Metagenomic next-generation sequencing of rectal swabs for the surveillance of antimicrobial-resistant organisms on the Illumina Miseq and Oxford MinION platforms.

Antimicrobial resistance (AMR) is a public health threat where efficient surveillance is needed to prevent outbreaks. Existing methods for detection of gastrointestinal colonization of multidrug-resistant organisms (MDRO) are limited to specific organisms or resistance mechanisms. Metagenomic next-generation sequencing (mNGS) is a more rapid and agnostic diagnostic approach for microbiome and resistome investigations. We determined if mNGS can detect MDRO from rectal swabs in concordance with standard microbiology results. We performed and compared mNGS performance on short-read Illumina MiSeq (N = 10) and long-read Nanopore MinION (N = 5) platforms directly from rectal swabs to detect vancomycin-resistant enterococci (VRE) and carbapenem-resistant Gram-negative organisms (CRO). We detected Enterococcus faecium (N = 8) and Enterococcus faecalis (N = 2) with associated van genes (9/10) in concordance with VRE culture-based results. We studied the microbiome and identified CRO, Pseudomonas aeruginosa (N = 1), Enterobacter cloacae (N = 1), and KPC-producing Klebsiella pneumoniae (N = 1). Nanopore real-time analysis detected the blaKPC gene in 2.3 min and provided genetic context (blaKPC harbored on pKPC_Kp46 IncF plasmid). Illumina sequencing provided accurate allelic variant determination (i.e., blaKPC-2) and strain typing of the K. pneumoniae (ST-15). We demonstrated an agnostic approach for surveillance of MDRO, examining advantages of both short- and long-read mNGS methods for AMR detection.

Evaluation of the Direct MacConkey Method for Identification of Carbapenem-Resistant Gram-Negative Organisms from Rectal Swabs: Reevaluating Zone Diameter Cutoffs.

The optimal method to screen for gastrointestinal colonization with carbapenem-resistant organisms (CRO) has yet to be established. The direct MacConkey (direct MAC) plate method demonstrates high sensitivity for CRO detection, but established zone diameter (ZD) criteria for ertapenem (≤27 mm) and meropenem (≤32 mm) result in high rates of false positives upon confirmatory testing. To increase specificity, we screened for CRO in two high-risk wards using the direct MAC plate method, recorded ZDs for each sample, and generated receiver operating characteristic (ROC) curves to evaluate the optimal ZD cutoff criteria. Of 6,868 swabs obtained over an 18-month period, 4,766 (69%) had growth on MAC plates, and 2,500 (36%) met criteria for further evaluation based on previously established ZDs around the carbapenem disks. A total of 812 (12%) swabs were confirmed positive for at least one CRO and included 213 (3%) carbapenemase-producing organisms (CPO), resulting in a specificity of 78% for the direct MAC plate method. Reducing the ertapenem and meropenem ZDs to ≤25 mm improved specificity to 83%, decreasing the confirmatory testing workload by 32%. The sensitivities with the lower ZD criteria were 89% for CRO and 94% for CPO, respectively. The direct MAC plate method criteria for CRO testing can be modified to balance the sensitivity and specificity of CRO while reducing the burden on clinical microbiology laboratories. These modifications can be particularly helpful in regions with a low CRO prevalence.

A Prospective Study of Acinetobacter baumannii Complex Isolates and Colistin Susceptibility Monitoring by Mass Spectrometry of Microbial Membrane Glycolipids.

Acinetobacter baumannii is a prevalent nosocomial pathogen with a high incidence of multidrug resistance. Treatment of infections due to this organism with colistin, a last-resort antibiotic of the polymyxin class, can result in the emergence of colistin-resistant strains. Colistin resistance primarily occurs via modifications of the terminal phosphate moieties of lipopolysaccharide-derived lipid A, which reduces overall membrane electronegativity. These modifications are readily identified by mass spectrometry (MS). In this study, we prospectively collected Acinetobacter baumannii complex clinical isolates from a hospital system in Pennsylvania over a 3-year period. All isolates were evaluated for colistin resistance using standard MIC testing by both agar dilution and broth microdilution, as well as genospecies identification and lipid A profiling using MS analyses. Overall, an excellent correlation between colistin susceptibility and resistance, determined by MIC testing, and the presence of a lipid A modification, determined by MS, was observed with a sensitivity of 92.9% and a specificity of 94.0%. Additionally, glycolipid profiling was able to differentiate A. baumannii complex organisms based on their membrane lipids. With the growth of MS use in clinical laboratories, a reliable MS-based glycolipid phenotyping method that identifies colistin resistance in A. baumannii complex clinical isolates, as well as other Gram-negative organisms, represents an alternative or complementary approach to existing diagnostics.

Antibiotic pressure on the acquisition and loss of antibiotic resistance genes in Klebsiella pneumoniae.

