Anna F. Lau

Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae

Single-molecule sequencing of bacteria at the NIH Clinical Center documents diverse plasmids encoding antibiotic resistance and their transfer between microbes. How Antibiotic Resistance Spreads Among Bacteria Antibiotic-resistant microbes are spreading at an alarming rate in health care facilities throughout the world. Conlan et al. use a new DNA sequencing method to take a close look at one way in which antibiotic resistance spreads. With single-molecule sequencing, the authors completely characterized individual plasmids, the circular bits of DNA that carry the genes for antibiotic resistance in bacteria. They focused on resistance to the carbapenems, a class of antibiotics that is often used for infections that do not respond to more conventional antimicrobial agents. By using this approach in their microbial surveillance program at the NIH Clinical Center, the authors found evidence that plasmids carrying carbapenemase genes moved from one microbial species to another within the hospital environment. They also used the technique to test hypotheses about patient-to-patient transmission and to characterize a previously undescribed carbapenemase-encoding plasmid carried by diverse bacterial species that could cause dangerous clinical infections. Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care–associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment.

Development of a Clinically Comprehensive Database and a Simple Procedure for Identification of Molds from Solid Media by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the rapid and highly accurate identification of clinical pathogens but has not been utilized extensively in clinical mycology due to challenges in developing an effective protein extraction method and the limited databases available. Here, we developed an alternate extraction procedure and constructed a highly stringent database comprising 294 individual isolates representing 76 genera and 152 species. To our knowledge, this is the most comprehensive clinically relevant mold database developed to date. When challenged with 421 blinded clinical isolates from our institution, by use of the BioTyper software, accurate species-level (score of ≥2.0) and genus-level (score of ≥1.7) identifications were obtained for 370 (88.9%) and 18 (4.3%) isolates, respectively. No isolates were misidentified. Of the 33 isolates (7.8%) for which there was no identification (score of <1.7), 25 were basidiomycetes not associated with clinical disease and 8 were Penicillium species that were not represented in the database. Our library clearly outperformed the manufacturers database that was obtained with the instrument, which identified only 3 (0.7%) and 26 (6.2%) isolates at species and genus levels, respectively. Identification was not affected by different culture conditions. Implementation into our routine workflow has revolutionized our mycology laboratory efficiency, with improved accuracy and decreased time for mold identification, eliminating reliance on traditional phenotypic features.

A Rapid Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method for Single-Plasmid Tracking in an Outbreak of Carbapenem-Resistant Enterobacteriaceae

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. Rapid methods for tracking plasmids carrying carbapenemase genes could greatly benefit infection control efforts. Here, we demonstrate that real-time, direct tracking of a single plasmid in a bacterial strain responsible for an outbreak is possible using a commercial matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system. In this case, we retrospectively tracked the blaKPC carbapenemase gene-bearing pKpQIL plasmid responsible for a CRE outbreak that occurred at the NIH Clinical Center in 2011. An ∼11,109-Da MS peak corresponding to a gene product of the blaKPC pKpQIL plasmid was identified and characterized using a combination of proteomics and molecular techniques. This plasmid peak was present in spectra from retrospectively analyzed K. pneumoniae outbreak isolates, concordant with results from whole-genome sequencing, and absent from a diverse control set of blaKPC -negative clinical Enterobacteriaceae isolates. Notably, the gene characterized here is located adjacent to the blaKPC Tn4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other blaKPC Tn4401-containing plasmids and suggests that this signature MS peak may be useful in tracking other plasmids conferring carbapenem resistance. Plasmid identification using this MALDI-TOF MS method was accomplished in as little as 10 min from isolated colonies and 30 min from positive (spiked) blood cultures, demonstrating the potential clinical utility for real-time plasmid tracking in an outbreak.

Plasmid Dynamics in KPC-Positive Klebsiella pneumoniae during Long-Term Patient Colonization

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli. Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae. Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists’ actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms. IMPORTANCE In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae. Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care. In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae. Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care.

Clinical Performance of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Detection of Certain blaKPC-Containing Plasmids

ABSTRACT Rapid detection of bla KPC-containing organisms can significantly impact infection control and clinical practices, as well as therapeutic choices. Current molecular and phenotypic methods to detect these organisms, however, require additional testing beyond routine organism identification. In this study, we evaluated the clinical performance of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to detect pKpQIL_p019 (p019)—an ∼11,109-Da protein associated with certain bla KPC-containing plasmids that was previously shown to successfully track a clonal outbreak of bla KPC-pKpQIL-Klebsiella pneumoniae in a proof-of-principle study (A. F. Lau, H. Wang, R. A. Weingarten, S. K. Drake, A. F. Suffredini, M. K. Garfield, Y. Chen, M. Gucek, J. H. Youn, F. Stock, H. Tso, J. DeLeo, J. J. Cimino, K. M. Frank, and J. P. Dekker, J Clin Microbiol 52:2804–2812, 2014, http://dx.doi.org/10.1128/JCM.00694-14). PCR for the p019 gene was used as the reference method. Here, blind analysis of 140 characterized Enterobacteriaceae isolates using two protein extraction methods (plate extraction and tube extraction) and two peak detection methods (manual and automated) showed sensitivities and specificities ranging from 96% to 100% and from 95% to 100%, respectively (2,520 spectra analyzed). Feasible laboratory implementation methods (plate extraction and automated analysis) demonstrated 96% sensitivity and 99% specificity. All p019-positive isolates (n = 26) contained bla KPC and were carbapenem resistant. Retrospective analysis of an additional 720 clinical Enterobacteriaceae spectra found an ∼11,109-Da signal in nine spectra (1.3%), including seven from p019-containing, carbapenem-resistant isolates (positive predictive value [PPV], 78%). Instrument tuning had a significant effect on assay sensitivity, highlighting important factors that must be considered as MALDI-TOF MS moves into applications beyond microbial identification. Using a large blind clinical data set, we have shown that spectra acquired for routine organism identification can also be analyzed automatically in real time at high throughput, at no additional expense to the laboratory, to enable rapid detection of potentially bla KPC-containing carbapenem-resistant isolates, providing early and clinically actionable results.

