistance, Bacterial/genetics , Humans , In Vitro Techniques , Linezolid , Microbial Sensitivity Tests , Models, Biological , Oxazolidinones/administration & dosage , Phenotype , Point Mutation , RNA, Bacterial/genetics , RNA, Ribosomal, 23S/genetics , Species SpecificityABSTRACT
BACKGROUND: Infections caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are increasing in frequency and are associated with high mortality rates. Circulation of CTX-M-type ESBLs in the community is of particular concern, because it may confound standard infection-control measures. METHODS: We analyzed the results of epidemiologic studies of infection caused by ESBL-producing Enterobacteriaceae in nonhospitalized patients from 6 centers in Europe, Asia, and North America. Risk factors for infection with an ESBL-producing organism were identified by univariate and multivariate analyses. RESULTS: A total of 983 patient-specific isolates were reviewed (890 [90.5%] of which were Escherichia coli, 68 [6.9%] of which were Klebsiella species, and 25 [2.5%] of which were Proteus mirabilis); 339 [34.5%] of the isolates produced ESBLs. CTX-M types were the most frequent ESBLs (accounting for 65%). Rates of co-resistance to ciprofloxacin among ESBL-producing isolates were high (>70%), but significant variation was seen among centers with respect to rates of resistance to gentamicin, amoxicillin-clavulanate, and trimethoprim-sulfamethoxazole. Similar risk factors for infection with an ESBL-producing organism were found in the different participating centers. Significant risk factors, identified by multivariate analysis, were recent antibiotic use, residence in a long-term care facility, recent hospitalization, age 65 years, and male sex (area under the receiver-operator characteristic [ROC] curve, 0.80). However, 34% of ESBL-producing isolates (115 of 336 isolates) were obtained from patients with no recent health care contact; the area under the ROC curve for the multivariate model for this group of patients was only 0.70, which indicated poorer predictive value. CONCLUSIONS: Community-acquired ESBL-producing Enterobacteriaceae are now prevalent worldwide, necessitating international collaboration. Novel approaches are required to adequately address issues such as empirical treatment for severe community-acquired infection and infection control.
Subject(s)
Enterobacteriaceae Infections/epidemiology , Enterobacteriaceae/enzymology , beta-Lactam Resistance , beta-Lactamases/biosynthesis , Community-Acquired Infections/epidemiology , Community-Acquired Infections/microbiology , Enterobacteriaceae/drug effects , Enterobacteriaceae Infections/microbiology , Female , Global Health , Health Surveys , Humans , Male , Microbial Sensitivity Tests , Multivariate Analysis , Prevalence , ROC Curve , Risk Factors , beta-Lactamases/geneticsABSTRACT
Gene transfer via bacterial integrons is a major pathway for facilitating the spread of antibiotic resistance genes across bacteria. Recently the mechanism underlying the recombination catalyzed by class 1 integron recombinase (IntI1) between attC and attI1 was highlighted demonstrating the involvement of a single-stranded intermediary on the attC site. However, the process allowing the generation of this single-stranded substrate has not been determined, nor have the active IntI1*DNA complexes been identified. Using the in vitro strand transfer assay and a crosslink strategy we previously described we demonstrated that the single-stranded attC sequences could be generated in the absence of other bacterial proteins in addition to IntI. This suggests a possible role for this protein in stabilizing and/or generating this structure. The mechanism of folding of the active IntI*DNA complexes was further analyzed and we show here that it involves a cooperative binding of the protein to each recombination site and the emergence of different oligomeric species specific for each DNA substrate. These findings provide further insight into the recombination reaction catalyzed by IntI1.
Subject(s)
Bacteria/enzymology , Integrases/metabolism , Recombination, Genetic , Base Sequence , Biocatalysis , Biopolymers/metabolism , Electrophoresis, Polyacrylamide Gel , Oligodeoxyribonucleotides , Protein BindingABSTRACT
A clinical strain of Klebsiella pneumoniae was found to possess the chromosomal gene bla(SHV-56), encoding a new inhibitor-resistant beta-lactamase with a pI of 7.6. SHV-56 is derived from SHV-11 by the single substitution K234R. This mutation therefore evidences a new critical site for inhibitor resistance among SHV enzymes.
