Janet A. Hindler

Colistin MIC Variability by Method for Contemporary Clinical Isolates of Multidrug-Resistant Gram-Negative Bacilli

ABSTRACT In vitro evaluation of colistin susceptibility is fraught with complications, due in part to the inherent cationic properties of colistin. In addition, no reference method has been defined against which to compare the results of colistin susceptibility testing. This study systematically evaluated the available methods for colistin MIC testing in two phases. In phase I, colistin MICs were determined in 107 fresh clinical isolates of multidrug-resistant (MDR) Gram-negative bacilli (GNB) by broth microdilution with polysorbate 80 (BMD-T), broth macrodilution (TDS), and the Etest. In phase II, 50 of these isolates, 10 of which were colistin resistant, were tested in parallel using BMD-T, TDS, agar dilution, broth microdilution without polysorbate 80 (BMD), and the TREK Gram-negative extra MIC format (GNXF) Sensititre. The Etest was also performed on these 50 isolates using Mueller-Hinton agar (MHA) from three different manufacturers. Colistin MIC results obtained from the five methods were compared to the MIC results obtained using BMD-T, the method that enables the highest nominal concentration of colistin in the test medium. Essential agreement ranged from 34% (BMD) to 83% (TDS), whereas categorical agreement was >90% for all methods except for BMD, which was 88%. Very major errors (VMEs) (i.e., false susceptibility) for the Etest were found in 47 to 53% of the resistant isolates, depending on the manufacturer of the MHA that was used. In contrast, VMEs were found for 10% (n = 1) of the resistant isolates by BMD and 0% of the isolates by the TDS, agar dilution, and Sensititre methods. Based on these data, we urge clinical laboratories to be aware of the variable results that can occur when using different methods for colistin MIC testing and, in particular, to use caution with the Etest.

First Report of Ceftazidime-Avibactam Resistance in a KPC-3-Expressing Klebsiella pneumoniae Isolate

ABSTRACT Ceftazidime-avibactam is the first antimicrobial approved by the U.S. FDA for the treatment of carbapenem-resistant Enterobacteriaceae. Avibactam, a non-β-lactam β-lactamase inhibitor, inactivates class A serine carbapenemases, including Klebsiella pneumoniae carbapenemase (KPC). We report a KPC-producing K. pneumoniae isolate resistant to ceftazidime-avibactam (MIC, 32/4 μg/ml) from a patient with no prior treatment with ceftazidime-avibactam.

New Delhi Metallo-β-Lactamase (NDM-1)-Producing Klebsiella pneumoniae: Case Report and Laboratory Detection Strategies

ABSTRACT The spread of antimicrobial resistance among Enterobacteriaceae is a significant clinical threat. We report the first case of an Enterobacteriaceae strain harboring the NDM-1 metallo-β-lactamase in a pediatric patient in the United States. We describe strategies for the detection of this novel resistance mechanism encountered in an isolate of Klebsiella pneumoniae.

Phenotypic and Molecular Characteristics of Carbapenem-Resistant Enterobacteriaceae in a Health Care System in Los Angeles, California, from 2011 to 2013

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) are a concern for health care in the United States but remain relatively uncommon in California. We describe the phenotype, clonality, and carbapenemase-encoding genes present in CRE isolated from patients at a Californian tertiary health care system. CRE for this study were identified by evaluating the antibiograms of Enterobacteriaceae isolated in the UCLA Health System from 2011 to 2013 for isolates that were not susceptible to meropenem and/or imipenem. The identification of these isolates was subsequently confirmed by matrix-associated laser desorption ionization–time of flight, and broth microdilution tests were repeated to confirm the CRE phenotype. Real-time PCR for bla KPC, bla SME, bla IMP, bla NDM-1, bla VIM, and bla OXA-48 was performed. Clonality was assessed by repetitive sequence-based PCR (repPCR) and multilocus sequence typing (MLST). Of 15,839 nonduplicate clinical Enterobacteriaceae isolates, 115 (0.73%) met the study definition for CRE. This number increased from 0.5% (44/8165) in the first half of the study to 0.9% (71/7674) in the second (P = 0.004). The most common CRE species were Klebsiella pneumoniae, Enterobacter aerogenes, and Escherichia coli. A carbapenemase-encoding gene was found in 81.7% (94/115) of CRE and included bla KPC (78.3%), bla NDM-1 (0.9%), and bla SME (2.6%). The majority of bla KPC genes were in K. pneumoniae isolates, which fell into 14 clonal groups on typing. bla KPC was identified in more than one species of CRE cultured from the same patient in four cases. Three bla SME-carrying Serratia marcescens isolates and one bla NDM-1 carrying Providencia rettgeri isolate were detected. CRE are increasing in California, and carbapenemases, particularly KPC, are a common mechanism for carbapenem resistance in this region.

