Multicenter Clinical and Molecular Epidemiological Analysis of Bacteremia Due to Carbapenem-Resistant Enterobacteriaceae (CRE) in the CRE Epicenter of the United States.

Although the New York/New Jersey (NY/NJ) area is an epicenter for carbapenem-resistant Enterobacteriaceae (CRE), there are few multicenter studies of CRE from this region. We characterized patients with CRE bacteremia in 2013 at eight NY/NJ medical centers and determined the prevalence of carbapenem resistance among Enterobacteriaceae bloodstream isolates and CRE resistance mechanisms, genetic backgrounds, capsular types (cps), and antimicrobial susceptibilities. Of 121 patients with CRE bacteremia, 50% had cancer or had undergone transplantation. The prevalences of carbapenem resistance among Klebsiella pneumoniae, Enterobacter spp., and Escherichia coli bacteremias were 9.7%, 2.2%, and 0.1%, respectively. Ninety percent of CRE were K. pneumoniae and 92% produced K. pneumoniae carbapenemase (KPC-3, 48%; KPC-2, 44%). Two CRE produced NDM-1 and OXA-48 carbapenemases. Sequence type 258 (ST258) predominated among KPC-producing K. pneumoniae (KPC-Kp). The wzi154 allele, corresponding to cps-2, was present in 93% of KPC-3-Kp, whereas KPC-2-Kp had greater cps diversity. Ninety-nine percent of CRE were ceftazidime-avibactam (CAZ-AVI)-susceptible, although 42% of KPC-3-Kp had an CAZ-AVI MIC of ≥4/4 µg/ml. There was a median of 47 h from bacteremia onset until active antimicrobial therapy, 38% of patients had septic shock, and 49% died within 30 days. KPC-3-Kp bacteremia (adjusted odds ratio [aOR], 2.58; P = 0.045), cancer (aOR, 3.61, P = 0.01), and bacteremia onset in the intensive care unit (aOR, 3.79; P = 0.03) were independently associated with mortality. Active empirical therapy and combination therapy were not associated with survival. Despite a decade of experience with CRE, patients with CRE bacteremia have protracted delays in appropriate therapies and high mortality rates, highlighting the need for rapid diagnostics and evaluation of new therapeutics.

A novel quantitative PCR detects Babesia infection in patients not identified by currently available non-nucleic acid amplification tests.

BACKGROUND: Ticks transmit Babesia microti, the causative agents of babesiosis in North America and Europe. Babesiosis is now endemic in Northeastern USA and affects people of all ages. Babesia species infect erythrocytes and can be transmitted through blood transfusion. Whole blood and blood products, which are not tested for Babesia, can cause transfusion-transmitted babesiosis (TTB) resulting in severe consequences in the immuno-compromised patients. The purpose of this study was epidemiological evaluation of babesiosis in a tick-infested state. RESULTS: We examined blood samples from 192 patients who visited clinics during the active tick-borne diseases season, using a newly developed qPCR assay that uses the specific molecular beacon probe. Due to the absence of clear symptomology, clinical laboratories did not test 131 samples by IFA, FISH or microscopic examination of Giemsa-stained blood smears. Babesia infection was detected in all age groups by FISH and microscopy; notably patients >40 years of age represented 64% of tested samples and 13% were younger patients. We tested all samples using qPCR and found that 38% were positive for Babesia. Of 28 samples that were positive by FISH, 27 (96%) were also positive by qPCR indicating high congruency between nucleic acid based tests. Interestingly, of 78 asymptomatic samples not tested by FISH, 22 were positive by our qPCR. Direct detection of Babesia relies upon microscopic examination of patient blood smears, which is labor intensive, difficult to scale up, requires specific expertise and is hence, often not performed. In fact, a clinical laboratory examined only 23 of 86 blood samples obtained from two different counties by microscopy. By considering individuals positive for Babesia infection when results from currently available microscopy, FISH or serological tests were positive, we found that our qPCR is highly sensitive (96.2%) and showed a specificity of 70.5% for Babesia. CONCLUSION: Robust qPCR using specific probes can be highly useful for efficient and appropriate diagnosis of babesiosis in patients in conjunction with conventional diagnostics, or as a stand-alone test, especially for donated blood screening. The use of a nucleic acid amplification test based screening of blood and blood products could prevent TTB.

Complete sequence of a bla(KPC)-harboring cointegrate plasmid isolated from Escherichia coli.

