We thank Gonzales-Luna and colleagues [1] for their comments. We agree that laboratories must have access to accurate and standardized methods for antimicrobial susceptibility testing (AST) results to be clinically meaningful. The reference method for performing Clostridioides difficile AST is agar dilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines [2]. The CLSI method for performing AST for anaerobic bacteria recommends that 5 μg/mL of hemin be incorporated into agar dilution plates and that the hemin stock solution should be protected from light and stored at 4°C–8°C for no longer than 1 month [2]. The susceptibility testing done by Gargis et al [3] was performed according to these guidelines, and the hemin stock solution was protected from light. | | Nevertheless, we read with interest the research in recent years [4–6] related to heme-dependent metronidazole resistance, including the reported association between isolates characterized as heme dependent and metronidazole resistant and the presence of a T to G mutation (PnimBG) in the −10 promoter region of the nitroimidazole reductase gene, nimB [5]. While Olaitan et al [5] found that not all heme-dependent metronidazole-resistant isolates contained the PnimBG mutation, Olaitan et al [5] indicate that most do; therefore, the presence of PnimBG may be predictive of resistance. We determined that the nimB mutation was present in 20% of our study isolates (116 of 593), of which 99% (115 of 116) belonged to RT027 (Table 1). The remaining isolate was RT014, the only RT014 isolate containing the PnimBG mutation among the 65 evaluated.

BACKGROUND: Antimicrobial susceptibility testing (AST) is not routinely performed for Clostridioides difficile and data evaluating minimum inhibitory concentrations (MICs) are limited. We performed AST and whole genome sequencing (WGS) for 593 C. difficile isolates collected between 2012-2017 through the Centers for Disease Control and Prevention's Emerging Infections Program. METHODS: MICs to six antimicrobial agents (ceftriaxone, clindamycin, meropenem, metronidazole, moxifloxacin, and vancomycin) were determined using the reference agar dilution method according to Clinical and Laboratory Standards Institute guidelines. WGS was performed on all isolates to detect the presence of genes or mutations previously associated with resistance. RESULTS: Among all isolates, 98.5% displayed a vancomycin MIC ≤ 2 μg/mL and 97.3% displayed a metronidazole MIC ≤ 2 μg/mL. Ribotype 027 (RT027) isolates displayed higher vancomycin MICs (MIC50: 2 μg/mL; MIC90: 2 μg/mL) than non-RT027 isolates (MIC50: 0.5 μg/mL; MIC90: 1 μg/mL) (P < 0.01). No vanA/B genes were detected. RT027 isolates also showed higher MICs to clindamycin and moxifloxacin and were more likely to harbor associated resistance genes or mutations. CONCLUSIONS: Elevated MICs to antibiotics used for treatment of C. difficile infection were rare and there was no increase in MICs over time. The lack of vanA/B genes or mutations consistently associated with elevated vancomycin MICs suggests there are multifactorial mechanisms of resistance. Ongoing surveillance of C. difficile using reference AST and WGS to monitor MIC trends and the presence of antibiotic resistance mechanisms is essential.

Oxacillinase (OXA)-48-like carbapenemases remain relatively uncommon in the United States. We performed phenotypic and genotypic characterization of 30 Enterobacteriaceae producing OXA-48-like carbapenemases that were recovered from patients during 2010-2014. Isolates were collected from 12 states and not associated with outbreaks, although we could not exclude limited local transmission. The alleles beta-lactamase OXA-181 (blaOXA-181) (43%), blaOXA-232 (33%), and blaOXA-48 (23%) were found. All isolates were resistant to ertapenem and showed positive results for the ertapenem and meropenem modified Hodge test and the modified carbapenem inactivation method; 73% showed a positive result for the Carba Nordmann-Poirel test. Whole-genome sequencing identified extended-spectrum beta-lactamase genes in 93% of isolates. In all blaOXA-232 isolates, the gene was on a ColKP3 plasmid. A total of 12 of 13 isolates harboring blaOXA-181 contained the insertion sequence DeltaISEcp1. In all isolates with blaOXA-48, the gene was located on a TN1999 transposon; these isolates also carried IncL/M plasmids.

Antimicrobial resistance is a threat to public health globally and leads to an estimated 23,000 deaths annually in the United States alone. Here, we report the genomic characterization of an unusual Klebsiella pneumoniae, nonsusceptible to all 26 antibiotics tested, that was isolated from a U.S. PATIENT: The isolate harbored four known beta-lactamase genes, including plasmid-mediated blaNDM-1 and blaCMY-6, as well as chromosomal blaCTX-M-15 and blaSHV-28, which accounted for resistance to all beta-lactams tested. In addition, sequence analysis identified mechanisms that could explain all other reported nonsusceptibility results, including nonsusceptibility to colistin, tigecycline, and chloramphenicol. Two plasmids, IncA/C2 and IncFIB, were closely related to mobile elements described previously and isolated from Gram-negative bacteria from China, Nepal, India, the United States, and Kenya, suggesting possible origins of the isolate and plasmids. This is one of the first K. pneumoniae isolates in the United States to have been reported to the Centers for Disease Control and Prevention (CDC) as nonsusceptible to all drugs tested, including all beta-lactams, colistin, and tigecycline.IMPORTANCE Antimicrobial resistance is a major public health threat worldwide. Bacteria that are nonsusceptible or resistant to all antimicrobials available are of major concern to patients and the public because of lack of treatment options and potential for spread. A Klebsiella pneumoniae strain that was nonsusceptible to all tested antibiotics was isolated from a U.S. PATIENT: Mechanisms that could explain all observed phenotypic antimicrobial resistance phenotypes, including resistance to colistin and beta-lactams, were identified through whole-genome sequencing. The large variety of resistance determinants identified demonstrates the usefulness of whole-genome sequencing for detecting these genes in an outbreak response. Sequencing of isolates with rare and unusual phenotypes can provide information on how these extremely resistant isolates develop, including whether resistance is acquired on mobile elements or accumulated through chromosomal mutations. Moreover, this provides further insight into not only detecting these highly resistant organisms but also preventing their spread.

