Amy J. Mathers

The Role of Epidemic Resistance Plasmids and International High-Risk Clones in the Spread of Multidrug-Resistant Enterobacteriaceae

SUMMARY Escherichia coli sequence type 131 (ST131) and Klebsiella pneumoniae ST258 emerged in the 2000s as important human pathogens, have spread extensively throughout the world, and are responsible for the rapid increase in antimicrobial resistance among E. coli and K. pneumoniae strains, respectively. E. coli ST131 causes extraintestinal infections and is often fluoroquinolone resistant and associated with extended-spectrum β-lactamase production, especially CTX-M-15. K. pneumoniae ST258 causes urinary and respiratory tract infections and is associated with carbapenemases, most often KPC-2 and KPC-3. The most prevalent lineage within ST131 is named fimH30 because it contains the H30 variant of the type 1 fimbrial adhesin gene, and recent molecular studies have demonstrated that this lineage emerged in the early 2000s and was then followed by the rapid expansion of its sublineages H30-R and H30-Rx. K. pneumoniae ST258 comprises 2 distinct lineages, namely clade I and clade II. Moreover, it seems that ST258 is a hybrid clone that was created by a large recombination event between ST11 and ST442. Epidemic plasmids with bla CTX-M and bla KPC belonging to incompatibility group F have contributed significantly to the success of these clones. E. coli ST131 and K. pneumoniae ST258 are the quintessential examples of international multidrug-resistant high-risk clones.

Molecular Dissection of an Outbreak of Carbapenem-Resistant Enterobacteriaceae Reveals Intergenus KPC Carbapenemase Transmission through a Promiscuous Plasmid

ABSTRACT 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 β-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 blaKPC. The remaining strains harbored distinct blaKPC 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 blaKPC 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. 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.

First Clinical Cases of OXA-48-Producing Carbapenem-Resistant Klebsiella pneumoniae in the United States: the “Menace” Arrives in the New World

ABSTRACT OXA-48 has emerged as a major carbapenemase associated with the Enterobacteriaceae in Europe, North Africa, and Asia. We report the first two clinical cases of OXA-48-type carbapenemase-producing Enterobacteriaceae in the United States from patients recently hospitalized in Saudi Arabia and India. Each is more carbapenem resistant than nearly all previously reported OXA-48-type-producing Enterobacteriaceae.

Fatal cross infection by carbapenem-resistant Klebsiella in two liver transplant recipients

Abstract: Members of the family Enterobacteriaceae including Klebsiella have re‐emerged as major pathogens in solid organ transplantation. The recent appearance and dissemination of carbapenemase‐producing Enterobacteriaceae in Europe and the northeastern United States represents a major challenge to the treatment of enteric gram‐negative bacterial infections in immunocompromised patients; however, few reports have detailed the outcomes of such infections. Here we report 2 cases of Klebsiella pneumoniae carbapenemase (KPC)‐producing Klebsiella infections in orthotopic liver transplant recipients, which were the index case and initial secondary case for an outbreak of KPC‐producing Enterobacteriaceae in our institution. In both instances, the pathogens were initially misidentified as being carbapenem sensitive, the infections recurred after cessation of directed therapy, and the patients ultimately succumbed to their infections.

Klebsiella pneumoniae carbapenemase (KPC) producing K. pneumoniae at a Single Institution: Insights into Endemicity from Whole Genome Sequencing

ABSTRACT The global emergence of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) multilocus sequence type ST258 is widely recognized. Less is known about the molecular and epidemiological details of non-ST258 K. pneumoniae in the setting of an outbreak mediated by an endemic plasmid. We describe the interplay of blaKPC plasmids and K. pneumoniae strains and their relationship to the location of acquisition in a U.S. health care institution. Whole-genome sequencing (WGS) analysis was applied to KPC-Kp clinical isolates collected from a single institution over 5 years following the introduction of blaKPC in August 2007, as well as two plasmid transformants. KPC-Kp from 37 patients yielded 16 distinct sequence types (STs). Two novel conjugative blaKPC plasmids (pKPC_UVA01 and pKPC_UVA02), carried by the hospital index case, accounted for the presence of blaKPC in 21/37 (57%) subsequent cases. Thirteen (35%) isolates represented an emergent lineage, ST941, which contained pKPC_UVA01 in 5/13 (38%) and pKPC_UVA02 in 6/13 (46%) cases. Seven (19%) isolates were the epidemic KPC-Kp strain, ST258, mostly imported from elsewhere and not carrying pKPC_UVA01 or pKPC_UVA02. Using WGS-based analysis of clinical isolates and plasmid transformants, we demonstrate the unexpected dispersal of blaKPC to many non-ST258 lineages in a hospital through spread of at least two novel blaKPC plasmids. In contrast, ST258 KPC-Kp was imported into the institution on numerous occasions, with other blaKPC plasmid vectors and without sustained transmission. Instead, a newly recognized KPC-Kp strain, ST941, became associated with both novel blaKPC plasmids and spread locally, making it a future candidate for clinical persistence and dissemination.

Nested Russian doll-like genetic mobility drives rapid dissemination of the carbapenem resistance gene blakpc

ABSTRACT The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance.

