Plasmid-mediated AmpC Β-lactamase Research Articles

Evaluation of Boronic Acid Disk Tests for Differentiating KPC-Possessing Klebsiella pneumoniae Isolates in the Clinical Laboratory

The worldwide increase in the occurrence and dissemination of KPC beta-lactamases among gram-negative pathogens makes critical the early detection of these enzymes. Boronic acid disk tests using different antibiotic substrates were evaluated for detection of KPC-possessing Klebsiella pneumoniae isolates. A total of 57 genotypically confirmed KPC-possessing K. pneumoniae isolates with varying carbapenem MICs were examined. To measure the specificity of the tests, 106 non-KPC-possessing isolates (89 K. pneumoniae and 17 Escherichia coli isolates) were randomly selected among those exhibiting reduced susceptibility to cefoxitin, expanded-spectrum cephalosporins, or carbapenems. As many as 56, 53, and 40 of the non-KPC-possessing isolates harbored extended-spectrum beta-lactamases, metallo-beta-lactamases, and plasmid-mediated AmpC beta-lactamases, respectively. By use of CLSI methodology and disks containing imipenem, meropenem, or cefepime, either alone or in combination with 400 microg of boronic acid, all 57 KPC producers gave positive results (sensitivity, 100%) whereas all 106 non-KPC producers were negative (specificity, 100%). The meropenem duplicate disk with or without boronic acid demonstrated the largest differences in inhibition zone diameters between KPC producers and non-KPC producers. By use of disks containing ertapenem, all isolates were correctly differentiated except for five AmpC producers that gave false-positive results (sensitivity, 100%; specificity, 95.3%). These practical and simple boronic acid disk tests promise to be very helpful for the accurate differentiation of KPC-possessing K. pneumoniae isolates, even in regions where different broad-spectrum beta-lactamases are widespread.

Colonisation dynamics and virulence of two clonal groups of multidrug-resistant Escherichia coli isolated from dogs

The study established the virulence potential of multidrug-resistant Escherichia coli (MDREC) isolates from nosocomial infections in hospitalised dogs. The isolates were resistant to fluoroquinolones, belonged to two distinct clonal groups (CG1 and CG2) and contained a plasmid-mediated AmpC (CMY-7) β-lactamase. CG1 isolates ( n = 14) possessed two of 36 assayed extraintestinal virulence genes ( iutA and traT) and belonged to phylogenetic group A, whereas CG2 isolates ( n = 19) contained four such genes ( iutA, ibeA, fimH and kpsMT K5) and belonged to group D. In a mouse gastrointestinal tract colonisation model, colonisation by index CG1 strain C1 was transient, in contrast to the index CG2 strain C2b, which persisted up to 40 days post-inoculation. In a mouse subcutaneous challenge model, both strains were less virulent than archetypal group B2 extraintestinal pathogenic E. coli (ExPEC) strain CFT073; strain C1 caused no systemic signs and strain C2b was lethal to only one of six mice. In a mouse urinary tract infection model, strain C2b colonised the mouse bladder over 2 logs higher compared to strain C1. Whilst both groups of canine MDREC appear less virulent than a reference human ExPEC strain, CG2 strains have greater capacity for colonisation and virulence.

Multiresistant Enterobacteriaceae: new threat of an old problem

The Enterobacteriaceae are among the most important causes of serious nosocomial and community-onset bacterial infections in humans, and resistance to antimicrobial agents in these species has become an increasingly relevant problem for healthcare providers. β-lactam and fluoroquinolone antibiotics are important drug classes used to treat infections caused by Enterobacteriaceae. Emerging resistance mechanisms against these agents have recently been described in Enterobacteriaceae and include the production of newer β-lactamases and plasmid-mediated quinolone resistance. The newer β-lactamases consist of the following: plasmid-mediated AmpC β-lactamases (e.g., ephamycin [CMY], CMY types), extended-spectrum β-lactamases (e.g., cefotaxime [CTX], CTX-M first isolated at Munich) and carbapenem-hydrolyzing enzymes (e.g., Klebsiella pneumoniae carbapenemase [KPC], KPC types and the metallo-β-lactamases). Recent developments in the epidemiology, clinical relevance and laboratory detection of infections caused by multiresistant Enterobacteriaceae with these new types of resistance mechanisms will be addressed in this review.

