Genomic Analysis of the Emergence and Rapid Global Dissemination of the Clonal Group 258 Klebsiella pneumoniae Pandemic.

Multidrug-resistant Klebsiella pneumoniae producing the KPC carbapenemase have rapidly spread throughout the world, causing severe healthcare-associated infections with limited antimicrobial treatment options. Dissemination of KPC-producing K. pneumoniae is largely attributed to expansion of a single dominant strain, ST258. In this study, we explore phylogenetic relationships and evolution within ST258 and its clonal group, CG258, using whole genome sequence analysis of 167 isolates from 20 countries collected over 17 years. Our results show a common ST258 ancestor emerged from its diverse parental clonal group around 1995 and likely acquired blaKPC prior to dissemination. Over the past two decades, ST258 has remained highly clonal despite diversity in accessory elements and divergence in the capsule polysaccharide synthesis locus. Apart from the large recombination event that gave rise to ST258, few mutations set it apart from its clonal group. However, one mutation occurs in a global transcription regulator. Characterization of outer membrane protein sequences revealed a profile in ST258 that includes a truncated OmpK35 and modified OmpK37. Our work illuminates potential genomic contributors to the pathogenic success of ST258, helps us better understand the global dissemination of this strain, and identifies genetic markers unique to ST258.

New Delhi metallo-ß-lactamase-producing carbapenem-resistant Escherichia coli associated with exposure to duodenoscopes.

IMPORTANCE: Carbapenem-resistant Enterobacteriaceae (CRE) producing the New Delhi metallo-ß-lactamase (NDM) are rare in the United States, but have the potential to add to the increasing CRE burden. Previous NDM-producing CRE clusters have been attributed to person-to-person transmission in health care facilities. OBJECTIVE: To identify a source for, and interrupt transmission of, NDM-producing CRE in a northeastern Illinois hospital. DESIGN, SETTING, AND PARTICIPANTS: Outbreak investigation among 39 case patients at a tertiary care hospital in northeastern Illinois, including a case-control study, infection control assessment, and collection of environmental and device cultures; patient and environmental isolate relatedness was evaluated with pulsed-field gel electrophoresis (PFGE). Following identification of a likely source, targeted patient notification and CRE screening cultures were performed. MAIN OUTCOMES AND MEASURES: Association between exposure and acquisition of NDM-producing CRE; results of environmental cultures and organism typing. RESULTS: In total, 39 case patients were identified from January 2013 through December 2013, 35 with duodenoscope exposure in 1 hospital. No lapses in duodenoscope reprocessing were identified; however, NDM-producing Escherichia coli was recovered from a reprocessed duodenoscope and shared more than 92% similarity to all case patient isolates by PFGE. Based on the case-control study, case patients had significantly higher odds of being exposed to a duodenoscope (odds ratio [OR], 78 [95% CI, 6.0-1008], P < .001). After the hospital changed its reprocessing procedure from automated high-level disinfection with ortho-phthalaldehyde to gas sterilization with ethylene oxide, no additional case patients were identified. CONCLUSIONS AND RELEVANCE: In this investigation, exposure to duodenoscopes with bacterial contamination was associated with apparent transmission of NDM-producing E coli among patients at 1 hospital. Bacterial contamination of duodenoscopes appeared to persist despite the absence of recognized reprocessing lapses. Facilities should be aware of the potential for transmission of bacteria including antimicrobial-resistant organisms via this route and should conduct regular reviews of their duodenoscope reprocessing procedures to ensure optimal manual cleaning and disinfection.

Carbapenem-resistant Klebsiella pneumoniae producing New Delhi metallo-ß-lactamase at an acute care hospital, Colorado, 2012.

OBJECTIVE: To investigate an outbreak of New Delhi metallo-ß-lactamase (NDM)-producing carbapenem-resistant Enterobacteriaceae (CRE) and determine interventions to interrupt transmission. DESIGN, SETTING, AND PATIENTS: Epidemiologic investigation of an outbreak of NDM-producing CRE among patients at a Colorado acute care hospital. METHODS: Case patients had NDM-producing CRE isolated from clinical or rectal surveillance cultures (SCs) collected during the period January 1, 2012, through October 20, 2012. Case patients were identified through microbiology records and 6 rounds of SCs in hospital units where they had resided. CRE isolates were tested by real-time polymerase chain reaction for blaNDM. Medical records were reviewed for epidemiologic links; relatedness of isolates was evaluated by pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS). Infection control (IC) was assessed through staff interviews and direct observations. RESULTS: Two patients were initially identified with NDM-producing CRE during July-August 2012. A third case patient, admitted in May, was identified through microbiology records review. SC identified 5 additional case patients. Patients had resided in 11 different units before identification. All isolates were highly related by PFGE. WGS suggested 3 clusters of CRE. Combining WGS with epidemiology identified 4 units as likely transmission sites. NDM-producing CRE positivity in certain patients was not explained by direct epidemiologic overlap, which suggests that undetected colonized patients were involved in transmission. CONCLUSIONS: A 4-month outbreak of NDM-producing CRE occurred at a single hospital, highlighting the risk for spread of these organisms. Combined WGS and epidemiologic data suggested transmission primarily occurred on 4 units. Timely SC, combined with targeted IC measures, were likely responsible for controlling transmission.