Subject(s)
Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/genetics , beta-Lactamases/chemistry , beta-Lactamases/genetics , Aged , Amino Acid Substitution , Base Sequence , DNA Primers/genetics , DNA, Bacterial/genetics , Female , Genes, Bacterial , Humans , Isoelectric Point , Kinetics , Klebsiella Infections/drug therapy , Klebsiella Infections/microbiology , Klebsiella pneumoniae/isolation & purification , Molecular Sequence Data , Mutagenesis, Insertional , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , beta-Lactam Resistance/genetics , beta-Lactamases/metabolismABSTRACT
BACKGROUND AND AIMS: Listeria monocytogenes and Staphylococcus aureus invade and multiply in THP-1 monocytes. Fluoroquinolones accumulate in these cells, but are less active against intracellular than extracellular forms of L. monocytogenes and S. aureus. We examined whether differentiation of THP-1 monocytes into adherent, macrophage-like cells increases fluoroquinolone uptake and activity. METHODS: THP-1 monocytes were differentiated with phorbol myristate acetate (PMA) and compared with unstimulated cells for: (i) moxifloxacin and levofloxacin accumulation; and (ii) activity against phagocytosed L. monocytogenes and S. aureus (5 h contact). RESULTS: The differentiation of THP-1 monocytes caused: (i) a 3- to 4-fold increase in moxifloxacin uptake and a significant increase in its activity against intracellular L. monocytogenes (from 1.3 log(10) to 2.1 log(10) cfu decrease compared with the post-phagocytosis inoculum), but not against S. aureus (1.0-1.2 log(10) cfu decrease throughout); and (ii) no change in levofloxacin accumulation and intracellular activity against either L. monocytogenes or S. aureus. CONCLUSIONS: Although differentiation of monocytes enhances the uptake and activity of moxifloxacin against L. monocytogenes, this cannot be extended to other intracellular bacteria and to levofloxacin. These results further demonstrate that antibiotic intracellular accumulation and activity are not necessarily linked and suggest that intracellular drug and pathogen combinations must be studied individually.
Subject(s)
Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Aza Compounds/metabolism , Aza Compounds/pharmacology , Levofloxacin , Listeria monocytogenes/drug effects , Monocytes/metabolism , Monocytes/microbiology , Ofloxacin/metabolism , Ofloxacin/pharmacology , Quinolines/metabolism , Quinolines/pharmacology , Staphylococcus aureus/drug effects , Cell Line, Tumor , Colony Count, Microbial , Cytoplasm/chemistry , Cytoplasm/microbiology , Fluoroquinolones , Humans , Microbial Viability , MoxifloxacinABSTRACT
OBJECTIVES: The aim of this study was to assess antibiotic resistance rates and mechanisms of beta-lactam and aminoglycoside resistance among isolates of Pseudomonas aeruginosa isolated in the extra-hospital setting (community and private healthcare centres). PATIENTS AND METHODS: During a 4 month period, 226 non-repetitive strains of P. aeruginosa were collected from patients residing in private healthcare centres (73.5%) or at home (26.5%). Resistance rates were evaluated by MIC determination, and beta-lactam and aminoglycoside resistance was analysed by phenotypic tests, PCR amplification, cloning and sequencing. RESULTS: Among the ticarcillin-resistant strains (38.1%), 33.7% overexpressed their chromosomal cephalosporinase, 27.9% produced acquired penicillinases (21 PSE-1, 2 OXA-21 and 1 TEM-2), 4.7% produced extended-spectrum beta-lactamases (ESBLs) (3 TEM-21 and 1 SHV-2a) and 45.3% possessed a non-enzymatic resistance (NER). Thus, 88.4% had a single mechanism of resistance, whereas 11.6% cumulated several mechanisms. No carbapenemases were detected among the 6.6% imipenem-resistant strains. With regard to aminoglycosides, 23.0% of the strains exhibited an acquired resistance to gentamicin (GEN), tobramycin (TOB), amikacin (AMK) or netilmicin (NET). Enzymatic resistance was more frequent (71.2%) than NER (34.6%). Various aminoglycoside modifying enzymes were associated with overlapping phenotypes: 36.5% strains produced AAC(6')-I with either a serine (GEN-TOB-NET) or a leucine (TOB-NET-AMK) at position 119, or both variants (GEN-TOB-NET-AMK); 21.2% expressed ANT(2'')-I (GEN-TOB), 7.7% AAC(3)-II (GEN-TOB-NET), 5.8% AAC(3)-I (GEN) and 1.9% AAC(6')-II (GEN-TOB-NET-AMK) or AACA7 (TOB-NET-AMK). CONCLUSIONS: Antibiotic resistance rates in P. aeruginosa were globally similar in general practice as in French hospitals. This first analysis of resistance mechanisms showed an unexpectedly high frequency of ESBLs and an unusual distribution of aminoglycoside modifying enzymes.