Prevalence and Mechanisms of Broad-Spectrum β-Lactam Resistance in Enterobacteriaceae : a Children's Hospital Experience

The objective of this study was to investigate the trends and patterns of resistance in beta-lactamase-producing members of the family Enterobacteriaceae in a children's hospital over a 9-year period (1999 to 2007). Clinically significant isolates of the Enterobacteriaceae were screened for patterns of broad-spectrum resistance to beta-lactams. The strains likely to be resistant were subsequently confirmed by an inhibitor-based disc test. The plasmid-mediated resistance determinants in these isolates were identified by PCR and by in vitro transformation, which successfully reproduced the AmpC phenotype unrestricted by the species of the host organisms. Among 8,048 Enterobacteriaceae isolates belonging to the four chromosomal ampC-negative or -nonfunctional genera, 86 (1.07%) isolates (56 Escherichia coli isolates, 22 Klebsiella species isolates, 1 Proteus mirabilis isolate, and 7 Salmonella species isolates) exhibited broad-spectrum beta-lactam resistance patterns. These organisms collectively produced three classes of beta-lactamases, including class A extended-spectrum beta-lactamases (n = 47), class C or AmpC beta-lactamases (n = 36, including 4 isolates that produced both class A and class C enzymes), and class A or B carbapenem-hydrolyzing beta-lactamases (n = 3). The proportion increased from 0.46% during the first 3 years to 1.84% during the last 3 years (relative risk [RR], 4.04; 95% confidence interval [CI], 2.28 to 7.42; P < 0.001). The increase was mainly due to the emergence of a plasmid-mediated bla(CMY-2) beta-lactamase, the incidence of which increased from 0.11% during the first 3 years to 0.96% during the last 3 years (RR, 9.11; 95% CI, 2.76 to 30.1; P = 0.001). Class A-type resistance increased slightly during the study period, from 0.35% during the first 3 years to 0.85% during the last 3 years (RR, 2.42; 95% CI, 1.15 to 5.07; P = 0.02). A Proteus mirabilis strain was documented to possess a novel bla(DHA) determinant. Of special concern, three carbapenemase-producing isolates were identified between 2003 and 2006. The infections caused by resistant isolates of the Enterobacteriaceae mainly affected hospitalized patients with underlying conditions; however, 19 (22%) episodes were of community onset in otherwise well children. The rate of resistance to broad-spectrum beta-lactams among isolates of the Enterobacteriaceae is increasing in children in both hospital- and community-acquired settings, and the resistance is driven largely by plasmid-mediated AmpC beta-lactamases. These data have important implications for empirical antimicrobial strategies targeting serious pediatric infections. Further study of this problem is warranted.

Emergence of a cefepime- and cefpirome-resistant Citrobacter freundii clinical isolate harbouring a novel chromosomally encoded AmpC β-lactamase, CMY-37

Citrobacter freundii strain 4306 was isolated from a urine specimen of a patient in March 2006 in Palestine. This strain showed a unique multidrug resistance phenotype, as it was resistant both to 7-α-methoxy- and oxyimino-cephalosporins, including cefepime, cefpirome and monobactams, in addition to quinolones, streptomycin and trimethoprim/sulfamethoxazole. Clavulanic acid did not act synergistically with cephalosporins by the double-disk synergy test. Molecular characterisation showed that the resistance to 7-α-methoxy- and oxyimino-cephalosporins was due to a novel AmpC β-lactamase, designated CMY-37, with an isoelectric point of ∼9.0. CMY-37 is a variant of C. freundii chromosomal AmpC enzymes with at least seven amino acid substitutions. One of these substitutions, L316I, is located within the R2 loop that is considered the hotspot region responsible for the extended substrate spectrum in class C β-lactamases. The bla CMY-37 gene was cloned and expressed in Escherichia coli TG1. CMY-37 is chromosomally encoded and is not associated with IS Ecp1-like element. Phylogenetic analysis suggested that CMY-37 is the origin of many plasmid-mediated AmpC β-lactamases. This study highlights the emergence of cefepime and cefpirome resistance in C. freundii owing to a new type of AmpC β-lactamase.