Horizontal transfer of bla(KPC)-harboring plasmids contributes significantly to the inter- and intraspecies spread of Klebsiella pneumoniae carbapenemase (KPC). Here we report the complete nucleotide sequence of a bla(KPC)-harboring IncFIA plasmid, pBK32533, from Escherichia coli. pBK32533 is a cointegrate plasmid comprising of a 72-kb sequence identical to that of the nonconjugative pBK30661 plasmid plus an additional 170-kb element that harbors the genes for plasmid transfer. pBK32533 demonstrates how bla(KPC) can be spread from a nonconjugative plasmid through cointegration.

Molecular survey of the dissemination of two blaKPC-harboring IncFIA plasmids in New Jersey and New York hospitals.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains have spread worldwide and become a major threat in health care facilities. Transmission of blaKPC, the plasmid-borne KPC gene, can be mediated by clonal spread and horizontal transfer. Here, we report the complete nucleotide sequences of two novel blaKPC-3-harboring IncFIA plasmids, pBK30661 and pBK30683. pBK30661 is 74 kb in length, with a mosaic plasmid structure; it exhibits homologies to several other plasmids but lacks the plasmid transfer operon (tra) and the origin of transfer (oriT) that are required for plasmid transfer. pBK30683 is a conjugative plasmid with a cointegrated plasmid structure, comprising a 72-kb element that highly resembles pBK30661 (>99.9% nucleotide identities) and an extra 68-kb element that harbors tra and oriT. A PCR scheme was designed to detect the distribution of blaKPC-harboring IncFIA (pBK30661-like and pBK30683-like) plasmids in a collection of clinical Enterobacteriaceae isolates from 10 hospitals in New Jersey and New York. KPC-harboring IncFIA plasmids were found in 20% of 491 K. pneumoniae isolates, and all carried blaKPC-3. pBK30661-like plasmids were identified mainly in the epidemic sequence type 258 (ST258) K. pneumoniae clone, while pBK30683-like plasmids were widely distributed in ST258 and other K. pneumoniae sequence types and among non-K. pneumoniae Enterobacteriaceae species. This suggests that both clonal spread and horizontal plasmid transfer contributed to the dissemination of blaKPC-harboring IncFIA plasmids in our area. Further studies are needed to understand the distribution of this plasmid group in other health care regions and to decipher the origins of pBK30661-like and pBK30683-like plasmids.

Comparative genomic analysis of KPC-encoding pKpQIL-like plasmids and their distribution in New Jersey and New York Hospitals.

The global spread of Klebsiella pneumoniae carbapenemase (KPC) is predominately associated with K. pneumoniae strains genotyped as sequence type 258 (ST258). The first ST258-associated plasmid, pKpQIL, was described in Israel in 2006, but its history in the northeastern United States remains unknown. Six pKpQIL-like plasmids from four K. pneumoniae isolates (three ST258 and one ST234), one Escherichia coli isolate, and one Enterobacter aerogenes isolate, collected from 2003 to 2010 in New York (NY) and New Jersey (NJ) hospitals, were completely sequenced. The sequences and overall sizes of the six plasmids are highly similar to those of pKpQIL; the major difference is that five of six NJ/NY strains harbor blaKPC-2, while pKpQIL contains blaKPC-3. Moreover, a 26.7-kb fragment was inverted in pKpQIL-234 (from ST234 K. pneumoniae), while a 14.5-kb region was deleted in pKpQIL-Ec (from ST131 E. coli). PCR screening of 284 other clinical K. pneumoniae isolates identified 101 (35.6%) harboring pKpQIL-like plasmids from 9 of 10 surveyed hospitals, demonstrating the wide dissemination of pKpQIL in this region of endemicity. Among the positive isolates, 87.1% were typed as ST258 and 88.1% carried blaKPC-2. The finding of pKpQIL-like plasmid in this study from strains that predate the initial report of KPC in Israel provides evidence that pKpQIL may have originated in the United States. Our findings demonstrate that pKpQIL plasmids are both spreading clonally in ST258 strains and spreading horizontally to different sequence types and species, further highlighting the clinical and public health concerns associated with carbapenem resistance.

Multiplex real-time PCR for detection of an epidemic KPC-producing Klebsiella pneumoniae ST258 clone.

We describe a multiplex real-time PCR assay capable of identifying both the epidemic Klebsiella pneumoniae ST258 clone and bla(KPC) carbapenemase genes in a single reaction. The assay displayed excellent sensitivity (100%) and specificity (100%) for identification of ST258 clone and bla(KPC) in a collection of 75 K. pneumoniae isolates comprising 41 sequence types. Our results suggest that this assay is an effective tool for surveillance of this clone among carbapenem-resistant K. pneumoniae clinical isolates.

Successful treatment of daptomycin-nonsusceptible methicillin-resistant Staphylococcus aureus bacteremia with the addition of rifampin to daptomycin.