Subject(s)
Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Community-Acquired Infections/microbiology , Drug Resistance, Bacterial , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , beta-Lactams/pharmacology , Acetyltransferases/biosynthesis , Adolescent , Adult , Aged , Aged, 80 and over , Bacterial Proteins/genetics , Child , Child, Preschool , Community Health Centers , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Female , France , Humans , Infant , Infant, Newborn , Male , Microbial Sensitivity Tests , Middle Aged , Nucleotidyltransferases/biosynthesis , Polymerase Chain Reaction , Pseudomonas aeruginosa/isolation & purification , Sequence Analysis, DNA , beta-Lactamases/biosynthesisABSTRACT
Gene inactivation and complementation experiments showed that the tripartite AheABC efflux pump of Aeromonas hydrophila extruded at least 13 substrates, including nine antibiotics. The use of phenylalanine-arginine-beta-naphthylamide (PAbetaN) revealed an additional system(s) contributing to intrinsic resistance. This is the first analysis of the role of multidrug efflux systems in Aeromonas spp.
Subject(s)
Aeromonas hydrophila/drug effects , Bacterial Proteins/metabolism , Drug Resistance, Multiple, Bacterial , Aeromonas hydrophila/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Biological Transport, Active , Dipeptides/pharmacology , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Sequence Analysis, DNA , Substrate SpecificityABSTRACT
IntI1 integrase is a tyrosine recombinase involved in the mobility of antibiotic resistance gene cassettes within bacterial class 1 integrons. Recent data have shown that its recombination specifically involves the bottom strand of the attC site, but the exact mechanism of the reaction is still unclear. An efficient in vitro assay is still required to better characterize the biochemical properties of the enzyme. In this report we describe for the first time an in vitro system partially reproducing the activity of a recombinant pure IntI1. This new assay, which constitutes the only available in vitro model of recombination by IntI1, was used to determine whether this enzyme might be the sole bacterial protein required for the recombination process. Results show that IntI1 possesses all the features needed for performing recombination between attI and attC sites. However, differences in the in vitro intermolecular recombination efficiencies were found according to the target sites and were correlated with DNA affinities of the enzyme but not with in vivo data. The differential affinity of the enzyme for each site, its capacity to bind to a single-stranded structure at the attC site and the recombination observed with single-stranded substrates unambiguously confirm that it constitutes an important intermediary in the reaction. Our data strongly suggest that the enzyme possesses all the functions for generating and/or recognizing this structure even in the absence of other cellular factors. Furthermore, the in vitro assay reported here constitutes a powerful tool for the analysis of the recombination steps catalyzed by IntI1, its structure-function studies and the search for specific inhibitors.
Subject(s)
Integrases/metabolism , Pseudomonas aeruginosa/enzymology , Vibrio cholerae/enzymology , Base Sequence , Catalysis , DNA Primers , Integrases/isolation & purification , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombination, GeneticABSTRACT
The synthesis of new 4-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]quinoxaline derivatives la-1 is described in five or six steps starting from various substituted nitroanilines 2a-e. The bioisostere 5-[2-(alkylamino)ethylthio]pyrrolo[1,2- a]thieno[3,2-e]pyrazine 1m was also prepared. The new derivatives were evaluated as efflux pump inhibitors (EPIs) in a model targeting the NorA system of Staphylococcus aureus. The antibiotic susceptibility of two strains overproducing NorA, SA-1199B and SA-1, was determined alone and in combination with the neo-synthesised compounds by the agar diffusion method and MIC determination, in comparison with reserpine and omeprazole taken as reference EPIs. A preliminary structure-activity relationship study firstly allowed to clarify the influence of the substituents at positions 7 and/or 8 of the pyrrolo[1,2-a]quinoxaline nucleus. Methoxy substituted compounds, 1b and 1g, were more potent EPIs than the unsubstituted compounds (1a and 1f), followed by chlorinated derivatives (1c-d and 1h). Moreover, the replacement of the N,N-diethylamino group (compounds 1a-e) by a bioisostere such as pyrrolidine (compounds 1f-h) enhanced the EPI activity, in contrast with the replacement by a piperidine moiety (compounds 1i-k). Finally, the pyrrolo[1,2-a]thieno[3,2-e]pyrazine compound 1m exhibited a higher EPI activity than its pyrrolo[1,2-a]quinoxaline analogue la, opening the way to further pharmacomodulation.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Drug Resistance, Multiple, Bacterial/drug effects , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Pyrazines/chemical synthesis , Pyrazines/pharmacology , Quinoxalines/chemical synthesis , Quinoxalines/pharmacology , Staphylococcus aureus/drug effects , Microbial Sensitivity Tests , Molecular Structure , Pyrazines/chemistry , Quinoxalines/chemistryABSTRACT
During a 2004 survey, 49 extended-spectrum-beta-lactamase-producing enterobacteria were collected in 20 French private health care centers and one local hospital. They included 12 CTX-M-producing Escherichia coli strains (1.8% versus 0.3% in a 1999 survey). Most of them belonged to the same clone and contained a bla(CTX-M-15) gene on similar conjugative plasmids.
