Karen F. Anderson

Evaluation of Methods To Identify the Klebsiella pneumoniae Carbapenemase in Enterobacteriaceae

ABSTRACT The Klebsiella pneumoniae carbapenem (KPC) β-lactamase occurs in Enterobacteriaceae and can confer resistance to all β-lactam agents including carbapenems. The enzyme may confer low-level carbapenem resistance, and the failure of susceptibility methods to identify this resistance has been reported. Automated and nonautomated methods for carbapenem susceptibility were evaluated for identification of KPC-mediated resistance. Ertapenem was a more sensitive indicator of KPC resistance than meropenem and imipenem independently of the method used. Carbapenemase production could be confirmed with the modified Hodge test.

Genetic Factors Associated with Elevated Carbapenem Resistance in KPC-Producing Klebsiella pneumoniae

ABSTRACT In the United States, the most prevalent mechanism of carbapenem resistance among Enterobacteriaceae is the production of a Klebsiella pneumoniae carbapenemase (KPC). KPC-producing isolates often exhibit a range of carbapenem MICs. To better understand the factors that contribute to overall carbapenem resistance, we analyzed 27 KPC-producing K. pneumoniae isolates with different levels of carbapenem resistance, 11 with low-level (i.e., meropenem or imipenem MIC ≤ 4 μg/ml), 2 with intermediate-level (i.e., meropenem and imipenem MIC = 8 μg/ml), and 14 with high-level (i.e., imipenem or meropenem MIC ≥ 16 μg/ml) carbapenem resistance, that were received from throughout the United States. Among 14 isolates that exhibited high-level carbapenem resistance, Western blot analysis indicated that 10 produced an elevated amount of KPC. These isolates either contained an increased blaKPC gene copy number (n = 3) or had deletions directly upstream of the blaKPC gene (n = 7). Four additional isolates lacked elevated KPC production but had high-level carbapenem resistance. Porin sequencing analysis identified 22 isolates potentially lacking a functional OmpK35 and three isolates potentially lacking a functional OmpK36. The highest carbapenem MICs were found in two isolates that lacked both functioning porins and produced elevated amounts of KPC. The 11 isolates with low-level carbapenem resistance contained neither an upstream deletion nor increased blaKPC copy number. These results suggest that both blaKPC copy number and deletions in the upstream genetic environment affect the level of KPC production and may contribute to high-level carbapenem resistance in KPC-producing K. pneumoniae, particularly when coupled with OmpK36 porin loss.

Detection of the Klebsiella pneumoniae Carbapenemase Type 2 Carbapenem-Hydrolyzing Enzyme in Clinical Isolates of Citrobacter freundii and K. oxytoca Carrying a Common Plasmid

ABSTRACT The Klebsiella pneumoniae carbapenemase (KPC) was detected in carbapenem-resistant isolates of Citrobacter freundii and Klebsiella oxytoca recovered from different patients in a Michigan hospital. Restriction analysis and hybridization with a KPC-specific probe showed the blaKPC-2 genes of these two genera of the family Enterobacteriaceae are carried on a common plasmid.

Prevalence and Risk Factors for Acquisition of Carbapenem-Resistant Enterobacteriaceae in the Setting of Endemicity

OBJECTIVE To describe the epidemiology of carbapenem-resistant Enterobacteriaceae (CRE) carriage and acquisition among hospitalized patients in an area of CRE endemicity. DESIGN Cohort study with a nested case-control study. SETTING Two acute care, academic hospitals in New York City. PARTICIPANTS All patients admitted to 7 study units, including intensive care, medical-surgical, and acute rehabilitation units. METHOD Perianal samples were collected from patients at admission and weekly thereafter to detect asymptomatic gastrointestinal carriage of CRE. A nested case-control study was performed to identify factors associated with CRE acquisition. Case patients were those who acquired CRE during a single hospitalization. Control subjects had no microbiologic evidence of CRE and at least 1 negative surveillance sample. Clinical data were abstracted from the medical record. RESULTS The prevalence of CRE in the study population was 5.4% (306 of 5,676 patients), and 104 patients met the case definition of acquisition during a single hospital stay. Mechanical ventilation (odds ratio [OR], 11.5), pulmonary disease (OR, 5.2), days of antibiotic therapy (OR, 1.04), and CRE colonization pressure (OR, 1.15) were independently associated with CRE acquisition. Pulsed-field gel electrophoresis analysis identified 87% of tested Klebsiella pneumoniae isolates as sharing related patterns (greater than 78% similarity), which suggests clonal transmission within and between the study hospitals. CONCLUSIONS Critical illness and underlying medical conditions, CRE colonization pressure, and antimicrobial exposure are important risk factors for CRE acquisition. Adherence to infection control practices and antimicrobial stewardship appear to be critical components of a CRE control program.

Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus

ABSTRACT We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests, ≤1 μg/ml [n = 60 strains], 2 μg/ml [n = 24], 4 μg/ml [n = 36], or 8 μg/ml [n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent ±1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs ≤ 2 μg/ml) grew on the VScr, but all strains for which the VA MICs were 8 μg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 μg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 μg/ml was difficult for most systems and methods, including the reference methods.

