Transmission of Carbapenem-Resistant Klebsiella pneumoniae in US Hospitals.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKp) is the most prevalent carbapenem-resistant Enterobacterales in the United States. We evaluated CRKp clustering in patients in US hospitals. METHODS: From April 2016 to August 2017, 350 patients with clonal group 258 CRKp were enrolled in the Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae, a prospective, multicenter, cohort study. A maximum likelihood tree was constructed using RAxML. Static clusters shared ≤21 single-nucleotide polymorphisms (SNP) and a most recent common ancestor. Dynamic clusters incorporated SNP distance, culture timing, and rates of SNP accumulation and transmission using the R program TransCluster. RESULTS: Most patients were admitted from home (n = 150, 43%) or long-term care facilities (n = 115, 33%). Urine (n = 149, 43%) was the most common isolation site. Overall, 55 static and 47 dynamics clusters were identified involving 210 of 350 (60%) and 194 of 350 (55%) patients, respectively. Approximately half of static clusters were identical to dynamic clusters. Static clusters consisted of 33 (60%) intrasystem and 22 (40%) intersystem clusters. Dynamic clusters consisted of 32 (68%) intrasystem and 15 (32%) intersystem clusters and had fewer SNP differences than static clusters (8 vs 9; P = .045; 95% confidence interval [CI]: -4 to 0). Dynamic intersystem clusters contained more patients than dynamic intrasystem clusters (median [interquartile range], 4 [2, 7] vs 2 [2, 2]; P = .007; 95% CI: -3 to 0). CONCLUSIONS: Widespread intrasystem and intersystem transmission of CRKp was identified in hospitalized US patients. Use of different methods for assessing genetic similarity resulted in only minor differences in interpretation.

Impact of a Rapid Molecular Test for Klebsiella pneumoniae Carbapenemase and Ceftazidime-Avibactam Use on Outcomes After Bacteremia Caused by Carbapenem-Resistant Enterobacterales.

BACKGROUND: Patients with bacteremia due to carbapenem-resistant Enterobacterales (CRE) experience delays until appropriate therapy and high mortality rates. Rapid molecular diagnostics for carbapenemases and new ß-lactam/ß-lactamase inhibitors may improve outcomes. METHODS: We conducted an observational study of patients with CRE bacteremia from 2016 to 2018 at 8 New York and New Jersey medical centers and assessed center-specific clinical microbiology practices. We compared time to receipt of active antimicrobial therapy and mortality between patients whose positive blood cultures underwent rapid molecular testing for the Klebsiella pneumoniae carbapenemase (KPC) gene (blaKPC) and patients whose cultures did not undergo this test. CRE isolates underwent antimicrobial susceptibility testing by broth microdilution and carbapenemase profiling by whole-genome sequencing. We also assessed outcomes when ceftazidime-avibactam and polymyxins were used as targeted therapies. RESULTS: Of 137 patients with CRE bacteremia, 89 (65%) had a KPC-producing organism. Patients whose blood cultures underwent blaKPC PCR testing (n = 51) had shorter time until receipt of active therapy (median: 24 vs 50 hours; P = .009) compared with other patients (n = 86) and decreased 14-day (16% vs 37%; P = .007) and 30-day (24% vs 47%; P = .007) mortality. blaKPC PCR testing was associated with decreased 30-day mortality (adjusted odds ratio: .37; 95% CI: .16-.84) in an adjusted model. The 30-day mortality rate was 10% with ceftazidime-avibactam monotherapy and 31% with polymyxin monotherapy (P = .08). CONCLUSIONS: In a KPC-endemic area, blaKPC PCR testing of positive blood cultures was associated with decreased time until appropriate therapy and decreased mortality for CRE bacteremia, and ceftazidime-avibactam is a reasonable first-line therapy for these infections.

CG258 Klebsiella pneumoniae isolates without ß-lactam resistance at the onset of the carbapenem-resistant Enterobacteriaceae epidemic in New York City.