Inhibitor-based methods for detection of derepressed hyperproduction and plasmid-mediated AmpC β-lactamases

Objective To investigate the reliability of using inhibitors including Phenylboronic acid (PBA)and Fqucloxacillin(FCC)in detecting derepressed hyperproduction and plasmid-mediated AmpC B-lactamases.Methods PBA and FCC were chosen as inhibitors and double-disk potentiation method and double-disk synergy method were used to detect positive and negative control strains of AmpC β-lactamases and 107 clinical isolates for AmpC β-lactamases production.The positive control strains included E.cloacae (029M),plasmid-mediated ACT-1 type of E.coli DH5a2919,MOX-1 type of k pheumoniae,LAT-2 type of E.coil.The negative control strains included E.cloacae 029(wild-type),E.coli SHV-1,E.coli SHV-2, E.coil SHV-5,E.coli TEM-1,E.coli TEM-3,k peumoniae SHV-18 and E.coli ATCC25922.We compared the results above with the three dimensional test(3-DT)to observe the accuracy in detecting AmpC-BLA.Results 3-DT together with PBA and FCC based inhibition tests showed the 4 positive control strains and the 9 negative control strains were determined as expected.AmpC-BILA was detected in 107 clinical isolates ofEnterobacteriaceaes.The positive rate of3-DTmethod is24.3%.The positive rates ofPBA.FCC double-disk potentiation method and double-disk synergy method are 30.8%(33/107),26.2%(28/107) and 23.4%(25/107),respectively.The conjugate results in two strains of P mirabilis and one strain of K.peumoniae were positive.They were all plasmid-mediated AmpC-Bi.A.There Was a higher false positive when using PBA and FCC-based double-disk potentiation method to detect the induction type of AmpC-BLA, but the accuracy of double-disk synergy method was high.Compared with the 3-DT,the coincidence rate using PBA and FCC-based double-disk synergy method is 99.1%.Conclusions Using PBA and FCC as inhibitors in the double-disk synergy test is a accurate and reliable method to detect AmpC-BLA regardless of derepressed hyperproduction type or plasmid-mediated type. Key words: beta-Lactamases; Boronic acids; Flucloxacillin; Microbial sensitivity tests

The prevalence of plasmid-mediated AmpC β-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae from five children’s hospitals in China

The purpose of this study was to investigate the prevalence of plasmid-mediated AmpC beta-lactamases in Escherichia coli and Klebsiella pneumoniae from five children's hospitals in China. A total of 494 E. coli and 637 K. pneumoniae isolates were collected from five children's hospitals in China from 2005 to 2006. The isolates with decreased susceptibility to cefoxitin were subjected to confirmation test with 3-aminophenyl boronic acid. Polymerase chain reaction (PCR) amplification of the blaAmpC, blaTEM, blaCTXM, and blaSHV genes and their gene sequencing were performed. Transconjugants were achieved by conjugation experiments. Plasmid-mediated AmpC beta-lactamases were found in 10.1% of K. pneumoniae (64/637) and in 2.0% of E. coli (10/494) strains. The proportion of plasmid-mediated AmpC-producing strains significantly increased from 2005 (2.6%) to 2006 (9.3%) (p<0.001). The DHA-1-producing isolates were the most prevalent type (93.2%, 69/74). The sequences of blaDHA-1 genes were all identical to those from the GenBank. Strains of blaCMY-2 were isolated from five isolates (6.8%), which were all from E. coli. One sequence of blaCMY-2 differs from blaCMY-2 in the GenBank. Eighteen of the 74 (24.3%) AmpC-producing K. pneumoniae and E. coli isolates coproduced an extended-spectrum beta-lactamase (ESBL). Cefoxitin resistance was transferred to 15 of the 74 positive strains (20.3%). Our study has demonstrated the occurrence of plasmid-mediated AmpC beta-lactamases in E. coli and K. pneumoniae in Chinese pediatric patients and DHA-1 type AmpC enzymes had the highest prevalent rate. The CMY-2 AmpC beta-lactamases from the children's hospitals in China in this study are the first reported. Hence, continuous surveillance of the prevalence and evolution of AmpC beta-lactamase is important.