Clinical Performance of Check-Direct CPE, a Multiplex PCR for Direct Detection of bla KPC, bla NDM and/or bla VIM, and bla OXA-48 from Perirectal Swabs

ABSTRACT We evaluated the clinical performance of Check-Direct CPE for carbapenemase detection directly from 301 perirectal swabs (258 patients) in a nonoutbreak setting. Culture of a PCR-confirmed, carbapenemase-containing organism, or history of colonization with such organism within the previous 2 weeks, was used as the reference standard. Check-Direct CPE demonstrated a sensitivity value, specificity value, positive predictive value (PPV), and negative predictive value (NPV) of 100% (all bla KPC), 88%, 21%, and 100%, respectively. False positives accounted for 79% (n = 34) of samples for which a cycle threshold (CT ) value was reached. Simulated studies to evaluate specimen pooling as an approach to minimize costs showed no difference in CT values for pooled groups of three or five that each contained a single specimen spiked with ∼1,500 CFU bla KPC Klebsiella pneumoniae; however, the detection rate dropped to 60% at a seeded concentration of ∼150 CFU. When data were pooled, CT values for bla KPC were higher for heavy-feces-containing than for light-feces-containing liquid-suspended specimens. Furthermore, CT values for liquid-suspended specimens were 4 to 5 CT values lower (i.e., represented greater sensitivity) than those seen in direct swab analysis. Culture was equivalent to or better than Check-Direct CPE for 13/15 (87%) isolates tested in a limit-of-detection analysis. Detection of a carbapenemase gene at a CT cutoff value of ≤35 was culture confirmed in 23/24 (96%) of cases; however, CT values of >35 overlapped broadly between culture-positive (n = 21) and culture-negative (n = 36) specimens. Check-Direct CPE will likely prove most useful in high-prevalence areas or in outbreak settings where rapid carbapenemase detection is critical for infection control management.

Performance of Matrix-Assisted Laser Desorption Ionization−Time of Flight Mass Spectrometry for Identification of Aspergillus, Scedosporium, and Fusarium spp. in the Australian Clinical Setting

ABSTRACT We developed an Australian database for the identification of Aspergillus, Scedosporium, and Fusarium species (n = 28) by matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS). In a challenge against 117 isolates, species identification significantly improved when the in-house-built database was combined with the Bruker Filamentous Fungi Library compared with that for the Bruker library alone (Aspergillus, 93% versus 69%; Fusarium, 84% versus 42%; and Scedosporium, 94% versus 18%, respectively).

Candida colonization as a risk marker for invasive candidiasis in mixed medical-surgical intensive care units: Development and evaluation of a simple, standard protocol

ABSTRACT Colonization with Candida species is an independent risk factor for invasive candidiasis (IC), but the minimum and most practicable parameters for prediction of IC have not been optimized. We evaluated Candida colonization in a prospective cohort of 6,015 nonneutropenic, critically ill patients. Throat, perineum, and urine were sampled 72 h post-intensive care unit (ICU) admission and twice weekly until discharge or death. Specimens were cultured onto chromogenic agar, and a subset underwent molecular characterization. Sixty-three (86%) patients who developed IC were colonized prior to infection; 61 (97%) tested positive within the first two time points. The median time from colonization to IC was 7 days (range, 0 to 35). Colonization at any site was predictive of IC, with the risk of infection highest for urine colonization (relative risk [RR] = 2.25) but with the sensitivity highest (98%) for throat and/or perineum colonization. Colonization of ≥2 sites and heavy colonization of ≥1 site were significant independent risk factors for IC (RR = 2.25 and RR = 3.7, respectively), increasing specificity to 71% to 74% but decreasing sensitivity to 48% to 58%. Molecular testing would have prompted a resistance-driven decision to switch from fluconazole treatment in only 11% of patients infected with C. glabrata, based upon species-level identification alone. Positive predictive values (PPVs) were low (2% to 4%) and negative predictive values (NPVs) high (99% to 100%) regardless of which parameters were applied. In the Australian ICU setting, culture of throat and perineum within the first two time points after ICU admission captures 84% (61/73 patients) of subsequent IC cases. These optimized parameters, in combination with clinical risk factors, should strengthen development of a setting-specific risk-predictive model for IC.

Complete Genome Sequence of a Klebsiella pneumoniae Isolate with Chromosomally Encoded Carbapenem Resistance and Colibactin Synthesis Loci.

ABSTRACT Klebsiella pneumoniae is an important nosocomial pathogen, and multidrug-resistant strains have become a worldwide concern. Here, we report the complete genome of a K. pneumoniae isolate with chromosomally integrated blaKPC genes and a colibactin synthesis locus.

Detection and Whole-Genome Sequencing of Carbapenemase-Producing Aeromonas hydrophila Isolates from Routine Perirectal Surveillance Culture

ABSTRACT Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a 6-week period and were without epidemiological links. Detection of the bla KPC-2 gene in one isolate prompted inclusion of non-Enterobacteriaceae in our surveillance culture workup. Whole-genome sequencing confirmed that the isolates were unrelated and provided data for Aeromonas reference genomes.