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.

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

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

Outbreak of carbapenem-resistant enterobacteriaceae at a long-term acute care hospital: sustained reductions in transmission through active surveillance and targeted interventions.

OBJECTIVE: To describe a Klebsiella pneumoniae carbapenemase (KPC)-producing carbapenem-resistant Enterobacteriaceae (CRE) outbreak and interventions to prevent transmission. DESIGN, SETTING, AND PATIENTS: Epidemiologic investigation of a CRE outbreak among patients at a long-term acute care hospital (LTACH). METHODS: Microbiology records at LTACH A from March 2009 through February 2011 were reviewed to identify CRE transmission cases and cases admitted with CRE. CRE bacteremia episodes were identified during March 2009-July 2011. Biweekly CRE prevalence surveys were conducted during July 2010-July 2011, and interventions to prevent transmission were implemented, including education and auditing of staff and isolation and cohorting of CRE patients with dedicated nursing staff and shared medical equipment. Trends were evaluated using weighted linear or Poisson regression. CRE transmission cases were included in a case-control study to evaluate risk factors for acquisition. A real-time polymerase chain reaction assay was used to detect the bla(KPC) gene, and pulsed-field gel electrophoresis was performed to assess the genetic relatedness of isolates. RESULTS: Ninety-nine CRE transmission cases, 16 admission cases (from 7 acute care hospitals), and 29 CRE bacteremia episodes were identified. Significant reductions were observed in CRE prevalence (49% vs. 8%), percentage of patients screened with newly detected CRE (44% vs. 0%), and CRE bacteremia episodes (2.5 vs. 0.0 per 1,000 patient-days). Cases were more likely to have received ß-lactams, have diabetes, and require mechanical ventilation. All tested isolates were KPC-producing K. pneumoniae, and nearly all isolates were genetically related. CONCLUSION: CRE transmission can be reduced in LTACHs through surveillance testing and targeted interventions. Sustainable reductions within and across healthcare facilities may require a regional public health approach.

An outbreak of Klebsiella pneumoniae late-onset sepsis in a neonatal intensive care unit in Guatemala.

BACKGROUND: Gram-negative bloodstream infections are an important cause of neonatal mortality. In October 2009, we investigated a Klebsiella spp outbreak in a neonatal intensive care unit in Guatemala. METHODS: Probable cases were defined as a Klebsiella spp isolated from blood in neonates aged <28 days in the neonatal intensive care unit between October 1 and November 10, 2009; confirmed cases were identified as Klebsiella pneumoniae. Clinical data were abstracted from medical charts. K pneumoniae isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and tested for antimicrobial susceptibility. Infection control practices were inspected. RESULTS: There were 14 confirmed cases. The median age at onset of infection was 3 days (range, 2-8 days). Nine patients died (64%). K pneumoniae isolates were resistant to multiple antimicrobials. PFGE revealed 2 distinct clusters. Breaches in infection control procedures included inappropriate intravenous solution use and inadequate hand hygiene and contact precautions. CONCLUSIONS: We report a K pneumoniae outbreak with high neonatal mortality in Guatemala. PFGE clustering suggested a common source possibly related to reuse of a single-use intravenous medication or solution. The risk for K pneumoniae bloodstream infections in neonates in low-resource settings where sharing of solutions is common needs to be emphasized.

Molecular dissection of an outbreak of carbapenem-resistant enterobacteriaceae reveals Intergenus KPC carbapenemase transmission through a promiscuous plasmid.

Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as major causes of health care-associated infections worldwide. This diverse collection of organisms with various resistance mechanisms is associated with increased lengths of hospitalization, costs of care, morbidity, and mortality. The global spread of CRE has largely been attributed to dissemination of a dominant strain of Klebsiella pneumoniae producing a serine ß-lactamase, termed K. pneumoniae carbapenemase (KPC). Here we report an outbreak of KPC-producing CRE infections in which the degree of horizontal transmission between strains and species of a promiscuous plasmid is unprecedented. Sixteen isolates, comprising 11 unique strains, 6 species, and 4 genera of bacteria, were obtained from 14 patients over the first 8 months of the outbreak. Of the 11 unique strains, 9 harbored the same highly promiscuous plasmid carrying the KPC gene bla(KPC). The remaining strains harbored distinct bla(KPC) plasmids, one of which was carried in a strain of Klebsiella oxytoca coisolated from the index patient and the other generated from transposition of the bla(KPC) element Tn4401. All isolates could be genetically traced to the index patient. Molecular epidemiological investigation of the outbreak was aided by the adaptation of nested arbitrary PCR (ARB-PCR) for rapid plasmid identification. This detailed molecular genetic analysis, combined with traditional epidemiological investigation, provides insights into the highly fluid dynamics of drug resistance transmission during the outbreak. IMPORTANCE The ease of horizontal transmission of carbapenemase resistance plasmids across strains, species, and genera of bacteria observed in this study has several important public health and epidemiological implications. First, it has the potential to promote dissemination of carbapenem resistance to new populations of Enterobacteriaceae, including organisms of low virulence, leading to the establishment of reservoirs of carbapenem resistance genes in patients and/or the environment and of high virulence, raising the specter of untreatable community-associated infections. Second, recognition of plasmid-mediated outbreaks, such as those described here, is problematic because analysis of resistance plasmids from clinical isolates is laborious and technically challenging. Adaptation of nested arbitrary PCR (ARB-PCR) to investigate the plasmid outbreak facilitated our investigation, and the method may be broadly applicable to other outbreaks due to other conserved mobile genetic elements. Whether infection control measures that focus on preventing transmission of drug-resistant clones are effective in controlling dissemination of these elements is unknown.

IMP-producing carbapenem-resistant Klebsiella pneumoniae in the United States.

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-ß-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.

Genetic factors associated with elevated carbapenem resistance in KPC-producing Klebsiella pneumoniae.

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 bla(KPC) gene copy number (n = 3) or had deletions directly upstream of the bla(KPC) 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 bla(KPC) copy number. These results suggest that both bla(KPC) 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.

A rapid antimicrobial susceptibility test for Bacillus anthracis.

An effective public health response to a deliberate release of Bacillus anthracis will require a rapid distribution of antimicrobial agents for postexposure prophylaxis and treatment. However, conventional antimicrobial susceptibility testing for B. anthracis requires a 16- to 20-h incubation period. To reduce this time, we have combined a modified broth microdilution (BMD) susceptibility testing method with real-time quantitative PCR (qPCR). The growth or inhibition of growth of B. anthracis cells incubated in 2-fold dilutions of ciprofloxacin (CIP) (0.015 to 16 microg/ml) or doxycycline (DOX) (0.06 to 64 microg/ml) was determined by comparing the fluorescence threshold cycle (C(T)) generated by target amplification from cells incubated with each drug concentration with the C(T) of the no-drug (positive growth) control. This DeltaC(T) readily differentiated susceptible and nonsusceptible strains. Among susceptible strains, the median DeltaC(T) values were > or = 7.51 cycles for CIP and > or = 7.08 cycles for DOX when drug concentrations were at or above the CLSI breakpoint for susceptibility. For CIP- and DOX-nonsusceptible strains, the DeltaC(T) was < 1.0 cycle at the breakpoint for susceptibility. When evaluated with 14 genetically and geographically diverse strains of B. anthracis, the rapid method provided the same susceptibility results as conventional methods but required less than 6 h, significantly decreasing the time required for the selection and distribution of appropriate medical countermeasures.

Emergence of blaKPC-containing Klebsiella pneumoniae in a long-term acute care hospital: a new challenge to our healthcare system.

OBJECTIVES: To characterize isolates of Klebsiella pneumoniae producing KPC carbapenemase (KPC-Kp) associated with an outbreak in a long-term acute care hospital (LTACH) in South Florida. METHODS: During 21 March to 20 April 2008, 241 K. pneumoniae isolates detected at Integrated Regional Laboratories (Ft. Lauderdale, FL) for which the ertapenem MICs were > or =4 mg/L were studied. PCR, cloning and sequence analysis were used to detect bla(KPC) and to characterize the beta-lactamase and outer membrane proteins (Omps). The expression level of KPC enzymes was studied by immunoblotting. Genetic relatedness of isolates was investigated with rep-PCR and PFGE. Clinical records of patients were investigated. RESULTS: Seven KPC-Kp strains were isolated from different patients located at a single LTACH, with a further three isolates being recovered from patients at different hospitals. All KPC-Kp isolates in patients from the LTACH and from one hospital patient were genetically related and shared PFGE patterns that clustered with known sequence type (ST) 258 strains. These strains were highly resistant to carbapenems (MICs > or = 32 mg/L) due to an increased level of KPC expression and loss of Omps. Rectal colonization was documented in all LTACH patients with KPC-Kp isolates. Treatment failures were common (crude mortality rate of 69%). Active surveillance and enhanced infection control practices terminated the KPC-Kp outbreak. CONCLUSIONS: The detection of KPC-Kp in an LTACH represents a serious infection control and therapeutic challenge in a new clinical setting. The speed at which the epidemic of KPC-Kp is spreading in our healthcare system mandates urgent action.