Prevalence of Plasmid-Mediated AmpC β-Lactamases in a Chinese University Hospital from 2003 to 2005: First Report of CMY-2-Type AmpC β-Lactamase Resistance in China

The aim of this study was to investigate the prevalences of plasmid-mediated AmpC beta-lactamases (PABLs) in isolates of Escherichia coli and Klebsiella spp. from a university hospital in China. A total of 1,935 consecutive nonrepeat clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca were collected between January 2003 and July 2005. The isolates with cefoxitin zone diameters less than 18 mm (screen positive) were selected for PCR of the bla(AmpC) genes and sequencing. Fifty-four (2.79%) isolates harbored PABLs, as demonstrated by PCR and isoelectric focusing. Sequence analysis revealed the presence of bla(DHA-1) and bla(CMY-2) genes. The Southern blot hybridization studies confirmed that bla(CMY-2) and bla(DHA-1) were located on plasmids. Based on species, PABLs were detected in 4.29% (29 isolates of DHA-1 and 1 isolate of CMY-2) of K. pneumoniae, 1.91% (11 isolates of DHA-1 and 12 isolates of CMY-2) of E. coli, and 3.03% (1 isolate of DHA-1) of K. oxytoca isolates. In contrast to our anticipation, the occurrence rate of DHA-1-producing K. pneumonia significantly decreased (P < 0.01), from 7.54% in 2003 to 2.72% in 2004. The results of random amplified polymorphic DNA analysis indicate that the prevalences of DHA-1-producing K. pneumoniae and CMY-2-producing E. coli strains were not due to epidemic strains. In conclusion, DHA-1 was the most prevalent acquired AmpC beta-lactamase in this collection of isolates from a medical center in China, and DHA-1-producing K. pneumoniae was the most prevalent bacterium harboring a PABL. To the best of our knowledge, this is the first report of CMY-2-type AmpC beta-lactamases in the Chinese mainland.

Prevalence of AmpC and other β-lactamases in enterobacteria at a large urban university hospital in Brazil

Production of extended-spectrum β-lactamases (ESBLs) has been reported in virtually all species of Enterobacteriaceae, which greatly complicates the therapy for infections caused by these organisms . However, the frequency of isolates producing AmpC β-lactamases, especially plasmid-mediated AmpC (pAmpC), is largely unknown. These β-lactamases confer resistance to extended-spectrum cephalosporins and aztreonam, a multidrug-resistant (MDR) profile. The aim of the present study was to determine the occurrence of ESBL and pAmpC β-lactamases in a hospital where MDR enterobacterial isolates recently emerged. A total of 123 consecutive enterobacterial isolates obtained from 112 patients at a university hospital in Rio de Janeiro, Brazil, during March to June 2001 were included in the study. ESBL was detected by the addition of clavulanate to cephalosporin containing disks and by double diffusion. AmpC production was evaluated by a modified tridimensional test and a modified Hodge test. The presence of plasmid-mediated ampC β-lactamase genes was evaluated by multiplex polymerase chain reaction. Sixty-five (53%) of 123 enterobacterial isolates were MDR obtained from 56 patients. ESBL production was detected in 35 isolates; 5 clonal Escherichia coli isolates exhibited high levels of chromosomal AmpC and ESBL production. However, no isolates contained pAmpC genes. Infection or colonization by MDR enterobacteria was not associated with any predominant resistant clones. A large proportion of hospital infections caused by ESBL-producing enterobacteria identified during the study period were due to sporadic infections rather than undetected outbreaks. This observation emphasizes the need to improve our detection methods for ESBL- and AmpC-producing organisms in hospitals where extended-spectrum cephalosporins are in wide use.