Objectives: To examine the epidemiology of ß-lactam resistance in 'clonal group 258' (CG258), a successful KPC clonal group, over 14 years. Methods: Isolates were collected from 1999 to 2013 for a study of antibiotic resistance in Enterobacteriaceae in New York City; 515 bloodstream isolates had antibiotic susceptibility data available and 436 were available for a CG258 PCR assay. The 56 resulting CG258 isolates were characterized by MLST, capsular type and ESBL and KPC carriage. KPC-positive isolates were assessed for common KPC plasmid types, KPC subtype and Tn4401 isoform. Results: RT-PCR revealed 56 isolates were CG258. Seventeen of the 56 CG258 isolates were phenotypically susceptible to all carbapenems (all KPC negative). Five out of 17 susceptible isolates were of the cps-2 (wzi154) capsule type; none was cps-1 (wzi29). Nineteen out of 28 KPC-2 isolates were cps-1 (wzi29) and 8/10 KPC-3 isolates carried cps-2 (wzi154); however, cps-2 (wzi154) predominated among KPC-2-positive isolates in 2003 and 2004. KPC-2 was first detected in 2003 and KPC-3 was first detected in 2006. KPC-harbouring plasmids pKpQIL (all Tn4401a) and pBK30683 (all Tn4401d) were detected in 16/38 and 6/38 carbapenem-resistant isolates, respectively. Discussion: CG258-lineage Klebsiella pneumoniae isolates were completely absent in 1999, but common in 2003. Twenty-one percent of CG258 isolates were susceptible to carbapenems in addition to lacking both common ESBL and blaKPC-mediated resistance. The cps-2 (wzi154) capsule type was common in both these susceptible isolates and in early KPC-2-harbouring isolates, suggesting it was the initial capsule type in CG258. Carbapenem-resistant isolates carried common KPC-harbouring plasmids with the same KPC and Tn4401 isoforms, suggesting frequent clonal spread.

Carbapenem-resistant Klebsiella pneumoniae vertebral osteomyelitis in a renal transplant recipient treated with ceftazidime-avibactam.

Ceftazidime-avibactam (CAZ-AVI) is a novel cephalosporin beta lactamase inhibitor combination that has shown activity against carbapenem-resistant Enterobactericeae. Data are limited on its utilization in the treatment of carbapenem-resistant Klebsiella pneumoniae osteomyelitis in solid organ transplant patients. We describe a case report on the use of CAZ-AVI in the treatment of vertebral osteomyelitis in a renal transplant recipient.

Genomic Characterization of Two KPC-Producing Klebsiella Isolates Collected in 1997 in New York City.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have now become a global public health threat. However, the origin of this pandemic and the characterization of pre-2003 blaKPC-harboring plasmids remain unknown. Here we used next-generation sequencing to characterize two KPC-2-producing K. pneumoniae and Kmichiganensis isolates collected from a New York City hospital in 1997. Although identified in two different Klebsiella species, the blaKPC-2 gene was harbored by Tn4401b transposons on two highly similar IncN plasmids.

A novel resistance mutation in eccC5 of the ESX-5 secretion system confers ofloxacin resistance in Mycobacterium tuberculosis.

BACKGROUND: Fluoroquinolone resistance in Mycobacterium tuberculosis is often conferred by DNA gyrase mutations. However, a substantial proportion of fluoroquinolone-resistant M. tuberculosis isolates do not have such mutations. METHODS: Ofloxacin-resistant and lineage-matched ofloxacin-susceptible M. tuberculosis isolates underwent WGS. Novel candidate resistance mutations were confirmed by Sanger sequencing and conferral of resistance was assessed via site-directed mutagenesis and allelic exchange. Ofloxacin MIC was determined by resazurin microtitre assay (REMA) and the effects on MICs of efflux pump inhibitors (CCCP, reserpine and verapamil) were determined. RESULTS: Of 26 ofloxacin-resistant isolates, 8 (31%) did not have resistance-conferring DNA gyrase mutations. The V762G mutation in Rv1783 (eccC5, encoding a protein in the ESX-5 membrane complex secretion system) was present on WGS in 8/26 (31%) resistant isolates and 0/11 susceptible isolates (P = 0.005). The mutation was identified in five isolates without DNA gyrase mutations and three isolates with such mutations; it was identified in both European-American and East Asian M. tuberculosis lineages. The ofloxacin MIC increased from 1 to 32 mg/L after introduction of the V762G mutation into M. tuberculosis H37Rv. In this strain with the V762G mutation, ofloxacin MIC did not change in the presence of efflux pump inhibitors. CONCLUSIONS: A novel V762G mutation in Rv1783 conferred ofloxacin resistance in M. tuberculosis by a mechanism other than drug efflux. This occurred in a substantial proportion of resistant isolates, particularly those without DNA gyrase mutations.

