ABSTRACT
The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an Escherichia coli clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did.
Subject(s)
Anti-Bacterial Agents/chemistry , Cephalosporins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Mutation , Plasmids/chemistry , beta-Lactamases/chemistry , Amino Acid Substitution , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Cephalosporins/metabolism , Cephalosporins/pharmacology , Cysteine/chemistry , Cysteine/metabolism , Escherichia coli/genetics , Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression , Genetic Loci , Humans , Hydrolysis , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Microbial Sensitivity Tests , Models, Molecular , Multilocus Sequence Typing , Plasmids/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Substrate Specificity , Tyrosine/chemistry , Tyrosine/metabolism , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
OBJECTIVES: To characterize the mechanisms implicated in fluoroquinolone (FQ) and expanded-spectrum cephalosporin (ESC) resistance in three clinical and seven faecal multidrug-resistant (MDR; resistant to at least three antimicrobial classes) Escherichia coli isolates from a dog with atopic dermatitis, also suffering from recurrent otitis, that had already been exposed to prolonged antimicrobial treatment and colonized for a long period. METHODS: MICs of FQs, ESCs and other antimicrobials were determined by the broth microdilution method. Phenotypic tests (efflux pump inhibition and combination disc tests) and isoelectric focusing were combined with genotypic analyses [PCRs, sequencing, conjugation, S1 nuclease PFGE, PCR-based replicon typing, plasmid multilocus sequence typing (pMLST) and PCR mapping] to characterize the molecular basis of FQ and ESC resistance. Isolates were further characterized by MLST and PFGE. RESULTS: Three otitis and five faecal isolates with enrofloxacin MICs of 32 to >128 mg/L displayed the GyrA:S83L+D87N/ParC:E62K/ParE:G545D pattern harbouring novel ParC and ParE substitutions, whereas the two remaining faecal isolates were susceptible or borderline resistant single-step mutants (GyrA:S83L pattern) and carried qnrS1. Efflux pump overexpression also contributed to FQ resistance and the MDR phenotype. The three otitis and five faecal isolates also exhibited cefoxitin/ceftazidime MICs of 32-64 mg/L and harboured blaCMY-2, adjusted to ISEcp1, on an IncI1/ST65 conjugative plasmid, previously described in Salmonella Heidelberg from poultry. Interestingly, all isolates shared an identical MLST type (ST212), with the otitis isolates showing indistinguishable patterns with the high-level resistant faecal E. coli isolates. CONCLUSIONS: The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a blaCMY-2-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.
Subject(s)
Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli/drug effects , Escherichia coli/genetics , beta-Lactamases/genetics , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Typing Techniques , Cefoxitin/pharmacology , Ceftazidime/pharmacology , Cephalosporins/pharmacology , DNA Gyrase/genetics , DNA Topoisomerase IV/genetics , Dermatitis, Atopic/microbiology , Dogs , Enrofloxacin , Escherichia coli/isolation & purification , Escherichia coli Infections/drug therapy , Feces/microbiology , Fluoroquinolones/pharmacology , Microbial Sensitivity Tests , Molecular Sequence Data , Multilocus Sequence Typing , Otitis/microbiology , Plasmids/genetics , beta-Lactam Resistance/geneticsABSTRACT
The virulence of a KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) strain representing those circulating in Greece was assessed in a mouse septicemia model. The strain was virtually avirulent (50% lethal dose, >10(8) and 5 × 10(7) CFU for immunocompetent and neutropenic animals, respectively). Also, it was highly susceptible to serum killing, rapidly phagocytosed in vitro, and classified as K41, which is not among the virulent capsular types. The findings indirectly support the notion that high ST258-associated mortality is largely due to inefficient antimicrobial treatment.
