Jennifer A. Black

AmpC Disk Test for Detection of Plasmid-Mediated AmpC β-Lactamases in Enterobacteriaceae Lacking Chromosomal AmpC β-Lactamases

ABSTRACT Although plasmid-mediated AmpC β-lactamases were first reported in the late 1980s, many infectious disease personnel remain unaware of their clinical importance. These enzymes are typically produced by isolates of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Salmonella spp. and are associated with multiple antibiotic resistance that leaves few therapeutic options. Plasmid-mediated AmpC β-lactamases have been associated with false in vitro susceptibility to cephalosporins. Many laboratories do not test for this resistance mechanism because current tests are inconvenient, subjective, lack sensitivity and/or specificity, or require reagents that are not readily available. In this study a new test, the AmpC disk test, based on filter paper disks impregnated with EDTA, was found to be a highly sensitive, specific, and convenient means of detection of plasmid-mediated AmpC β-lactamases in organisms lacking a chromosomally mediated AmpC β-lactamase. Using cefoxitin insusceptibility as a screen, the test accurately distinguished AmpC and extended-spectrum β-lactamase production and differentiated AmpCs from non-β-lactamase mechanisms of cefoxitin insusceptibility, such as reduced outer membrane permeability. The test is a potentially useful diagnostic tool. It can provide important infection control information and help to ensure that infected patients receive appropriate antibiotic therapy.

Prevalence of Newer β-Lactamases in Gram-Negative Clinical Isolates Collected in the United States from 2001 to 2002

ABSTRACT Newer β-lactamases such as extended-spectrum β-lactamases (ESBLs), transferable AmpC β-lactamases, and carbapenemases are associated with laboratory testing problems of false susceptibility that can lead to inappropriate therapy for infected patients. Because there appears to be a lack of awareness of these enzymes, a study was conducted during 2001 to 2002 in which 6,421 consecutive, nonduplicate clinical isolates of aerobically growing gram-negative bacilli from patients at 42 intensive care unit (ICU) and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. From these isolates, 746 screen-positive isolates (11.6%) were referred to a research facility and investigated to determine the prevalence of ESBLs in all gram-negative isolates, transferable AmpC β-lactamases in Klebsiella pneumoniae, and carbapenemases in Enterobacteriaceae. The investigations involved phenotypic tests, isoelectric focusing, β-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular analyses. ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae) and were found in species other than those currently recommended for ESBL testing by the CLSI (formerly NCCLS). These isolates occurred at 74% of the ICU sites and 43% of the non-ICU sites. Transferable AmpC β-lactamases were detected in 3.3% of K. pneumoniae isolates and at 16 of the 63 sites (25%) with no difference between ICU and non-ICU sites. Three sites submitted isolates that produced class A carbapenemases. No class B or D carbapenemases were detected. In conclusion, organisms producing ESBLs and transferable AmpC β-lactamases were widespread. Clinical laboratories must be able to detect important β-lactamases to ensure optimal patient care and infection control.

Occurrence of Newer β-Lactamases in Klebsiella pneumoniae Isolates from 24 U.S. Hospitals

ABSTRACT Despite the discovery of novel β-lactamases such as extended-spectrum β-lactamases (ESBLs), imported AmpC, and carbapenem-hydrolyzing β-lactamases at least a decade ago, there remains a low level of awareness of their importance and how to detect them. There is a need to increase the levels of awareness of clinical laboratories about the detection of newer β-lactamases. Therefore, a study was conducted in 2000 to investigate the occurrence of these β-lactamases in Klebsiella pneumoniae isolates at 24 U.S. medical centers. To enhance the likelihood of detecting imported AmpC and carbapenem-hydrolyzing β-lactamases, participating laboratories were permitted to include archived strains (1996 to 2000) that were intermediate or resistant to either cefoxitin or imipenem. The β-lactamase production of 408 isolates positive by screening of 1,123 isolates was investigated by ESBL phenotypic confirmation tests; and for AmpC and carbapenem-hydrolyzing β-lactamases, three-dimensional tests, isoelectric focusing, β-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular tests were used. ESBL-producing isolates were detected at 18 of the 24 sites (75%), imported AmpC-producing isolates were detected at 10 sites (42%), inducible imported AmpC-producing isolates were detected at 3 sites (12.5%), and a molecular class A carbapenem-hydrolyzing enzyme was detected at 1 site (4%). No class B or D carbapenem-hydrolyzing enzymes were detected. ESBLs and imported AmpC β-lactamases were detected at a significant number of sites, indicating widespread penetration of these enzymes into U.S. medical institutions. Because these enzymes may significantly affect therapeutic outcomes, it is vital that clinical laboratories be aware of them and be able to detect their occurrence.

