Inhibitor-Based Methods for Detection of Plasmid-Mediated AmpC β-Lactamases in Klebsiella spp., Escherichia coli , and Proteus mirabilis

Abstract

Non-beta-lactam inhibitor-based methods were evaluated for detecting plasmid-mediated AmpC ␤-lactamases in Klebsiella spp., Escherichia coli, and Proteus mirabilis. Using CLSI methodology and disks containing cefotetan alone and in combination with 400 g of boronic acid, 9 of 10 positive control strains and 54 of 55 AmpC-PCR-positive clinical isolates were detected. Importantly 71% and 40% of these clinical isolates were susceptible by routine testing to ceftriaxone and ceftazidime, respectively. Boronic acid disks also enhanced detection of expanded-spectrum ␤-lactamases in AmpC producers.Pai et al. recently demonstrated a high rate of clinical failure among patients who were infected in the bloodstream with AmpC-␤-lactamase-producing Klebsiella pneumoniae and who received initial antimicrobial therapy, especially cephalosporin treatment (21). Of eight patients who were infected with organisms harboring DHA-1-like or CMY-1-like ␤-lactamases and who received initial and definitive therapy with cefotaxime or ceftazidime and no imipenem, six died. In contrast, 12 of 14 patients who were infected with organisms harboring one of the same two ␤-lactamases but who received imipenem as definitive therapy were cured. Clearly detection of AmpCproducing organisms is important to ensure effective therapeutic intervention and optimal clinical outcome. Imported ampC genes have also been found on the plasmids of other organisms that usually do not harbor these genes, such as Klebsiella oxytoca, Escherichia coli, and Proteus mirabilis (24). Some organisms may harbor plasmid-mediated expanded-spectrum ␤-lactamases (ESBLs) and AmpC ␤-lactamases.Currently, no guidelines for detection of plasmid-mediated AmpC-producing organisms or organisms harboring multiple ␤-lactamases are available. A study was designed to detect AmpC ␤-lactamase-producing isolates of Klebsiella spp., E. coli, and P. mirabilis by methods that closely resemble the phenotypic confirmatory tests for ESBLs (4). Two novel methods that use boronic acid, a known class-C enzyme inhibitor (7), were first tested against positive and negative control strains and then applied as confirmatory tests to clinical isolates.Positive control strains produced the following plasmid-mediated AmpC ␤-lactamases: MOX-1, LAT-2, DHA-1, DHA-2, ACC-1, MIR-1, ACT-1, FOX-1, and FOX-5b. This group included one or more enzymes from each of six families of plasmid-mediated AmpC ␤-lactamases recently selected by Perez-Perez and Hanson, who developed a PCR technique to identify family-specific ampC genes (23). Strains used as negative controls included an OMP F(-) strain, an OMP C(-) strain, and strains that produced the following ␤-lactamases: CTX-M-5, CTX-M-9, TEM-3, TEM-12, SHV-2, SHV-5, SHV-18 (K. pneumoniae ATCC 700603), OXA-3, and KPC-2. A K1-hyperproducing strain of K. oxytoca was also tested.Clinical isolates were recovered at the McGuire Veterans Affairs Medical Center and identified by the Vitek and the API 20E systems (bioMerieux Vitek, Hazelwood, Mo.). Isolates were screened for cef...

Extended-spectrum β-lactamase/AmpC-producing uropathogenic Escherichia coli from HIV patients: do they have a low virulence score?

“…PCR-based detection of ESBL gene families, bla CTX-M , bla TEM and bla SHV , was carried out using previously described primers (Chang et al, 2001;Pagani et al, 2003;Yagi et al, 2000). Phenotypic detection of AmpC b-lactamase production was performed by an inhibitor (boronic acid)-based method (Coudron, 2005) and further confirmed by AmpC disc test (Black et al, 2005). E. coli ATCC 25922 was used as the negative control.…”

AmpC and Extended Spectrum Beta Lactamase Producing Isolates of E. coli, Klebsiella spp. and P. mirabilis in a Tertiary Care Center and their Sensitivity to Other Antibiotics

“…Despite this fact, coproduction of AmpC and ESBLs has been documented in many studies (Grover et al, 2013;Doi and Paterson, 2007;Bakthavatchalu et al, 2013;Mohanty et al, 2010). Prevalence of AmpC producers have been reported to be from 8% to as high as 41.3% in various studies from India and from 1.15% to as high as 46.7% in various studies from other countries (Peter-Getzlaff et al, 2011;Grover et al, 2013;Bakthavatchalu et al, 2013;Polsfuss et al, 2011;Li et al, 2003;Singhal et al, 2005;Manoharan et al, 2012;Maraskolhr et al, 2014;Rudresh and Nagarathnamma, 2011;Tan et al, 2009;Helmy and Wasfi, 2014;Ingram et al, 2011;Yilmaz et al, 2013;Coudron, 2005).…”

Validação do teste de inibição pelo ácido aminofenilborônico para triagem de Klebsiella pneumoniae carbapenemases (KPC)

“…Esse composto é descrito como inibidor reversível das enzimas do tipo KPC (18) . O método foi descrito por Coudron (5) em 2005 e consiste em um teste de disco combinado. A sinergia do ácido fenilborônico (PBA) com os antibióticos foi aplicada para a identificação fenotípica do primeiro isolado produtor de KPC na Grécia.…”

“…Controle dos discos sem APBA também foram realizados. Os discos foram deixados para secar por 30 minutos e armazenados a -20ºC por até três semanas (5) .…”