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.

We characterized 9 New Delhi metallo-beta-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 beta-lactams, fluoroquinolones, and aminoglycosides, demonstrated MICs ≤1 microg/mL of colistin and polymyxin, and yielded positive metallo-beta-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 beta-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-beta-lactamase plasmids, including 2 from the same patient, were diverse.

The emergence and spread of carbapenem resistant Enterobacteriaceae (CRE) producing acquired carbapenemases has 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-beta-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. healthcare 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, a MBL carbapenemase not previously published as present among Enterobacteriaceae in the United States.

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

Staphylococcus aureus clinical isolates with vancomycin MICs of 2 mug/mL have been associated with vancomycin therapeutic failure and the heteroresistant vancomycin-intermediate S. aureus (hVISA) phenotype. Population analysis profile (PAP) with an area under the curve (AUC) ratio of ≥ 0.9 compared to 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 mug/mL by reference broth microdilution and screened for hVISA using PAP-AUC (21/140 [15%] hVISA) were tested by additional methods to detect hVISA. Methods included: 1) Etest macromethod using vancomycin and teicoplanin test strips, Brain Heart Infusion (BHI) agar and a 2.0 McFarland inoculum; 2) Etest GRD using vancomycin-teicoplanin double-sided gradient test strips on Mueller Hinton Agar (MHA) with 5% sheep's blood and 0.5 McFarland inoculum; and 3) BHI screen agar plates containing 4mug/mL vancomcyin and 16 g/L 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 read at 48 h. Etest macromethod was 57% sensitive and 96% specific, Etest GRD was 57% sensitive and 97% specific, BHI screen agar was 90% sensitive and 95% specific with 0.5 McFarland inoculum and 100% sensitive and 68% specific with 2.0 McFarland inoculum. BHI screen agar with 4 mug/mL vancomycin and casein and 0.5 McFarland inoculum had the best sensitivity and specificity combination, was easy to perform and may be useful for clinical detection of hVISA.

Of the 9 vancomycin-resistant Staphylococcus aureus (VRSA) cases reported to date in the literature, 7 occurred in Michigan (MI). In 5 out of 7 of the 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 and analysis of the geographical distribution of VRE containing this plasmid may explain the observed prevalence of VRSA in MI. A total of 1,641 VRE from three separate collections were tested for the Inc18-like vanA plasmid by PCR for traA, repR, and vanA. All VRE from 2 MI institutions (N= 386), and between 60 to 70 VRE (N=883) from 17 institutions in 13 other states were tested. Fifteen isolates (3.9%) from MI 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 isolates (0.6%) from outside of MI were positive (3 E. faecalis [2.7%] and 3 E. faecium [0.4%]). Fourteen of the 15 plasmid-positive isolates from MI had the same Tn1546 insertion site location as those of the VRSA-associated Inc18 plasmid whereas 5 out of 6 plasmid-positive isolates from outside of MI differed in this characteristic. The one exception was an E. faecium isolate with a pulsed-field gel electrophoresis (PFGE) pattern that was indistinguishable from a plasmid-positive isolate from MI. Most plasmid-positive E. faecalis demonstrated diverse patterns by PFGE, with the exception of three pairs with indistinguishable patterns, suggesting the plasmid is mobile. Although VRE with the VRSA-associated Inc18-like vanA plasmid were more common in MI, they remain rare. Periodic surveillance of VRE for the plasmid may be useful in predicting occurrence of VRSA.

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 impenem MIC ≤ 4 mug/ml), 2 with intermediate-level (i.e., meropenem and imipenem MIC = 8 mug/ml), and 14 with high-level carbapenem resistance (i.e., imipenem or meropenem MIC ≥ 16 mug/ml) that were received from throughout the U.S. Among 14 isolates that exhibited high-level carbapenem resistance, Western blot analysis indicated that 10 produced an elevated amount of KPC. These isolates contained either an increased blaKPC gene copy number (n = 3) or the presence of 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 contained elevated KPC production. 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.

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, <or=1 microg/ml [n = 60 strains], 2 microg/ml [n = 24], 4 microg/ml [n = 36], or 8 microg/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 <or= 2 microg/ml) grew on the VScr, but all strains for which the VA MICs were 8 microg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 microg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 microg/ml was difficult for most systems and methods, including the reference methods.