Multiple genotypic changes in hypersusceptible strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients do not always correlate with the phenotype

OBJECTIVES Although Pseudomonas aeruginosa from cystic fibrosis patients are well known for their antibiotic resistance, isolates that are highly susceptible to multiple drug classes have also been encountered. In this study, hypersusceptible P. aeruginosa isolates were analysed for changes in intrinsic resistance mechanisms to explain the observed phenotype. METHODS P. aeruginosa strains PA30 and PA431 were isolated from the sputa of cystic fibrosis patients and susceptibilities were determined by agar dilution. Isolates were genetically unrelated by PFGE analysis. Expression of efflux pumps, porins, a chromosomal cephalosporinase and a gene, glmS, previously implicated in hypersusceptibility were evaluated by real-time RT-PCR, outer membrane protein analysis and beta-lactamase hydrolysis assays. RESULTS PA30 was hypersusceptible to beta-lactams, fluoroquinolones and antimetabolites, with MICs at least 4-fold lower than those for the prototype strain PAO1, while PA431 was hypersusceptible to beta-lactams and antimetabolites. Both isolates overproduced the porin OprF but showed down-regulation in the production of the carbapenem channel OprD despite carbapenem hypersusceptibility. PA30 had decreased expression of the mexAB-oprM pump involved with intrinsic antibiotic resistance but overexpressed the mexCD-oprJ and mexEF-oprN efflux systems normally associated with acquired resistance. PA431 showed down-regulation of oprM, the last gene in the mexAB-oprM operon, but overexpressed the mexXY pump. The ampC beta-lactamase was weakly inducible in strain PA30, corresponding to cefoxitin hypersusceptibility. CONCLUSIONS The changes in expression of several intrinsic mechanisms in the hypersusceptible strains did not correlate with the observed phenotype. These data highlight the complex interactions of resistance mechanisms in P. aeruginosa and their roles in drug susceptibility.

Use of β-Lactamase Inhibitors in Disk Tests To Detect Plasmid-Mediated AmpC β-Lactamases

ABSTRACT Seeking a simple disk test for detection of organisms producing plasmid-mediated AmpC β-lactamases, we evaluated the diagnostic utility of the β-lactamase inhibitors 48-1220 (Ro 48-1220) and LN-2-128. Using NCCLS disk methodology, inhibition zone diameters were determined for five β-lactam antibiotics tested alone and in combination with 20 μg of either 48-1220 or LN-2-128. Using an increase of ≥4 mm in zone diameter in the presence of an inhibitor as a positive test, cefotetan with LN-2-128 and 48-1220 was adequate for the detection of organisms producing plasmid-mediated AmpCs (specificity of 90% and sensitivity of 100%).

In Vitro Activities of DX-619 and Comparison Quinolones against Gram-Positive Cocci

ABSTRACT The in vitro activity of the novel quinolone DX-619 was compared to those of currently available quinolones against U.S. clinical isolates of Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp., Streptococcus pyogenes, and Streptococcus pneumoniae. DX-619 was the most potent quinolone overall, indicating possible utility as an anti-gram-positive quinolone.

In Vitro Activity of Retapamulin and Antimicrobial Susceptibility Patterns in a Longitudinal Collection of Methicillin-Resistant Staphylococcus aureus Isolates from a Veterans Affairs Medical Center.

ABSTRACT Mupirocin is a topical antimicrobial used to decolonize patients who carry methicillin-resistant Staphylococcus aureus (MRSA), and the topical agent retapamulin may be a potential alternative therapy. The goal of this study was to determine the in vitro activity of retapamulin as well as a panel of 15 antimicrobial agents, including mupirocin, for 403 MRSA isolates collected longitudinally from a naive population at the Veterans Affairs Puget Sound Health Care System. The MICs for retapamulin had a unimodal distribution, ranging from 0.008 to 0.5 μg/ml. One isolate had an MIC of >16 μg/ml, was also resistant to clindamycin and erythromycin, and was recovered from the nares of a patient undergoing hemodialysis. Twenty-four isolates (6%) and 11 isolates (3%) demonstrated low-level resistance (MICs of 8 to 64 μg/ml) and high-level resistance (MICs of ≥512 μg/ml), respectively, to mupirocin. Isolates were recovered from 10 patients both before and after mupirocin therapy. Of those, isolates from 2 patients demonstrated MIC changes postmupirocin therapy; in both cases, however, strain typing demonstrated that the pre- and postmupirocin strains were different. A total of 386 isolates (96%) had vancomycin MICs of ≤1.0 μg/ml; 340 isolates (84%) were resistant to levofloxacin, 18 isolates (4.5%) were resistant to trimethoprim-sulfamethoxazole, and 135 isolates (33%) had elevated MICs of 4 μg/ml for linezolid. The baseline levels of resistance were low for mupirocin (9%) and even lower for retapamulin (0.25%) Although the use of mupirocin is currently the standard therapy for decolonization practices, the activity of retapamulin warrants its consideration as an alternative therapy in MRSA decolonization regimens.

In Vitro Activity of DX-619, a Novel Des-Fluoro(6) Quinolone, against a Panel of Streptococcus pneumoniae Mutants with Characterized Resistance Mechanisms

ABSTRACT The in vitro activities of DX-619 and four other quinolones were compared against Streptococcus pneumoniae mutants that contained a variety of alterations within the quinolone resistance-determining regions. DX-619 was the most potent quinolone and was least affected by the mutations.

Activity of faropenem against resistant isolates of Streptococcus pneumoniae

An in vitro study of the activity of 9 agents against 181 US pediatric isolates of Streptococcus pneumoniae identified imipenem and faropenem as the most active agents. Overall, faropenem was the most potent oral agent inhibiting 98% of isolates at 1 microg/mL.

Activity of oral agents against pediatric isolates of Streptococcus pneumoniae

An in vitro study of the activity of 10 oral agents against 153 pediatric isolates of Streptococcus pneumoniae identified moxifloxacin and levofloxacin as the most active agents regardless of penicillin or macrolide susceptibility. Moxifloxacin inhibited all strains at 0.25 microg/ml and was 8- to 16-fold more potent than levofloxacin.