Subject(s)
Enterobacteriaceae Infections/epidemiology , Enterobacteriaceae Infections/microbiology , Enterobacteriaceae/drug effects , Enterobacteriaceae/enzymology , beta-Lactamases/metabolism , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Enterobacteriaceae/genetics , France/epidemiology , Health Surveys , Hospitals, Private , Molecular Sequence Data , Plasmids/genetics , beta-Lactamases/geneticsABSTRACT
An isocratic high-performance liquid chromatography (HPLC) method with on-line extraction has been developed to determine linezolid in Mueller-Hinton broth. The loading mobile phase consisting of water-acetonitrile 99:1 (v/v) allowed retention of the analyte on a LiChrocart 4-4 pre-column filled with a LiChrospher 100 RP-8, 5 microm. The transfer of the analyte by a backflush mode to a 150 mm x 4.6 mm I.D. Kromasil C8 5 microm column was performed using a mobile phase of water-acetonitrile 80:20 (v/v). UV detection at 254 nm allowed a quantification limit of 0.39 microg/mL with a 50-microL sample size. The method was successfully applied to in vitro pharmacokinetic-pharmacodynamic studies.
Subject(s)
Acetamides/analysis , Anti-Infective Agents/analysis , Chromatography, High Pressure Liquid/methods , Culture Media/chemistry , Oxazolidinones/analysis , Acetamides/isolation & purification , Acetamides/pharmacokinetics , Anti-Infective Agents/isolation & purification , Anti-Infective Agents/pharmacokinetics , Calibration , Linezolid , Oxazolidinones/isolation & purification , Oxazolidinones/pharmacokinetics , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, UltravioletABSTRACT
OBJECTIVES: The aim of this study was to determine the intracellular activity of moxifloxacin against a reference strain and a clinical strain and to study the factors compromising the intracellular activity of moxifloxacin. METHODS: The bactericidal activity of moxifloxacin at therapeutic concentrations was studied against extracellular (broth) and intracellular (infected THP-1 monocytes) forms of Staphylococcus aureus and compared with that of levofloxacin. The activity of moxifloxacin was also evaluated in the presence of alkalinizing agents, in intracellular salt medium mimicking the phagolysosomal environment and in cell lysate. RESULTS: Moxifloxacin, bactericidal against two S. aureus strains (ATCC 25923 and a clinical isolate, Sa2669) in broth, accumulated over 6-fold in monocytes. Against intracellular bacteria, moxifloxacin displayed a markedly reduced activity, not better than levofloxacin, with a maximal reduction of 1 log(10) cfu at 5 h. Cellular accumulation of moxifloxacin was not modified by the addition of efflux pump inhibitors or lysosomal alkalinizing agents. Alkalinization of phagolysosomes significantly enhanced intracellular killing by moxifloxacin. The bactericidal activity of moxifloxacin, abolished in the intracellular salt medium, was partially restored when the pH was raised from 5.0 to 7.4. The binding to intracellular components (35%) did not influence the activity of moxifloxacin. In all cases, surviving bacteria remained fully susceptible to the antibiotic. CONCLUSIONS: The defeat of intracellular activity of moxifloxacin against S. aureus appeared to be more substantially related to cellular parameters (acidic pH and composition of the phagolysosomes) than to the intrinsic activity of the drug and to pharmacokinetic properties.