Emerging Resistance, New Antimicrobial Agents … but No Tests! The Challenge of Antimicrobial Susceptibility Testing in the Current US Regulatory Landscape

Accurate and timely performance of antimicrobial susceptibility testing (AST) by the clinical laboratory is paramount to combating antimicrobial resistance. The ability of laboratories in the United States to effectively perform ASTs is challenged by several factors. Some, such as new resistance mechanisms and the associated evolution of testing recommendations and breakpoints, are inevitable. Others are entirely man-made. These include unnecessarily strict US Food and Drug Administration (FDA) limitations on how commercial AST systems can be used for diagnostic testing, the absence of up-to-date performance data on these systems, and the lack of commercially available FDA-cleared tests for newer antimicrobial agents or for older agents with updated breakpoints. This viewpoint will highlight contemporary AST challenges faced by the clinical laboratory, and propose some solutions.

Multisite Evaluation of Cepheid Xpert Carba-R Assay for Detection of Carbapenemase-Producing Organisms in Rectal Swabs

ABSTRACT Rapid identification of patients who are colonized with carbapenemase-producing organisms (CPO) is included in multiple national guidelines for containment of these organisms. In a multisite study, we evaluated the performance of the Cepheid Xpert Carba-R assay, a qualitative diagnostic test that was designed for the rapid detection and differentiation of the bla KPC, bla NDM, bla VIM, bla OXA-48, and bla IMP-1 genes from rectal swab specimens. A double rectal swab set was collected from 383 patients admitted at four institutions (2 in the United States, 1 in the United Kingdom, 1 in Spain). One swab was used for reference culture (MacConkey broth containing 1 mg/liter of meropenem and subcultured to a MacConkey agar plate with a 10-μg meropenem disk) and for sequencing of DNA obtained from carbapenem-nonsusceptible isolates for carbapenemase identification. The other swab was used for the Xpert Carba-R assay. In addition to the clinical rectal swabs, 250 contrived specimens (108 well-characterized CPO and 142 negative controls spiked onto negative rectal swabs) were tested. Overall, 149/633 (23.5%) samples were positive by the Xpert Carba-R assay. In 6 samples, multiple targets were detected (4 VIM/OXA-48, 1 IMP-1/NDM, and 1 NDM/KPC). The Xpert Carba-R assay detected 155 targets (26 IMP-1, 30 VIM, 27 NDM, 33 KPC, 39 OXA-48) within a time range of 32 to 48 min. The sensitivity, specificity, and positive and negative predictive values of the Xpert Carba-R assay compared to those of the reference culture and sequencing results were 96.6% (95% confidence interval [CI], 92.2% to 98.9%), 98.6% (95% CI, 97.1% to 99.4%), 95.3%, and 99.0%, respectively. The Cepheid Xpert Carba-R assay is an accurate and rapid test to identify rectal colonization with CPO, which can guide infection control programs to limit the spread of these organisms.

Ciprofloxacin-Resistant Haemophilus Influenzae Infection in a Patient with Chronic Lung Disease

OBJECTIVE: To report a case of ciprofloxacin-resistant Haemophilus influenzae infection in a patient with chronic lung disease who was exposed to multiple courses of antimicrobial therapy. CASE SUMMARY: The patient suffered recurrent pulmonary infections and developed bronchiectasis as a consequence of longstanding, severe, combined immunodeficiency disease. He had received ciprofloxacin on several occasions for treatment and prophylaxis of recurrent pulmonary infections. On a recent admission his usual H. influenzae isolate, which had been highly susceptible to ciprofloxacin (minimum inhibitory concentration [MIC] ≤0.06 mg/L) on previous admissions, was resistant to ciprofloxacin and ofloxacin (MIC 8 and 16 mg/L, respectively). The patient responded to treatment with ceftizoxime and was discharged with oral cefixime, which was to be taken for a total of two weeks. DISCUSSION: Rare isolates of H. influenzae resistant to ofloxacin and lomefloxacin have been noted in Europe and Asia; however, none resistant to the fluoroquinolones have been previously reported in the US, and no resistance has been reported to ciprofloxacin. We believe that repetitive, cycling exposure to ciprofloxacin may have induced the resistance that developed in this patients flora. CONCLUSIONS: Fluoroquinolones may be added to the list of drugs to which H. influenzae have become resistant. Only judicious use of these drugs will preserve their activity against important pathogens in community-acquired infections.

