Modified Hodge and EDTA-disk synergy tests to screen metallo-β-lactamase-producing strains of Pseudomonas and Acinetobactet species

The objectives of the study were to investigate the distribution of cphA-related genes (cphA) encoding a CphA metallo-b-lactamase (MBL) among 51 consecutive Aeromonas blood isolates and to compare different phenotypic methods for detecting CphA. The presence of cphA was detected by PCR. Four phenotypic methods, the imipenem-EDTA combined disc test, imipenem-EDTA MBL Etest, agar dilution test and modified Hodge test (MHT), were used to detect imipenem susceptibility and MBL production. The results showed that 35 (69 %) blood isolates had cphA. All (100 %) of 16 Aeromonas aquariorum isolates and 12 Aeromonas veronii isolates, and 4 (80 %) of 5 Aeromonas hydrophila isolates, carried cphA, but none of 15 Aeromonas caviae isolates did. With the standard inocula, irrespective of the presence or absence of cphA, all but one (50, 98 %) isolates were susceptible to imipenem tested by disc diffusion, Etest and agar dilution (10 4 c.f.u. spot inocula), and did not exhibit MBL production by the imipenem-EDTA combined disc test and MBL Etest. By the agar dilution test using large inocula (10 7 c.f.u.), 34 (97 %) of 35 cphA + isolates had imipenem MICs of ¢16 mg ml "1 , higher than the susceptible breakpoint (4 mg ml "1 ), and demonstrated positive results for the MHT, while one cphA + and all 17 cphA " isolates had imipenem MICs of ¡4 mg ml "1 . In conclusion, the distribution of cphA among aeromonads is species-specific, found in A. aquariorum, A. veronii and A. hydrophila, and the MHT may be a phenotypic screening test for CphA production.

Section: Discussionmentioning

confidence: 99%

Distribution and phenotypic and genotypic detection of a metallo-β-lactamase, CphA, among bacteraemic Aeromonas isolates

Chen

et al. 2012

“…Hodge and imipenem and EDTA-sodium mercaptoacetic acid double-disc synergy (IEDDS) tests were performed on agar plates as described previously (Lee et al, 2001(Lee et al, , 2010a. The MICs of imipenem and meropenem were determined using Etest strips (AB Biodisk) on Mueller-Hinton agar plates with or without a fixed concentration of clavulanic acid (CA; 4 mg ml 21 ; Sigma-Aldrich) as an inhibitor of ESBLs, aminophenylboronic acid (APB; 300 mg ml 21 ; Sigma-Aldrich) as an inhibitor of AmpC b-lactamase, or Phe-Arg-b-naphthylamide (PAbN; 20 mg ml 21 ; SigmaAldrich) as an efflux pump inhibitor (Song et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

“…At the same time, CA and PAbN did not affect the MICs, indicating that ESBLs and PAbN-inhibited efflux pumps may not play a role in carbapenem resistance. All isolates exhibited negative results in Hodge and IEDDS tests (Lee et al, 2001(Lee et al, , 2010a, indicating that they did not produce carbapenemases.…”

Section: Antimicrobial Susceptibilitiesmentioning

confidence: 99%

Resistance to carbapenems in sequence type 11 Klebsiella pneumoniae is related to DHA-1 and loss of OmpK35 and/or OmpK36