Objectives: In this study, we characterize a concurrent disseminated infection with a virulent hypermucoviscous (HMV) Klebsiella pneumoniae and an OXA-181-producing XDR K. pneumoniae from a patient with recent hospitalization in India. During exposure to meropenem therapy, the highly susceptible HMV K. pneumoniae became resistant to carbapenems, consistent with the acquisition of blaOXA-181. Methods: Twelve K. pneumoniae isolates were recovered from the patient and the hospital room environment over a 3 month hospitalization. Phenotypic and molecular studies were completed to characterize the isolates. Oxford Nanopore and Illumina MiSeq WGS were performed to study phylogeny (MLST and SNPs), plasmids and virulence genes and demonstrate changes in the organism's resistome that occurred over time. Results: WGS revealed that the HMV K. pneumoniae belonged to ST23 and harboured an IncH1B virulence plasmid, while the XDR K. pneumoniae belonged to ST147 and possessed two MDR plasmids (IncR and IncFII), the blaOXA-181-bearing ColKP3 plasmid and chromosomal mutations conferring the XDR phenotype. Sequential isolates demonstrated plasmid diversification (fusion of the IncR and IncFII plasmids), mobilization of resistance elements (ompK35 inactivation by ISEcp1-blaCTX-M-15 mobilization, varying numbers of resistance genes on plasmid scaffolds) and chromosomal mutations (mutations in mgrB) leading to further antibiotic resistance that coincided with antibiotic pressure. Importantly, the HMV strain in this study was unable to preserve the carbapenem-resistant phenotype without the selective pressure of meropenem. Conclusions: To the best of our knowledge, we are the first to report a carbapenem-resistant HMV K. pneumoniae strain in the USA. Ultimately, this case demonstrates the role of antibiotic pressure in the acquisition and loss of important genetic elements.

Carbapenemase Detection among Carbapenem-Resistant Glucose-Nonfermenting Gram-Negative Bacilli.

Accurate detection of carbapenemase-producing glucose-nonfermenting Gram-negative bacilli (CPNFs), including Pseudomonas aeruginosa and Acinetobacter baumannii, is necessary to prevent their dissemination within health care settings. We performed a method comparison study of 11 phenotypic carbapenemase detection assays to evaluate their accuracy for the detection of CPNFs. A total of 96 carbapenem-resistant glucose-nonfermenting isolates were included, of which 29% produced carbapenemases. All CPNFs were molecularly characterized to identify ß-lactamase genes. A total of 86% of the carbapenemase-producing P. aeruginosa isolates produced class B carbapenemases. Several assays performed with a sensitivity of >90% for the detection of carbapenemase-producing P. aeruginosa, including all rapid chromogenic assays and the modified carbapenem inactivation method. Most included assays, with the exception of the Manual Blue Carba assay, the Modified Carba NP assay, the boronic acid synergy test, and the metallo-ß-lactamase Etest, had specificities of >90% for detecting carbapenemase-producing P. aeruginosa Class D carbapenemases were the most prevalent carbapenemases among the carbapenemase-producing A. baumannii strains, with 60% of the carbapenemase-producing A. baumannii isolates producing acquired OXA-type carbapenemases. Although several assays achieved >90% specificity in identifying carbapenemase-producing A. baumannii, no assays achieved a sensitivity of greater than 90%. Our findings suggest that the available phenotypic tests generally appear to have excellent sensitivity and specificity for detecting carbapenemase-producing P. aeruginosa isolates. However, further modifications to existing assays or novel assays may be necessary to accurately detect carbapenemase-producing A. baumannii.

Comparison of 11 Phenotypic Assays for Accurate Detection of Carbapenemase-Producing Enterobacteriaceae.

Early identification of carbapenemase-producing Enterobacteriaceae (CPE) is essential to prevent their dissemination within health care settings. Our objective was to evaluate the accuracy of 11 phenotypic assays for the detection of CPE. Two collections of carbapenem-resistant Enterobacteriaceae (CRE) isolates were evaluated, including 191 retrospective isolates (122 CP-CRE and 69 non-CP isolates) as well as 45 prospective clinical isolates (15 CP-CRE and 30 non-CP-CRE) obtained over a 3-month period. The sensitivity and specificity of each test was determined, with molecular genotype serving as the gold standard. Among the retrospective cohort, sensitivities ranged from 72% for the boronic acid synergy test for the detection of KPC producers to ≥98% for the modified Carba NP, the Rapidec Carba NP, the manual Blue Carba, and the modified carbapenem inactivation method for the detection of any CPE. Sensitivity differed among tests across enzyme classes. All assays had excellent specificity exceeding 95%, with the exception of the boronic acid synergy test (88%) and modified Hodge test (91%). Prospectively, 45 CRE isolates were encountered over a 3-month period, including 15 CPE (33%) and 30 non-CP-CRE (67%). Results from the prospective cohort were similar. However, a decrease in specificity was observed across most tests, likely due to restricted inclusion of non-CP-CRE to assess the specificity of the assays. Overall, accuracy of CPE detection varied across phenotypic tests. Local epidemiology of CP genotypes, turnaround time, and ease of incorporation into the laboratory workflow should be considered when selecting a phenotypic assay for clinical use.

Determining the Optimal Carbapenem MIC That Distinguishes Carbapenemase-Producing and Non-Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae.

Carbapenemase-producing (CP) Enterobacteriaceae are largely responsible for the rapid spread of carbapenem-resistant Enterobacteriaceae (CRE). Distinguishing CP-CRE from non-CP-CRE has important infection control implications. In a cohort of 198 CRE isolates, for isolates that remained susceptible or intermediate to some carbapenem antibiotics, an ertapenem MIC of 0.5 µg/ml and meropenem, imipenem, and doripenem MICs of 2 µg/ml were best able to distinguish CP-CRE from non-CP-CRE isolates.