Diagnosis and Treatment of Extended-Spectrum and AmpC β-Lactamase–Producing Organisms

To review the laboratory diagnosis of extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase-producing bacteria and evaluate potential treatment options. A PubMed search, restricted to English-language articles, was conducted (1966-May 2007) using the search terms ESBL, AmpC, diagnosis, detection, carbapenem, ertapenem, fluoroquinolone, cephalosporin, cefepime, tigecycline, and colistin. Additional references were identified through review of bibliographies of identified articles. All studies that evaluated laboratory methods for the detection of ESBLs and AmpC beta-lactamases and/or the treatment of these organisms were reviewed. All articles that were deemed to be clinically pertinent were included and critically evaluated. Numerous laboratory techniques are available for the detection of ESBLs. In contrast, laboratory techniques for detection of AmpC beta-lactamases are limited, particularly for plasmid-mediated AmpC beta-lactamases. Routine microbiologic testing may not detect ESBLs or AmpC beta-lactamases. Optimal antibiotic treatment options are derived from limited observational studies and case reports. Randomized clinical trials evaluating appropriate antibiotic treatment options are lacking. In vitro susceptibility does not always correlate with clinical outcomes. The use of imipenem was associated with the lowest incidence of mortality in patients with bacteremia due to ESBL-producing organisms. Laboratory detection of ESBLs for most organisms is possible with Clinical and Laboratory Standards Institute-recommended testing. However, these tests can be associated with both false negative and false positive results, particularly with organisms that harbor both ESBL- and plasmid-mediated AmpC beta-lactamases. No established guidelines exist for the detection of AmpC beta-lactamases. Imipenem and meropenem are superior to other antibiotics for the treatment of serious infections due to ESBL and AmpC beta-lactamase-producing gram-negative bacteria. While in vitro data demonstrate that tigecycline, ertapenem, and colistin might be potential choices, clinical experience is lacking.

Multiplex Asymmetric PCR-Based Oligonucleotide Microarray for Detection of Drug Resistance Genes Containing Single Mutations in Enterobacteriaceae

A multiplex asymmetric PCR (MAPCR)-based microarray method was developed for the detection of 10 known extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC beta-lactamase genes in gram-negative bacteria and for the typing of six important point mutations (amino acid positions 35, 43, 130, 179, 238, and 240) in the bla(SHV) gene. The MAPCR is based on a two-round reaction to promote the accumulation of the single-stranded amplicons amenable for microarray hybridization by employing multiple universal unrelated sequence-tagged primers and elevating the annealing temperature at the second round of amplification. A strategy to improve the discrimination efficiency of the microarray was constituted by introducing an artificial mismatch into some of the allele-specific oligonucleotide probes. The microarray assay correctly identified the resistance genes in both the reference strains and some 111 clinical isolates, and these results were also confirmed for some isolates by direct DNA sequence analysis. The resistance genotypes determined by the microarray correlated closely with phenotypic MIC susceptibility testing. This fast MAPCR-based microarray method should prove useful for undertaking important epidemiological studies concerning ESBLs and plasmid-mediated AmpC enzymes and could also prove invaluable as a preliminary screen to supplement phenotypic testing for clinical diagnostics.

Identification of plasmid-mediated extended-spectrum and AmpC β-lactamases in Enterobacter spp. isolated from dogs

The genetic determinants involved in reduced susceptibility to third-generation cephalosporins and aztreonam were identified in ten canine Enterobacter isolates associated with opportunistic infections in three veterinary hospitals in Brisbane, Australia. All isolates were evaluated by a combination of phenotypic (broth microdilution and disc susceptibility, modified disc diffusion and IEF) and genotypic (PFGE, plasmid analysis, Southern blot hybridization, bacterial conjugation, PCR and sequencing) methods to investigate genetic relatedness and to identify plasmid-mediated resistance genes, in particular beta-lactamase genes responsible for extended-spectrum cephalosporin resistance. The ten canine isolates were genotypically diverse based on PFGE and belonged to either Enterobacter cloacae or Enterobacter hormaechei on the basis of 16S rRNA gene sequence analysis. Plasmid profiles were also diverse. Nine isolates contained a transmissible blaSHV-12-carrying plasmid (approximately 140 kb) that also conferred resistance to chloramphenicol, gentamicin, spectinomycin, tetracycline, trimethoprim and sulfonamides. In all plasmid-mediated extended-spectrum beta-lactamase (ESBL)-producing isolates including transconjugants, blaSHV-12 was shown to reside in a approximately 6.5 kb plasmid fragment. The remaining isolate that was not an ESBL producer possessed an AmpC beta-lactamase gene (blaCMY-2) on a approximately 93 kb transmissible plasmid. This plasmid did not contain any other antimicrobial resistance genes. Additional plasmid-mediated beta-lactamases identified in some isolates included bla(TEM) and blaOXA-10. This is the first report of canine Enterobacter isolates containing transmissible plasmid-mediated blaSHV-12 and blaCMY-2 resistance genes. Therefore, Enterobacter isolated from opportunistic infections in dogs may be an important reservoir of plasmid-mediated resistance genes, which could potentially be spread to other members of the Enterobacteriaceae.