Subject(s)
Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , beta-Lactamases/metabolism , Animals , Drug Resistance, Multiple, Bacterial , Female , Humans , Kaplan-Meier Estimate , Mice , Mice, Inbred ICR , Sepsis/drug therapy , Sepsis/microbiologyABSTRACT
Klebsiella pneumoniae strains co-producing klebsiella pneumoniae carbapenemase (KPC) and verona integron-encoded metallo-beta-lactamase (VIM) are frequently isolated in Greece and have also occurred in other European countries. Conventional combined disc tests exhibit low sensitivity against these emerging pathogens. We have evaluated modifications of the KPC/Metallo-ß-Lactamase Confirmation kit (ROSCO) exhibiting high diagnostic value against KPC, VIM and KPC + VIM producers. The key changes were the inclusion of additional combined tablets containing meropenem plus two inhibitors (dipicolinic acid (1000 µg per tablet) for metallo-ß-lactamases and a boronic acid derivative for KPCs) and the replacement of aminophenylboronic acid by phenylboronic acid (400 µg per tablet).
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests/methods , beta-Lactamases/metabolism , Bacterial Proteins/isolation & purification , Cloxacillin/pharmacology , Europe , Greece , Imipenem/pharmacology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/metabolism , Meropenem , Picolinic Acids/pharmacology , Reproducibility of Results , Thienamycins/pharmacology , beta-Lactamases/isolation & purificationABSTRACT
VIM-27 metallo-ß-lactamase, an Ala(57) â Ser variant of VIM-1, was identified in three Klebsiella pneumoniae isolates belonging to sequence type 147. bla(VIM-27) was part of a class 1 integron carried by non-self-transferable plasmids. Kinetic parameters and MIC determinations indicated that VIM-27 hydrolyzed most ß-lactams, especially imipenem and cefoxitin, less effectively than VIM-1.
Subject(s)
Klebsiella pneumoniae/enzymology , beta-Lactamases/chemistry , beta-Lactamases/metabolism , Anti-Bacterial Agents/pharmacology , Cefoxitin/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Imipenem/pharmacology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Microbial Sensitivity Tests , Molecular Sequence Data , Molecular Structure , beta-Lactamases/geneticsABSTRACT
The sequence of pR3521, a self-transmissible plasmid from Escherichia coli, was determined. pR3521 (110,416 bp) comprised a contiguous IncB sequence (84,034 bp) sharing extensive similarities with IncI replicons and an acquired region (26,382 bp) carrying sequences of diverse origin, containing bla(ACC-4), bla(SCO-1), bla(TEM-1b) (two copies), strA, strB, sul2, and aacC2.
Subject(s)
Escherichia coli/genetics , Plasmids/genetics , Sequence Analysis, DNA/methods , beta-Lactamases/genetics , Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Molecular Sequence DataABSTRACT
The nucleotide sequence of pNL194, a VIM-1-encoding plasmid, is described in this study. pNL194 (79,307 bp) comprised an IncN-characteristic segment (38,940 bp) and a mosaic structure (40,367 bp) including bla(VIM-1), aacA7, aadA1, aadA2, dfrA1, dfrA12, aphA1, strA, strB, and sul1. Tn1000 or Tn5501 insertion within fipA probably facilitated recruitment of additional mobile elements carrying resistance genes.
Subject(s)
Bacterial Proteins/genetics , Klebsiella pneumoniae/genetics , Plasmids/genetics , beta-Lactamases/genetics , Models, Genetic , Molecular Sequence DataSubject(s)
Bacterial Proteins/metabolism , Cross Infection/epidemiology , Disease Outbreaks , Klebsiella Infections/epidemiology , Klebsiella pneumoniae/enzymology , beta-Lactamases/metabolism , Anti-Bacterial Agents/pharmacology , Greece/epidemiology , Humans , Imipenem/pharmacology , Klebsiella Infections/microbiology , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/drug effects , Meropenem , Thienamycins/pharmacologyABSTRACT
GES-13 beta-lactamase, a novel GES variant possessing Lys-104 and Asn-170, was identified in Pseudomonas aeruginosa. bla(GES-13) was the single gene cassette of a class 1 integron probably located in the chromosome. GES-13 efficiently hydrolyzed broad-spectrum cephalosporins and aztreonam. Imipenem was a potent inhibitor of GES-13 but was not hydrolyzed at measurable rates.