OBJECTIVE: To report a case in which daptomycin-nonsusceptible methicillin-resistant Staphylococcus aureus (MRSA) bacteremia was successfully treated with the addition of rifampin to daptomycin. CASE SUMMARY: An 84-year-old male presented with fever and chills following cystoscopy. After culturing was conducted, the patient received single doses of vancomycin and gentamicin and then continued on vancomycin plus ceftazidime. Blood cultures grew MRSA, with vancomycin and daptomycin minimum inhibitory concentrations (MICs) of < or =1 microg/mL and 0.25 microg/mL, respectively. Vancomycin was continued, with trough concentrations maintained >15 microg/mL, but results of blood cultures remained positive. On day 10, therapy was switched to daptomycin 6 mg/kg/day, but culture results remained positive. On day 13, testing for vancomycin heteroresistance was negative, with the MIC unchanged. The vancomycin MIC remained unchanged on day 19, but the daptomycin MIC had increased to 2 microg/mL. Rifampin 300 mg orally twice daily was added on day 20; blood cultures obtained 2 days later were sterile. The patient was discharged to complete a 6-week course of antibiotics and was doing well 4 months following therapy. DISCUSSION: Analysis of MRSA isolates obtained on days 1 and 19 showed an increase in the daptomycin MIC from 0.25 to 2 microg/mL. Because intervening isolates were not available for susceptibility testing, it is not possible to associate this increase with exposure to either vancomycin or daptomycin. Although in vitro synergy was not seen in this case, addition of rifampin to daptomycin therapy resolved the bacteremia. CONCLUSIONS: In patients with persistent MRSA bacteremia, isolates should be retested for susceptibility to both daptomycin and vancomycin, including assessment for vancomycin heteroresistance. Addition of rifampin to daptomycin may be effective for persistent MRSA bacteremia, even if daptomycin MICs are elevated. Prospective studies are needed to define the role of combination therapy.

Efficient detection of symptomatic and asymptomatic patient samples for Babesia microti and Borrelia burgdorferi infection by multiplex qPCR.

BACKGROUND: Tick-borne infections have been increasing steadily over the years, with co-infections with Borrelia burgdorferi and Babesia microti/divergens emerging as a serious health problem. B. burgdorferi is a spirochetal bacterium that causes Lyme disease while protozoan pathogens belonging to Babesia species are responsible for babesiosis. Currently used serological tests do not always detect acute Lyme disease or babesiosis, and fail to differentiate cured patients from those who get re-infected. This is a major problem for proper diagnosis particularly in regions endemic for tick-borne diseases. Microscopy based evaluation of babesiosis is confirmatory but is labor intensive and insensitive such that many asymptomatic patients remain undetected and donate blood resulting in transfusion transmitted babesiosis. RESULTS: We conducted multiplex qPCR for simultaneous diagnosis of active Lyme disease and babesiosis in 192 blood samples collected from a region endemic for both diseases. We document qPCR results obtained from testing of each sample three times to detect infection with each pathogen separately or together. Results for Lyme disease by qPCR were also compared with serological tests currently used for Lyme disease when available. Considering at least two out of three test results for consistency, 18.2% of patients tested positive for Lyme disease, 18.7% for co-infection with B. burgdorferi and B. microti and 6.3% showed only babesiosis. CONCLUSIONS: With an 80% sensitivity for detection of Lyme disease, and ability to detect co-infection with B. microti, multiplex qPCR can be employed for diagnosis of these diseases to start appropriate treatment in a timely manner.

Finegoldia magna (formerly Peptostreptococcus magnus): an overlooked etiology for toxic shock syndrome?

Finegoldia magna is an anaerobic Gram positive coccus, previously classified as Peptostreoptococcus magnus. It is normal flora of the gastrointestinal and genitourinary tract, and can be isolated from skin and the oral cavity and is often regarded as a contaminant in cultures. As the most frequently isolated anaerobic coccus, it is implicated in a range of mono- and polymicrobial infections, including skin and skin structure, bone and joint (native and prosthetic joints), infective endocarditis (native and prosthetic valves), necrotizing pneumonia, mediastinitis and meningitis. Recently, whole genome sequencing furthered the understanding of the pathogenicity of this organism by elucidating both chromosomally encoded and mobile plasmid mediated virulence factors. Although no cases of toxic shock syndrome have been attributed to F. magna, we present a case of a fatal monomicrobial F. magna bacteremia and hypothesize that superantigen activity, mediated via Protein L binding the variable domain of the κ light chains of IgG, resulted in the syndrome observed in our patient. Additionally, we suspect the overall significance of this pathogen is underestimated and with more sensitive detection methods, this organism will be identified more frequently in clinical cultures and associated with true infection.