High proportion of heteroresistance in gyrA and gyrB in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates.

Heteroresistance is the coexistence of populations with differing nucleotides at a drug resistance locus within a sample of organisms. Although Sanger sequencing is the gold standard for sequencing, it may be less sensitive than deep sequencing for detecting fluoroquinolone heteroresistance in Mycobacterium tuberculosis. Twenty-seven fluoroquinolone monoresistant and 11 fluoroquinolone-susceptible M. tuberculosis isolates were analyzed by Sanger and Illumina deep sequencing. Individual sequencing reads were analyzed to detect heteroresistance in the gyrA and gyrB genes. Heteroresistance to fluoroquinolones was identified in 10/26 (38%) phenotypically fluoroquinolone-resistant samples and 0/11 (P = 0.02) fluoroquinolone-susceptible controls. One resistant sample was excluded because of contamination with the laboratory strain M. tuberculosis H37Rv. Sanger sequencing revealed resistance-conferring mutations in 15 isolates, while deep sequencing revealed mutations in 20 isolates. Isolates with fluoroquinolone resistance-conferring mutations by Sanger sequencing all had at least those same mutations identified by deep sequencing. By deep sequencing, 10 isolates had a single fixed (defined as >95% frequency) mutation, while 10 were heteroresistant, 5 of which had a single unfixed (defined as <95% frequency) mutation and 5 had multiple unfixed mutations. Illumina deep sequencing identified a higher proportion of fluoroquinolone-resistant M. tuberculosis isolates with heteroresistance than did Sanger sequencing. The heteroresistant isolates frequently demonstrated multiple mutations, but resistant isolates with fixed mutations each had only a single mutation.

Blastomycosis Endocarditis: Case Report and Literature Review.

We report successful treatment of a case of disseminated blastomycosis originating in the right lung, with involvement of the right pleural space, multiple ribs and vertebral bodies, and the pericardium and mitral valve endocarditis. The 22-year-old patient presented with a 13-month history of right lower lobe pneumonia associated with fevers, night sweats, rib pain, and 27-kg weight loss. Pathology examination revealed Blastomyces from multiple biopsies of inflammatory masses in the right thorax. After a 4-week induction with liposomal amphotericin followed by oral itraconazole, the patient had complete resolution of the clinical and laboratory findings of blastomycosis.

Environmental Contamination with Candida Species in Multiple Hospitals Including a Tertiary Care Hospital with a Candida auris Outbreak.