Escherichia coli ST131: The quintessential example of an international multiresistant high-risk clone.

Escherichia coli ST131 emerged during the early to mid-2000s is an important human pathogen, has spread extensively throughout the world, and is responsible for the rapid increase in antimicrobial resistance among E. coli. ST131 is known to cause extraintestinal infections, being fluoroquinolone resistant, and is associated with ESBL production most often due to CTX-M-15. Recent molecular epidemiologic studies using whole-genome sequencing and phylogenetic analysis have demonstrated that the H30 ST131 lineage emerged in early 2000s that was followed by the rapid expansion of its sublineages H30-R and H30-Rx. Escherichia coli ST131 clearly has all of the essential characteristics that define a high-risk clone and might be the quintessential example of an international multiresistant high-risk clone. We urgently need rapid cost-effective detection methods for E. coli ST131, as well as well-designed epidemiological and molecular studies to understand the dynamics of transmission, risk factors, and reservoirs for ST131. This will provide insight into the emergence and spread of this multiresistant sequence type that will hopefully lead to information essential for preventing the spread of ST131.

Modified Hodge Test versus Indirect Carbapenemase Test: Prospective Evaluation of a Phenotypic Assay for Detection of Klebsiella pneumoniae Carbapenemase (KPC) in Enterobacteriaceae

ABSTRACT The currently recommended phenotypic test for the detection of carbapenemase-producing members of the family Enterobacteriaceae is the modified Hodge test (MHT). However, the MHT lacks specificity. Here we demonstrate an alternative phenotypic test, the indirect carbapenemase test, for the detection of bla KPC-producing isolates that has specificity superior to that of the MHT for non-Klebsiella Enterobacteriaceae.

Control of Simultaneous Outbreaks of Carbapenemase-Producing Enterobacteriaceae and Extensively Drug-Resistant Acinetobacter baumannii Infection in an Intensive Care Unit Using Interventions Promoted in the Centers for Disease Control and Prevention 2012 Carbapenemase-Resistant Enterobacteriaceae Toolkit

OBJECTIVE We describe the efficacy of enhanced infection control measures, including those recommended in the Centers for Disease Control and Preventions 2012 carbapenem-resistant Enterobacteriaceae (CRE) toolkit, to control concurrent outbreaks of carbapenemase-producing Enterobacteriaceae (CPE) and extensively drug-resistant Acinetobacter baumannii (XDR-AB). DESIGN Before-after intervention study. SETTING Fifteen-bed surgical trauma intensive care unit (ICU). METHODS We investigated the impact of enhanced infection control measures in response to clusters of CPE and XDR-AB infections in an ICU from April 2009 to March 2010. Polymerase chain reaction was used to detect the presence of blaKPC and resistance plasmids in CRE. Pulsed-field gel electrophoresis was performed to assess XDR-AB clonality. Enhanced infection-control measures were implemented in response to ongoing transmission of CPE and a new outbreak of XDR-AB. Efficacy was evaluated by comparing the incidence rate (IR) of CPE and XDR-AB before and after the implementation of these measures. RESULTS The IR of CPE for the 12 months before the implementation of enhanced measures was 7.77 cases per 1,000 patient-days, whereas the IR of XDR-AB for the 3 months before implementation was 6.79 cases per 1,000 patient-days. All examined CPE shared endemic blaKPC resistance plasmids, and 6 of the 7 XDR-AB isolates were clonal. Following institution of enhanced infection control measures, the CPE IR decreased to 1.22 cases per 1,000 patient-days (P = .001), and no more cases of XDR-AB were identified. CONCLUSIONS Use of infection control measures described in the Centers for Disease Control and Preventions 2012 CRE toolkit was associated with a reduction in the IR of CPE and an interruption in XDR-AB transmission.

Clinical microbiology costs for methods of active surveillance for Klebsiella pneumoniae carbapenemase-producing Enterobacteriaceae.

OBJECTIVE To compare direct laboratory costs of different methods for perirectal screening for carbapenemase-producing Enterobacteriaceae (CPE) colonization. DESIGN Cost-benefit analysis. SETTING A university hospital and affiliated long-term acute care hospital (LTACH). PARTICIPANTS Inpatients from the hospital or LTACH. METHODS Perirectal samples were collected from inpatients at risk for exposure to CPE. In 2009, we compared the accuracy of the Centers for Disease Control and Prevention (CDC)-recommended CPE screening method with similar methods incorporating a chromogenic agar (CA). We then performed a cost projection analysis using 2012 screening results for the CA method, the CDC method, and a molecular assay with wholesale pricing based on the 2009 analysis. Comparisons of turnaround and personnel time were also performed. RESULTS A total of 185 (2.7%) of 6,860 samples were confirmed as CPE positive during 2012. We previously found that the CDC protocol had a lower sensitivity than the CA method and predicted that the CDC protocol would have missed 92 of the CPE-positive screening results, whereas the modified protocol using CA would have missed 26, assuming similar prevalence and performance. Turnaround time was 3 days using the CDC and CA-modified protocols compared with 1 day for molecular testing. The estimated annual total program cost and total technologists hours would be the following: CA-modified protocol,