Regional dissemination of KPC-producing Klebsiella pneumoniae.

Production of a Klebsiella pneumoniae carbapenemase (KPC) is the most common mechanism of carbapenem resistance in the United States; however, until now, KPC-producing isolates have not been found in western Michigan. Molecular typing of two KPC-producing K. pneumoniae isolates from Michigan showed their similarity to other Midwestern isolates. They were also unrelated to the dominant sequence type observed throughout the United States, multilocus sequence type 258. This could represent regional dissemination of another KPC-producing K. pneumoniae strain.

Molecular epidemiology of KPC-producing Klebsiella pneumoniae isolates in the United States: clonal expansion of multilocus sequence type 258.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become more common in the United States and throughout the world. We used pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to examine the molecular epidemiology of KPC-producing K. pneumoniae isolates sent to the Centers for Disease Control and Prevention (CDC) for reference testing from 1996 to 2008. A dominant strain, sequence type 258 (ST 258), was found and likely accounts for 70% of the CDC's K. pneumoniae PFGE database. Isolates with PFGE patterns related to ST 258 were identified in 10 of the 19 U.S. states currently reporting KPC-producing K. pneumoniae, in addition to one isolate from Israel. KPC subtyping and analysis of the surrounding genetic environment were subsequently performed on 23 representative isolates. Thirteen isolates identified as ST 258 possessed either bla(KPC-2) or bla(KPC-3) and some variability in the Tn4401 element upstream of the bla(KPC) gene. Escherichia coli DH10B was successfully transformed by electroporation with KPC-encoding plasmid DNA from 20 of the 23 isolates. Restriction analysis of plasmid DNA prepared from transformants revealed a diversity of band patterns, suggesting the presence of different plasmids harboring the bla(KPC) gene, even among isolates of the same ST.

RGD-functionalized bioengineered spider dragline silk biomaterial.

Spider silk fibers have remarkable mechanical properties that suggest the component proteins could be useful biopolymers for fabricating biomaterial scaffolds for tissue formation. Two bioengineered protein variants from the consensus sequence of the major component of dragline silk from Nephila clavipes were cloned and expressed to include RGD cell-binding domains. The engineered silks were characterized by CD and FTIR and showed structural transitions from random coil to insoluble beta-sheet upon treatment with methanol. The recombinant proteins were processed into films and fibers and successfully used as biomaterial matrixes to culture human bone marrow stromal cells induced to differentiate into bone-like tissue upon addition of osteogenic stimulants. The recombinant spider silk and the recombinant spider silk with RGD encoded into the protein both supported enhanced the differentiation of human bone marrow derived mesenchymal stem cells (hMSCs) to osteogenic outcomes when compared to tissue culture plastic. The recombinant spider silk protein without the RGD displayed enhanced bone related outcomes, measured by calcium deposition, when compared to the same protein with RGD. Based on comparisons to our prior studies with silkworm silks and RGD modifications, the current results illustrate the potential to bioengineer spider silk proteins into new biomaterial matrixes, while also highlighting the importance of subtle differences in silk sources and modes of presentation of RGD to cells in terms of tissue-specific outcomes.

Novel nanocomposites from spider silk-silica fusion (chimeric) proteins.

Silica skeletal architectures in diatoms are characterized by remarkable morphological and nanostructural details. Silk proteins from spiders and silkworms form strong and intricate self-assembling fibrous biomaterials in nature. We combined the features of silk with biosilica through the design, synthesis, and characterization of a novel family of chimeric proteins for subsequent use in model materials forming reactions. The domains from the major ampullate spidroin 1 (MaSp1) protein of Nephila clavipes spider dragline silk provide control over structural and morphological details because it can be self-assembled through diverse processing methods including film casting and fiber electrospinning. Biosilica nanostructures in diatoms are formed in aqueous ambient conditions at neutral pH and low temperatures. The R5 peptide derived from the silaffin protein of Cylindrotheca fusiformis induces and regulates silica precipitation in the chimeric protein designs under similar ambient conditions. Whereas mineralization reactions performed in the presence of R5 peptide alone form silica particles with a size distribution of 0.5-10 microm in diameter, reactions performed in the presence of the new fusion proteins generate nanocomposite materials containing silica particles with a narrower size distribution of 0.5-2 microm in diameter. Furthermore, we demonstrate that composite morphology and structure could be regulated by controlling processing conditions to produce films and fibers. These results suggest that the chimeric protein provides new options for processing and control over silica particle sizes, important benefits for biomedical and specialty materials, particularly in light of the all aqueous processing and the nanocomposite features of these new materials.