Subject(s)
Genetic Variation , Pseudomonas aeruginosa/enzymology , beta-Lactamases , Anti-Bacterial Agents/pharmacology , Aztreonam/pharmacology , Cephalosporins/pharmacology , Humans , Integrons/genetics , Microbial Sensitivity Tests , Molecular Sequence Data , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Sequence Analysis, DNA , beta-Lactamases/biosynthesis , beta-Lactamases/genetics , beta-Lactams/pharmacologyABSTRACT
Two CMY-2 derivatives, CMY-31 (Gln(215)-->Arg) from Salmonella enterica serotype Newport and CMY-36 (Ala(77)-->Cys and Gln(193)-->Glu) from Klebsiella pneumoniae, were characterized. Both cephalosporinases functionally resembled CMY-2. bla(CMY) alleles occurred as parts of a putative transposon comprising ISEcp1B and a Citrobacter freundii-derived sequence carried by ColE1-like plasmids similar to CMY-5-encoding pTKH11 from Klebsiella oxytoca.
Subject(s)
Cephalosporinase/genetics , Plasmids , beta-Lactamases/genetics , Alleles , Amino Acid Sequence , Amino Acid Substitution , Arginine/metabolism , Base Sequence , Citrobacter freundii/enzymology , Cysteine/metabolism , DNA Transposable Elements , DNA, Bacterial/genetics , Escherichia coli/genetics , Genes, Bacterial , Glutamine/metabolism , Humans , Hydrolysis , Inhibitory Concentration 50 , Isoelectric Point , Kinetics , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Models, Genetic , Models, Molecular , Molecular Sequence Data , Salmonella enterica/classification , Salmonella enterica/genetics , Salmonella enterica/isolation & purification , Sequence Analysis, DNA , Species Specificity , Substrate Specificity , Transformation, Genetic , beta-Lactamase InhibitorsABSTRACT
A novel class A beta-lactamase (SCO-1) encoded by an 80-kb self-transferable plasmid from Escherichia coli is described. The interaction of SCO-1 with beta-lactams was similar to that of the CARB-type enzymes. Also, SCO-1 exhibited a 51% amino acid sequence identity with the RTG subgroup of chromosomal carbenicillinases (RTG-1, CARB-5, and CARB-8).
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Escherichia coli/enzymology , Penicillinase/metabolism , Plasmids , beta-Lactamases/metabolism , beta-Lactams/pharmacology , Amino Acid Sequence , Anti-Bacterial Agents/metabolism , Escherichia coli/genetics , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Penicillinase/chemistry , Penicillinase/genetics , Sequence Analysis, DNA , beta-Lactam Resistance , beta-Lactamases/chemistry , beta-Lactamases/genetics , beta-Lactams/metabolismABSTRACT
The in-vivo activity of imipenem against VIM-1-producing Klebsiella pneumoniae (VPKP) was assessed in a thigh infection model in neutropenic mice. Animals were infected with three VPKP isolates (imipenem MICs 2, 4 and 32 mg/L, respectively) and a susceptible clinical isolate (MIC 0.125 mg/L) that did not produce any beta-lactamase with broad-spectrum activity. Bacterial density at the site of infection was determined after imipenem treatment (30 and 60 mg/kg every 2 h for 24 h). The log(10) reduction in CFU/thigh was greatest for the wild-type isolate, intermediate for the two imipenem-susceptible VPKP isolates, and lowest for the imipenem-resistant VPKP isolate. Whilst in-vivo imipenem activity appeared reduced against in-vitro susceptible VIM-1 producers compared with a VIM-1-negative control, an increased drug dosage could moderate this reduction.