P669 Epidemiology and genotypes of plasmid-mediated AmpC β-lactamase produced by clinical isolates of Klebsiella pneumoniae in Korea

P669 Epidemiology and genotypes of plasmid-mediated AmpC β-lactamase produced by clinical isolates of Klebsiella pneumoniae in Korea

P1027 Plasmid-mediated AmpC β-lactamases in Enterobacteri-aceae lacking a chromosomal ampC gene and E. coli: prevalence at a Swiss university hospital and distribution of the different molecular types in the northern part of Switzerland

P1027 Plasmid-mediated AmpC β-lactamases in Enterobacteri-aceae lacking a chromosomal ampC gene and E. coli: prevalence at a Swiss university hospital and distribution of the different molecular types in the northern part of Switzerland

Detection of Extended-Spectrum β-Lactamases by Using Boronic Acid as an AmpC β-Lactamase Inhibitor in Clinical Isolates of Klebsiella spp. and Escherichia coli

We evaluated highly sensitive methods using boronic acid (BA) to detect extended-spectrum beta-lactamase (ESBL) production. A total of 182 clinical isolates of Klebsiella spp. (n = 118) and Escherichia coli (n = 64) were analyzed: 62 harbored only ESBLs, 80 harbored both ESBLs and plasmid-mediated AmpC beta-lactamases (pAmpCs), and 40 harbored only pAmpCs. The CLSI confirmatory test detected all isolates that produce only ESBLs but detected 85% of isolates that produce both enzymes. When a >/=5-mm increase in the zone diameter of either the cefotaxime (CTX) or the ceftazidime (CAZ) disk in the presence of both clavulanic acid (CA) and BA was considered to be a positive result, the test detected all isolates that harbor ESBLs (+/- pAmpCs) but showed frequent false-positive results (50%) for isolates that produce only pAmpCs. Meanwhile, when a >/=3-mm increase in the zone diameter of either the CTX/BA or the CAZ/BA disk in the presence of CA was considered to be a positive result, the test also detected all isolates that harbor ESBLs (+/- pAmpCs) and showed less frequent false-positive results (5%) in isolates that produce only pAmpCs. The latter new interpretive guideline has enhanced detection of ESBLs in clinical isolates of Klebsiella spp. and Escherichia coli and allowed detection of an ESBL even when potentially masked by a pAmpC.

Reduced imipenem susceptibility in Klebsiella pneumoniae clinical isolates with plasmid-mediated CMY-2 and DHA-1 β-lactamases co-mediated by porin loss

We investigated the resistance mechanisms and clonality among 42 imipenem-non-susceptible Klebsiella pneumoniae isolated at a tertiary care hospital in Korea. Two isolates had bla VIM-2 alleles, whereas bla CMY-2- and bla DHA-1-like alleles were detected in 24 and 16 isolates, respectively, with these enzymes confirmed by sequencing for representative isolates. Transfer of bla CMY-2 and bla DHA-1 was achieved by conjugation. Addition of 300 mg/L 3-aminophenylboronic acid (APB) reduced the minimum inhibitory concentration for 90% of the organisms (MIC 90) of imipenem and meropenem eight- and four-fold, respectively, for the bla CMY-2- and bla DHA-1-positive isolates, confirming the role of these enzymes in resistance. SDS-PAGE of outer membrane proteins for representative isolates showed lack or greatly diminished expression of OmpK35 and OmpK36 porins. Pulsed-field gel electrophoresis of XbaI-restricted genomic DNA revealed two closely related clusters among 23 bla CMY-2-positive isolates, whereas those with bla DHA-1 were more heterogeneous. In conclusion, reduced imipenem susceptibility among K. pneumoniae at this Korean hospital was largely co-mediated by production of plasmid-mediated AmpC β-lactamases along with lack or greatly diminished expression of OmpK35 and OmpK36 porins.