Subject(s)
Anti-Bacterial Agents/pharmacology , Aza Compounds/pharmacology , Quinolines/pharmacology , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Cell Line , Extracellular Space/drug effects , Fluoroquinolones , Humans , Hydrogen-Ion Concentration , Levofloxacin , Lysosomes/drug effects , Lysosomes/metabolism , Monocytes/metabolism , Monocytes/microbiology , Moxifloxacin , Ofloxacin/pharmacologyABSTRACT
Over a 12-year period, 68 Shigella strains (31 S. sonnei, 30 S. flexneri, 4 S. dysenteriae, and 3 S. boydii strains) were collected in a French University Hospital from the stools of patients who generally had a recent history of travel to various parts of the world (91%), particularly Africa (67%). These strains were often resistant (streptomycin, spectinomycin, trimethoprim, tetracycline, and sulfonamides, 66 to 84%; ampicillin and chloramphenicol, 34 to 38%; nalidixic acid, 4%) and even multiresistant (87%), and they generally carried integrons (81%) of class 1 (21%), class 2 (47%), or both (13%). Class 1 integrons were associated with ampicillin resistance due to the production of an OXA-30 beta-lactamase in S. flexneri and S. dysenteriae. Class 2 integrons were associated with trimethoprim resistance in S. sonnei. Class 1 and class 2 integrons were inserted within transposons Tn21 and Tn7, respectively, themselves located on the bacterial chromosome, except in one strain. Class 1 integrons showed an atypical organization consisting of the insertion sequence IS1 at the 3' end instead of the typical 3' conserved segment and two blaOXA-30 and aadA1 gene cassettes, despite the absence of epidemiological relationships between the strains, and an apparently functional integrase. Class 2 integrons showed the same albeit classical organization with the three dfrA1, sat, and aadA1 gene cassettes. Occasionally, the 3' end was deleted and the aadA1 gene cassette was unexpressed. Thus, integrons contributed only in part to the multidrug resistance of the Shigella strains. The highly conserved organization of integrons might be related to their location within mobile genetic superstructures.
Subject(s)
Drug Resistance, Bacterial/genetics , Integrons/genetics , Shigella/classification , Shigella/genetics , Anti-Bacterial Agents/pharmacology , DNA, Bacterial/genetics , Humans , Molecular Sequence Data , Shigella/drug effects , Shigella/isolation & purificationABSTRACT
A series of 11 pyrrolo[1,2-a]quinoxaline derivatives, 1a to 1k, sharing structural analogies with omeprazole, a eukaryotic efflux pump inhibitor (EPI) used as an antiulcer agent, was synthesized. Their inhibitory effect was evaluated using Staphylococcus aureus strain SA-1199B overexpressing NorA. By determinations of the MIC of norfloxacin in the presence of these EPIs devoid of intrinsic antibacterial activity and used at 128 microg/ml, and by the checkerboard method, compound 1e (MIC decrease, 16-fold; fractional inhibitory concentration index [SigmaFIC], 0.18) appeared to be more active than compounds 1b to 1d, reserpine, and omeprazole (MIC decrease, eightfold; SigmaFIC, 0.31), followed by compounds 1a and 1f (MIC decrease, fourfold; SigmaFIC, 0.37) and 1g to 1k (MIC decrease, twofold; SigmaFIC, 0.50 to 0.56). By time-kill curves combining norfloxacin (1/4 MIC) and the most efficient EPIs (128 microg/ml), compound 1e persistently restored the bactericidal activity of norfloxacin (inoculum reduction, 3 log(10) CFU/ml at 8 and 24 h), compound 1f led to a delayed but progressive decrease in the number of viable cells, and compounds 1b to 1d and omeprazole acted synergistically (inoculum reduction, 3 log(10) CFU/ml at 8 h but further regrowth), while compound 1a and reserpine slightly enhanced norfloxacin activity. The bacterial uptake of norfloxacin monitored by high-performance liquid chromatography confirmed that compounds 1a to 1f increased antibiotic accumulation, as did reserpine and omeprazole. Since these EPIs did not disturb the Deltapsi and DeltapH, they might directly interact with the pump. A structure-activity relationships study identified the benzimidazole nucleus of omeprazole as the main structural element involved in efflux pump inhibition and highlighted the critical role of the chlorine substituents in the stability and efficiency of compounds 1e to 1f. However, further pharmacomodulation is required to obtain therapeutically applicable derivatives.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Drug Resistance, Multiple, Bacterial/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Omeprazole/chemical synthesis , Omeprazole/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Anti-Bacterial Agents/metabolism , Culture Media , Indicators and Reagents , Kinetics , Microbial Sensitivity Tests , Norfloxacin/metabolism , Structure-Activity RelationshipABSTRACT