BACKGROUND: Environmental sources have been implicated as a potential source for exogenous acquisition of Candida species, particularly the emerging multidrug-resistant Candida auris. However, limited information is available on environmental reservoirs of Candida species in healthcare facilities. METHODS: During a 6-month period, cultures for Candida species were collected from high-touch surfaces in patient rooms and from portable equipment in 6 US acute care hospitals in 4 states. Additional cultures were collected from sink drains and floors in one of the hospitals and from high-touch surfaces, portable equipment, and sink drains in a hospital experiencing an outbreak due to C. auris. Candida species were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectometry. RESULTS: Candida species were recovered from patient rooms in 4 of the 6 hospitals. Seven of 147 patient room cultures (4.8%) and 1 of 57 (1.8%) portable equipment cultures were positive, with the most common species being C. parapsilosis. For the hospital where additional sites were sampled, Candida species were recovered from 8 of 22 (36.4%) hospital room floors and 4 of 17 (23.5%) sink drains. In the facility with a C. auris outbreak, Candida species were frequently recovered from sink drains (20.7%) and high-touch surfaces (15.4%), but recovery of C. auris was uncommon (3.8% of high-touch surfaces, 3.4% of sink drains, and 0% of portable equipment) and only present in rooms that currently or recently housed a patient with C. auris. CONCLUSION: Candida species often contaminate surfaces in hospitals and may be particularly common on floors and in sink drains. However, C. auris contamination was uncommon in a facility experiencing an outbreak, suggesting that current cleaning and disinfection practices can be effective in minimizing environmental contamination.

Carbapenem-Resistant Enterobacteriaceae Infections in Patients on Renal Replacement Therapy.

BACKGROUND: Patients on chronic intermittent renal replacement therapy (RRT) are at risk for infection with carbapenem-resistant Enterobacteriaceae (CRE). However, the impact of RRT on outcomes after CRE infections remains to be defined. Here we perform a comparison of outcomes for CRE-infected patients with preserved renal function compared with CRE-infected patients on RRT. METHODS: Cases and controls were defined from a prospective cohort of CRE-infected patients from the Consortium on Resistance against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE). Cases were defined as CRE-infected patients on RRT at hospital admission, while controls were defined as CRE-infected patients with serum creatinine <2 mg/dL and not receiving RRT at admission. Risk factors for 28-day in-hospital mortality were assessed using multivariable logistic regression. An ordinal ranking of outcomes by desirability analysis was performed. RESULTS: Patients on RRT were more likely to have diabetes mellitus and cardiac disease than controls. Urinary sources of infection were less common in the RRT group. In RRT patients, 28-day in-hospital mortality was increased as compared with controls: 22/71 (31%) vs 33/295 (11%). RRT remained significantly associated with 28-day in-hospital mortality after adjustment for source of infection, prehospitalization origin, and severity of illness (adjusted odds ratio, 2.27; 95% confidence interval [CI], 1.09-4.68; P = .03). Using univariable desirability of outcome ranking analysis, RRT status was associated with a 68% (95% CI, 61%-74%) chance of a worse disposition outcome. CONCLUSIONS: Chronic RRT in CRE-infected patients is associated with increased in-hospital mortality and worse disposition outcomes at 28 days.

T-cell hybridomas from HLA-transgenic mice as tools for analysis of human antigen processing.

The study of antigen processing and presentation by human antigen presenting cells (APC) has been limited by difficulties of producing and maintaining human T-cell clones. Murine T-cell hybridomas have advantages for detecting specific peptide-MHC complexes on APC. Human antigen-specific immortalized T-cell lines have not been successfully produced. We report and validate the use of transgenic mice with human MHC genes for HLA-A2, DR1 and DR4 to produce murine T-cell hybridomas that are restricted to human HLA alleles and respond to human macrophages, dendritic cells (DC), and B-cell lines. Hybridomas restricted by human MHC-I and -II specific for influenza matrix protein, tetanus toxoid, diphtheria antigen CRM(197), and various M. tuberculosis antigens were produced. Epitope specificity was determined for several hybridomas. T hybridomas recognized peptide-MHC complexes on fixed APC for analysis of kinetics or susceptibility to inhibitors of antigen processing. T hybridomas restricted by human MHC represent convenient and powerful tools for the study of antigen processing by human APC.

The purinergic P2X7 receptor is not required for control of pulmonary Mycobacterium tuberculosis infection.

The importance in vivo of P2X7 receptors in control of virulent Mycobacterium tuberculosis was examined in a low-dose aerosol infection mouse model. P2X7(-/-) mice controlled infection in lungs as well as wild-type mice, suggesting that the P2X7 receptor is not required for control of pulmonary M. tuberculosis infection.