Subject(s)
Anti-Bacterial Agents/pharmacology , Imipenem/pharmacology , Klebsiella pneumoniae/enzymology , Muscular Diseases/drug therapy , Animals , Anti-Bacterial Agents/blood , Dose-Response Relationship, Drug , Female , Imipenem/blood , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/isolation & purification , Mice , Mice, Inbred ICR , Microbial Sensitivity Tests , Muscular Diseases/microbiology , Neutropenia/blood , Time Factors , beta-Lactamases/biosynthesisSubject(s)
Anti-Bacterial Agents/pharmacology , Aztreonam/pharmacology , Carbapenems/pharmacology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Drug Resistance, Bacterial , Greece , Humans , In Vitro Techniques , Klebsiella Infections/drug therapy , Klebsiella Infections/microbiology , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , beta-Lactamases/biosynthesisABSTRACT
OBJECTIVES: To elucidate the mechanisms responsible for the diversity of beta-lactam resistance phenotypes among isolates of a VIM-1-producing Klebsiella pneumoniae (VPKP) strain that is endemic in Greek hospitals. METHODS: Five VPKP clinical isolates were studied. MICs of beta-lactams were determined by agar dilution. PFGE of XbaI-digested genomic DNA was used for typing. Profiles of outer membrane proteins (OMPs) were determined by SDS-PAGE. Selected isolates were transformed with a plasmid encoding the Omp36K porin. beta-Lactamase activities were analysed by IEF and imipenem hydrolysis was assessed by spectrophotometry. VIM-1-encoding, self-transmissible plasmids were characterized by replicon typing, RFLP and hybridization with bla(VIM)- and IS26-specific probes. Characterization of integrons was performed by PCR, cloning and sequencing. RESULTS: Isolates exhibited highly similar PFGE patterns. Imipenem MICs were 2, 4, 16, 32 and 64 mg/L. The isolate with the highest imipenem MIC (Vipm-64) lacked a 36 kDa OMP. Expression of a cloned OmpK36 in this isolate reduced the imipenem MIC to susceptibility levels. Imipenem-hydrolysing activity was significantly higher in Vipm-16 as compared with the other isolates that expressed similar amounts of VIM-1. All isolates transferred beta-lactam resistance to Escherichia coli through conjugative, IncN plasmids that exhibited differences in the RFLP and hybridization patterns with bla(VIM)- and IS26-specific probes. The Vipm-16 plasmid, mediating the higher imipenem MICs among transconjugants, carried two copies of bla(VIM-1). Cloning and sequencing showed In-e541-like integrons truncated at the 5'CS by insertion of IS26 elements at two different positions. CONCLUSIONS: A VIM-1-producing strain of K. pneumoniae has evolved through OMP alterations and rearrangements in the bla(VIM-1)-carrying plasmid probably mediated by IS26, generating isolates with imipenem MICs ranging from susceptibility to resistance.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Outer Membrane Proteins/genetics , Carbapenems/pharmacology , Genes, Bacterial , Klebsiella pneumoniae/genetics , beta-Lactam Resistance/genetics , beta-Lactamases/genetics , Cloning, Molecular , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/enzymology , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Plasmids , Polymerase Chain ReactionSubject(s)
Drug Resistance, Multiple, Bacterial , Klebsiella pneumoniae/drug effects , beta-Lactamases/biosynthesis , Anti-Bacterial Agents/pharmacology , Colistin/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Greece , Humans , Klebsiella Infections/microbiology , Klebsiella pneumoniae/classification , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Prostaglandins EABSTRACT
CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli emerged recently in Cameroon. CTX-M-15 was encoded by two different multiresistance plasmids, of which one carried an ISEcp1-bla(CTX-M-15) element flanked by a 5-bp target site duplication and inserted within a Tn2-derived sequence. A truncated form of this element in the second plasmid was identified.