Gatifloxacin (GAT) is a new 8-methoxy fluoroquinolone with enhanced activity against gram-positive cocci. Its activity was studied in an in vitro pharmacokinetic-pharmacodynamic model against five Staphylococcus aureus strains, either susceptible to ciprofloxacin or exhibiting various levels and mechanisms of ciprofloxacin (CIP) resistance: the ATCC 25923 reference strain (MICs of CIP and GAT: 0.5 and 0.1 microg/ml, respectively), its efflux mutant SA-1 (16 and 0.5 microg/ml; mutation in the norA promoter region), and three clinical strains, Sa2102 (2 and 0.2 microg/ml), Sa2667 (4 and 0.5 microg/ml), and Sa2669 (16 and 1 microg/ml), carrying mutations in the grlA (Ser80Tyr or Phe) and gyrA (Ser84Ala) quinolone resistance-determining regions (QRDRs) for Sa2669. Plasmatic pharmacokinetic profiles after daily 1-h perfusion of 400 mg for 48 h were accurately simulated. Thus, mean maximum concentration of drug in serum values for the two administration intervals were 5.36 and 5.80 microg/ml, respectively, and the corresponding half-life at beta-phase values were 8.68 and 7.80 h (goodness of fit coefficient, >0.98). Therapeutic concentrations of GAT allowed the complete eradication of the susceptible strain within 12 h (difference between the bacterial counts at the beginning of the treatment and at a defined time: -2.18 at the 1-h time point [t(1)] and -6.80 at t(24) and t(48); the bacterial killing and regrowth curve from 0 to 48 h was 30.2 h x log CFU/milliliter). However, mutants (M) with GAT MICs increased by 4- to 40-fold were selected from the other strains. They acquired mutations either supplementary (MSa2102 and MSa2667) or different (Ala84Val for MSa2669) in gyrA or in both gyrA and grlA QRDRs (MSA-1). MSa2667 additionally overproduced efflux system(s) without norA promoter modification. Thus, GAT properties should allow the total elimination of ciprofloxacin-susceptible S. aureus, but resistant mutants might emerge from strains showing reduced susceptibility to older fluoroquinolones independently of the first-step mutation(s).
Subject(s)
Anti-Infective Agents/pharmacology , Ciprofloxacin/pharmacology , Fluoroquinolones/pharmacology , Fluoroquinolones/pharmacokinetics , Staphylococcus aureus/drug effects , Area Under Curve , Computer Simulation , Drug Resistance, Bacterial/genetics , Gatifloxacin , Half-Life , In Vitro Techniques , Microbial Sensitivity Tests , Mutation , Reference Standards , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purificationABSTRACT
OBJECTIVES: Recent studies have raised the question of whether the intracellular activity of quinolones is optimal with respect to their cellular accumulation. The aim of this study was to compare the intracellular and extracellular activities of a commonly used quinolone, levofloxacin, and to examine the causes of the possible inconsistency between intracellular and extracellular effects. METHODS: The bactericidal activity of levofloxacin at therapeutic levels, alone or in combination with various efflux-pump inhibitors or alkalinizing agents, was studied against Staphylococcus aureus ATCC 25923 in Mueller-Hinton (MH) broth and in a THP-1 monocytic cell model, using intracellular salt medium (ISM) mimicking the phagolysosomal environment, and in cell lysate. RESULTS: Levofloxacin accumulation was 2-fold higher in uninfected than in infected cells. Intracellular activity was significantly lower than extracellular activity (decrease in the inoculum of < or = 1 log10 cfu/mL at 4 or 8 mg/L versus > or = 2 log10 units at > or = 1 mg/L in MH broth over 5 h). Persisters remained fully susceptible to the drug. The efflux pump inhibitors verapamil and gemfibrozil did not affect killing of intracellular bacteria, although gemfibrozil increased cellular accumulation of levofloxacin 1.7-fold. The lysosomotropic alkalinizing agents chloroquine and ammonium chloride significantly enhanced intracellular killing by levofloxacin. The bactericidal activity of levofloxacin, abolished in ISM, was partially restored when the pH was neutralized from 5.0 to 7.4. Binding to intracellular components (20%) substantially decreased the efficiency of levofloxacin. CONCLUSIONS: Levofloxacin exhibited substantially lower intracellular activity than extracellular activity. Cellular compartmentalization of the drug, phagolysosomal environment and antibiotic binding to cellular components most likely contribute to this failure.