Use of boronic acid disk methods to detect the combined expression of plasmid-mediated AmpC β-lactamases and extended-spectrum β-lactamases in clinical isolates of Klebsiella spp., Salmonella spp., and Proteus mirabilis

A study using boronic acid (BA), an AmpC enzyme inhibitor, was designed to detect the combined expression of plasmid-mediated AmpC β-lactamases (pAmpCs) and extended-spectrum β-lactamases (ESBLs) in bacterial isolates naturally lacking chromosomal ampC genes. A total of 122 Klebsiella spp., Salmonella spp., and Proteus mirabilis isolates producing or nonproducing pAmpCs and/or ESBLs were analyzed. Detection of genes encoding ESBLs and AmpCs was confirmed by polymerase chain reaction (PCR) followed by sequencing of PCR products. A ≥5-mm increase in zone diameter for i) cefoxitin (FOX) and/or cefotetan (CTT) containing BA versus FOX and/or CTT alone was considered positive for AmpC; ii) ceftazidime (CAZ)-clavulanate (CA) and/or cefotaxime (CTX)–CA tested in combination with BA versus CAZ and/or CTX containing BA was considered positive for ESBL. The disk tests of FOX and/or CTT alone and with BA detected 98.4% of organisms producing pAmpCs. All of the 21 pAmpC and ESBL coproducers were accurately detected ESBL by the disk tests of CTX–CA and/or CAZ–CA containing BA and CTX and/or CAZ containing BA. In conclusion, The BA disk test using Clinical and Laboratory Standards Institute methodology is simple and very efficient method to detect pAmpC and ESBL in organisms naturally lacking chromosomal AmpC enzymes. In particular, the method accurately detects the isolates that harbor both AmpCs and ESBLs.

Association of QnrB Determinants and Production of Extended-Spectrum β-Lactamases or Plasmid-Mediated AmpC β-Lactamases in Clinical Isolates of Klebsiella pneumoniae

Clinical isolates of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases or plasmid-mediated AmpC beta-lactamases were screened for qnrA and qnrB genes. QnrB was present in 54 of 54 DHA-1-producing K. pneumoniae isolates and 10 of 45 SHV-12-producing ones, suggesting that the distribution of plasmids conferring resistance to extended-spectrum cephalosporins and quinolones in clinical isolates of K. pneumoniae is widespread.

Dissemination of 16S rRNA methylase-mediated highly amikacin-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea

Novel 16S rRNA methylase-mediated high-level resistance to amikacin and arbekacin has been reported recently in clinical isolates of Gram-negative bacilli only from several countries. We tested amikacin- or arbekacin-nonsusceptible Gram-negative bacilli isolated in 2003 and 2005 at a tertiary-care hospital in Korea by polymerase chain reaction to detect 16S rRNA methylase genes. armA alleles were detected in 14 isolates of Klebsiella pneumoniae, 10 other species of Enterobacteriaceae, and 16 Acinetobacter baumannii, whereas the rmtB allele was detected in 1 K. pneumoniae isolate. The resistance 1st detected in 2003 persisted in 2005. 16S rRNA methylase-producing isolates were highly resistant to arbekacin and amikacin, and were mostly coresistant to levofloxacin. Most K. pneumoniae isolates also produced extended-spectrum β-lactamases and plasmid-mediated AmpC β-lactamases, and most A. baumannii isolates were nonsusceptible to carbapenems.

Effect of Porins and Plasmid-Mediated AmpC β-Lactamases on the Efficacy of β-Lactams in Rat Pneumonia Caused by Klebsiella pneumoniae

The in vivo activities of imipenem, meropenem, and cefepime were studied in a model of rat pneumonia caused by a plasmid-mediated AmpC beta-lactamase ACT-1-producing Klebsiella pneumoniae strain (K. pneumoniae strain 12) and a derivative porin-deficient mutant (K. pneumoniae strain 12dp). No differences between these activities were seen with K. pneumoniae 12. Only meropenem showed an activity slightly better than that of imipenem with K. pneumoniae 12dp.