Dissemination of an Enterococcus Inc18-Like vanA Plasmid Associated with Vancomycin-Resistant Staphylococcus aureus

ABSTRACT Of the 9 vancomycin-resistant Staphylococcus aureus (VRSA) cases reported to date in the literature, 7 occurred in Michigan. In 5 of the 7 Michigan VRSA cases, an Inc18-like vanA plasmid was identified in the VRSA isolate and/or an associated vancomycin-resistant Enterococcus (VRE) isolate from the same patient. This plasmid may play a critical role in the emergence of VRSA. We studied the geographical distribution of the plasmid by testing 1,641 VRE isolates from three separate collections by PCR for plasmid-specific genes traA, repR, and vanA. Isolates from one collection (phase 2) were recovered from surveillance cultures collected in 17 hospitals in 13 states. All VRE isolates from 2 Michigan institutions (n = 386) and between 60 and 70 VRE isolates (n = 883) from the other hospitals were tested. Fifteen VRE isolates (3.9%) from Michigan were positive for an Inc18-like vanA plasmid (9 E. faecalis [12.5%], 3 E. faecium [1.0%], 2 E. avium, and 1 E. raffinosus). Six VRE isolates (0.6%) from outside Michigan were positive (3 E. faecalis [2.7%] and 3 E. faecium [0.4%]). Of all E. faecalis isolates tested, 6.0% were positive for the plasmid, compared to 0.6% for E. faecium and 3.0% for other spp. Fourteen of the 15 plasmid-positive isolates from Michigan had the same Tn1546 insertion site location as the VRSA-associated Inc18-like plasmid, whereas 5 of 6 plasmid-positive isolates from outside Michigan differed in this characteristic. Most plasmid-positive E. faecalis isolates demonstrated diverse patterns by PFGE, with the exception of three pairs with indistinguishable patterns, suggesting that the plasmid is mobile in nature. Although VRE isolates with the VRSA-associated Inc18-like vanA plasmid were more common in Michigan, they remain rare. Periodic surveillance of VRE isolates for the plasmid may be useful in predicting the occurrence of VRSA.

New Delhi metallo-β-lactamase-producing Enterobacteriaceae, United States.

We characterized 9 New Delhi metallo-β-lactamase–producing Enterobacteriaceae (5 Klebsiella pneumoniae, 2 Escherichia coli, 1 Enterobacter cloacae, 1 Salmonella enterica serovar Senftenberg) isolates identified in the United States and cultured from 8 patients in 5 states during April 2009–March 2011. Isolates were resistant to β-lactams, fluoroquinolones, and aminoglycosides, demonstrated MICs ≤1 µg/mL of colistin and polymyxin, and yielded positive metallo-β-lactamase screening results. Eight isolates had blaNDM-1, and 1 isolate had a novel allele (blaNDM-6). All 8 patients had recently been in India or Pakistan, where 6 received inpatient health care. Plasmids carrying blaNDM frequently carried AmpC or extended spectrum β-lactamase genes. Two K. pneumoniae isolates and a K. pneumoniae isolate from Sweden shared incompatibility group A/C plasmids with indistinguishable restriction patterns and a common blaNDM fragment; all 3 were multilocus sequence type 14. Restriction profiles of the remaining New Delhi metallo-β-lactamase plasmids, including 2 from the same patient, were diverse.

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.

Comparison of Detection Methods for Heteroresistant Vancomycin-Intermediate Staphylococcus aureus, with the Population Analysis Profile Method as the Reference Method

ABSTRACT Staphylococcus aureus clinical isolates with vancomycin MICs of 2 μg/ml have been associated with vancomycin therapeutic failure and the heteroresistant vancomycin-intermediate S. aureus (hVISA) phenotype. A population analysis profile (PAP) with an area under the curve (AUC) ratio of ≥0.9 for the AUC of the clinical isolate versus the AUC for hVISA strain Mu3 is most often used for determining hVISA, but it is time-consuming and labor-intensive. A collection of 140 MRSA blood isolates with vancomycin MICs of 2 μg/ml by reference broth microdilution and screened for hVISA using PAP-AUC (21/140 [15%] hVISA) were tested by additional methods to detect hVISA. The methods included (i) Etest macromethod using vancomycin and teicoplanin test strips, brain heart infusion (BHI) agar, and a 2.0 McFarland inoculum; (ii) Etest glycopeptide resistance detection (GRD) using vancomycin-teicoplanin double-sided gradient test strips on Mueller-Hinton agar (MHA) with 5% sheep blood and a 0.5 McFarland inoculum; and (iii) BHI screen agar plates containing 4 μg/ml vancomycin and 16 g/liter casein using 0.5 and 2.0 McFarland inocula. Each method was evaluated using PAP-AUC as the reference method. The sensitivity of each method for detecting hVISA was higher when the results were read at 48 h. The Etest macromethod was 57% sensitive and 96% specific, Etest GRD was 57% sensitive and 97% specific, and BHI screen agar was 90% sensitive and 95% specific with a 0.5 McFarland inoculum and 100% sensitive and 68% specific with a 2.0 McFarland inoculum. BHI screen agar with 4 μg/ml vancomycin and casein and a 0.5 McFarland inoculum had the best sensitivity and specificity combination, was easy to perform, and may be useful for clinical detection of hVISA.

IMP-Producing Carbapenem-Resistant Klebsiella pneumoniae in the United States

ABSTRACT The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) producing acquired carbapenemases have created a global public health crisis. In the United States, CRE producing the Klebsiella pneumoniae carbapenemase (KPC) are increasingly common and are endemic in some regions. Metallo-β-lactamase (MBL)-producing CRE have recently been reported in the United States among patients who received medical care in countries where such organisms are common. Here, we describe three carbapenem-resistant K. pneumoniae isolates recovered from pediatric patients at a single U.S. health care facility, none of whom had a history of international travel. The isolates were resistant to carbapenems but susceptible to aztreonam, trimethoprim-sulfamethoxazole, and fluoroquinolones. The three isolates were closely related to each other by pulsed-field gel electrophoresis and contained a common plasmid. PCR and sequence analysis confirmed that these isolates produce IMP-4, an MBL carbapenemase not previously published as present among Enterobacteriaceae in the United States.