In Vitro Activity of Daptomycin in Combination with β-Lactams, Gentamicin, Rifampin, and Tigecycline against Daptomycin-Nonsusceptible Enterococci

ABSTRACT Enterococci that are nonsusceptible (NS; MIC > 4 μg/ml) to daptomycin are an emerging clinical concern. The synergistic combination of daptomycin plus beta-lactams has been shown to be effective against vancomycin-resistant Enterococcus (VRE) species in vitro. This study systematically evaluated by in vitro time-kill studies the effect of daptomycin in combination with ampicillin, cefazolin, ceftriaxone, ceftaroline, ertapenem, gentamicin, tigecycline, and rifampin, for a collection of 9 daptomycin-NS enterococci that exhibited a broad range of MICs and different resistance-conferring mutations. We found that ampicillin plus daptomycin yielded the most consistent synergy but did so only for isolates with mutations to the liaFSR system. Daptomycin binding was found to be enhanced by ampicillin in a representative isolate with such mutations but not for an isolate with mutation to the yycFGHIJ system. In contrast, ampicillin enhanced the killing of the LL-37 human antimicrobial peptide against daptomycin-NS E. faecium with either the liaFSR or yycFGHIJ mutation. Antagonism was noted only for rifampin and tigecycline and only for 2 or 3 isolates. These data add support to the growing body of evidence indicating that therapy combining daptomycin and ampicillin may be helpful in eradicating refractory VRE infections.

Performance of Vitek 2 for Antimicrobial Susceptibility Testing of Staphylococcus spp. and Enterococcus spp.

ABSTRACT Vitek 2 (bioMérieux, Inc., Durham, NC) is a widely used commercial antimicrobial susceptibility testing system. We compared MIC results obtained by Vitek 2 to those obtained by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) reference method for 134 staphylococcal and 84 enterococcal clinical isolates. Nineteen agents were evaluated, including all those available on Vitek 2 for testing staphylococci and enterococci. The resistance phenotypes tested included methicillin-resistant Staphylococcus aureus (MRSA) (n = 58), S. aureus with inducible clindamycin resistance (ICR) (n = 30), trimethoprim-sulfamethoxazole-resistant MRSA (n = 10), vancomycin-resistant Enterococcus (n = 37), high-level gentamicin-resistant Enterococcus (n = 15), linezolid-resistant Enterococcus (n = 5), and daptomycin-nonsusceptible Enterococcus faecalis (n = 6). For the staphylococci, there was 98.9% categorical agreement (CA). There was one very major error (VME) for gentamicin in a Staphylococcus hominis isolate, six VMEs for inducible clindamycin in S. aureus isolates, and two major errors (ME) for daptomycin in an S. aureus and a Staphylococcus epidermidis isolate. For enterococci, there was 97.3% CA. Two VMEs were observed for daptomycin in isolates of E. faecalis and 2 ME, 1 for high-level gentamicin resistance and 1 for nitrofurantoin, in E. faecium isolates. Overall, there was 98.3% CA and 99% essential agreement for the testing of staphylococci and enterococci by the Vitek 2. With the exception of detecting ICR in S. aureus, Vitek 2 performed reliably for antimicrobial susceptibility testing of staphylococci and enterococci.

In vitro activity of A-56268 (TE-031), a new macrolide antibiotic, compared with that of erythromycin and other antimicrobial agents.

A-56268 is a new macrolide antibiotic that resembles erythromycin in its spectrum of activity. A-56268 and erythromycin had identical activities against Streptococcus pyogenes, group B, C, and G streptococci, and viridans group streptococci. Erythromycin and A-56268 had similar activities against Staphylococcus aureus, coagulase-negative staphylococci, and group D enterococci. Like erythromycin, A-56268 was ineffective in inhibiting oxacillin-resistant S. aureus, oxacillin-resistant, coagulase-negative staphylococci, and penicillin-resistant viridans group streptococci. Haemophilus influenzae, Neisseria gonorrhoeae, and Legionella spp. were inhibited by A-56268 at concentrations similar to those of erythromycin.