Shin

Bae

Kim

et al. 2012

Resistance to carbapenems in sequence type 11 Klebsiella pneumoniae is related to DHA-1 and loss of OmpK35 and/or OmpK36 This study investigated the molecular mechanisms of carbapenem resistance in clinical isolates of Klebsiella pneumoniae from Korea and the clinical outcomes of resistant K. pneumoniae infection. Sixteen K. pneumoniae isolates showing resistance to carbapenems collected from a tertiary-care hospital were examined for the mechanisms of carbapenem resistance. PCR and sequencing experiments detected the bla DHA-1 AmpC b-lactamase gene in all 16 clinical isolates, whilst the bla genes of extended-spectrum b-lactamases were detected in 12 of 16 isolates. SDS-PAGE experiments indicated that all the isolates lacked the 35 kDa and/or 36 kDa outer-membrane proteins (OMPs). Sequence analysis of the corresponding OMP genes revealed various alterations. PFGE analysis demonstrated that there were no major clonal relationships among the K. pneumoniae isolates. However, multilocus sequence typing experiments showed that all isolates shared the same sequence type (ST), ST11, except for one isolate of ST48. The crude mortality rate of infected patients was 81.8 %. Carbapenem resistance was mainly due to a combination of DHA-1 AmpC b-lactamase coupled with the loss of OmpK35 and/or OmpK36 and was associated with poor clinical outcome. INTRODUCTIONCarbapenems are widely used and are often the only therapeutic option for treating serious infections caused by Klebsiella pneumoniae that produces extended-spectrum b-lactamases (ESBLs) and/or AmpC b-lactamases. The recent emergence of carbapenem-resistant K. pneumoniae in various countries is therefore worrying, as antimicrobial treatment options are limited.Carbapenem resistance in K. pneumoniae has been ascribed mainly to enzymic degradation by plasmid-borne carbapenemases of classes A (GES-2, -4, -5 and -6, and KPC enzymes), B (IMP-1 and -8, and VIM-1, -2 and -12) and D (OXA-48) (Poirel et al., 2004a;Queenan & Bush, 2007). However, some K. pneumoniae strains have acquired carbapenem resistance despite their lack of carbapenemase production. Loss of outer-membrane proteins (OMPs), including OmpK35 and OmpK36, plays a role in carbapenem resistance in K. pneumoniae strains harbouring ESBLs METHODSPatients and strains. This study was conducted between 2007 and 2008 at a tertiary-care teaching hospital with 2000 beds in Seoul, Korea. During this period, K. pneumoniae was isolated from 3278 patients, 16 (0.5 %) of which had K. pneumoniae clinical isolates that exhibited resistance to carbapenems. The isolates were identified using a Vitek GNI card (bioMérieux). Clinical characteristics were investigated through electronic medical record review. Data including age, sex, underlying disease, number of days from hospital admission to identification of K. pneumoniae infection, prior antibiotic treatment, anatomical site of isolation and clinical outcome were obtained.Antimicrobial susceptibility testing. Antibiotic-containing discs (Becton Dickinson) were used for routine an...

“…In order to analyse the production of class B and D carbapenemase, carbapenem-resistant isolates were first screened by a modified Hodge test and an imipenem-EDTA double disc synergy test (Lee et al, 2001). PCR was performed to detect bla OXA , bla IMP and bla VIM genes using specific primers as previously described (Song et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

Clinical and microbiological characterization of carbapenem-resistant Acinetobacter baumannii bloodstream infections

Song

Cheong

Choi

et al. 2011

The incidence of carbapenem-resistant Acinetobacter baumannii infection is increasing, which might be associated with high morbidity and mortality among critically ill patients with limited therapeutic options. This study was conducted to evaluate the clinical and microbiological features of carbapenem-resistant A. baumannii bacteraemia. The medical records of 28 adult patients with this bacteraemia admitted to Korea University Guro Hospital, from January 2005 through December 2010, were reviewed. Using the 28 bloodstream isolates, we intended to detect genes encoding carbapenemases, and investigate the inoculum effect on each of the antimicrobial agents rifampicin, imipenem, colistin and tigecycline. With one blood isolate from a patient with pneumonia, rifampicin-inducible resistance was examined using the experimental mouse pneumonia model. Out of 28 carbapenem-resistant A. baumannii bloodstream infections (BIs), the most common primary focus was the central venous catheter (35.7 %) and then the lung (32.1 %). The 30 day overall mortality was 53.6 %; in most cases (80 %) the patients died within 10 days after the onset of the bacteraemia. By univariate analysis, inappropriate antimicrobial therapy (73.3 vs 30.8 %, P50.02), mechanical ventilation (53.3 vs 15.4 %, P50.04) and a high Pitt bacteraemia score (4.9±1.9 vs 2.2±1.2, P,0.01) were statistically significant risk factors for mortality, while only a high Pitt bacteraemia score (odds ratio 2.6; 95 % confidence interval 1.1-6.5) was independently associated with 30 day mortality by multivariate analysis. All 28 isolates had the bla OXA-51 -like gene with upstream ISAbaI, 2 of which additionally had the bla OXA-58 -like gene and the bla OXA-23 -like gene. Inoculum effect and rifampicin inducible resistance were not detected. Considering the rapid progression to death in carbapenemresistant A. baumannii BIs, early empirical antibiotic therapy would be warranted based on the local microbiological data in each hospital.