Genetic Basis of Emerging Vancomycin, Linezolid, and Daptomycin Heteroresistance in a Case of Persistent Enterococcus faecium Bacteremia.

Whole-genome sequencing was used to examine a persistent Enterococcus faecium bacteremia that acquired heteroresistance to three antibiotics in response to prolonged multidrug therapy. A comparison of the complete genomes before and after each change revealed the emergence of known resistance determinants for vancomycin and linezolid and suggested that a novel mutation in fabF, encoding a fatty acid synthase, was responsible for daptomycin nonsusceptibility. Plasmid recombination contributed to the progressive loss of vancomycin resistance after withdrawal of the drug.

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.

An improved SELEX technique for selection of DNA aptamers binding to M-type 11 of Streptococcus pyogenes.

Streptococcus pyogenes is a clinically important pathogen consisting of various serotypes determined by different M proteins expressed on the cell surface. The M type is therefore a useful marker to monitor the spread of invasive S. pyogenes in a population. Serotyping and nucleic acid amplification/sequencing methods for the identification of M types are laborious, inconsistent, and usually confined to reference laboratories. The primary objective of this work is to develop a technique that enables generation of aptamers binding to specific M-types of S. pyogenes. We describe here an in vitro technique that directly used live bacterial cells and the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. Live S. pyogenes cells were incubated with DNA libraries consisting of 40-nucleotides randomized sequences. Those sequences that bound to the cells were separated, amplified using polymerase chain reaction (PCR), purified using gel electrophoresis, and served as the input DNA pool for the next round of SELEX selection. A specially designed forward primer containing extended polyA20/5Sp9 facilitated gel electrophoresis purification of ssDNA after PCR amplification. A counter-selection step using non-target cells was introduced to improve selectivity. DNA libraries of different starting sequence diversity (10(16) and 10(14)) were compared. Aptamer pools from each round of selection were tested for their binding to the target and non-target cells using flow cytometry. Selected aptamer pools were then cloned and sequenced. Individual aptamer sequences were screened on the basis of their binding to the 10 M-types that were used as targets. Aptamer pools obtained from SELEX rounds 5-8 showed high affinity to the target S. pyogenes cells. Tests against non-target Streptococcus bovis, Streptococcus pneumoniae, and Enterococcus species demonstrated selectivity of these aptamers for binding to S. pyogenes. Several aptamer sequences were found to bind preferentially to the M11 M-type of S. pyogenes. Estimated binding dissociation constants (Kd) were in the low nanomolar range for the M11 specific sequences; for example, sequence E-CA20 had a Kd of 7±1 nM. These affinities are comparable to those of a monoclonal antibody. The improved bacterial cell-SELEX technique is successful in generating aptamers selective for S. pyogenes and some of its M-types. These aptamers are potentially useful for detecting S. pyogenes, achieving binding profiles of the various M-types, and developing new M-typing technologies for non-specialized laboratories or point-of-care testing.

Whole-genome sequencing identifies emergence of a quinolone resistance mutation in a case of Stenotrophomonas maltophilia bacteremia.

Whole-genome sequences for Stenotrophomonas maltophilia serial isolates from a bacteremic patient before and after development of levofloxacin resistance were assembled de novo and differed by one single-nucleotide variant in smeT, a repressor for multidrug efflux operon smeDEF. Along with sequenced isolates from five contemporaneous cases, they displayed considerable diversity compared against all published complete genomes. Whole-genome sequencing and complete assembly can conclusively identify resistance mechanisms emerging in S. maltophilia strains during clinical therapy.

The Effects of SELEX Conditions on the Resultant Aptamer Pools in the Selection of Aptamers Binding to Bacterial Cells.

Aptamers of high affinity and specificity have a wide range of analytic and clinical applications. Selection of DNA or RNA aptamer molecules usually involves systematic evolution of ligands via exponential enrichment (SELEX), in which a random DNA or RNA library is incubated with a target molecule, and the oligonucleotides that bind the target are then separated from the nonbinders, PCR amplified, and used as refined libraries in the next round of selection. Conventional SELEX methodologies require the use of purified target molecules and their immobilization onto a solid support. However, purified targets from cells are not always available, and fixing the target to a support may alter its conformation. To overcome these problems, we have developed a SELEX technique using live bacterial cells in suspension as targets, for selecting DNA aptamers specific to cell-surface molecules. Through the selection of aptamers binding to Lactobacillus acidophilus and Streptococcus pyogenes, we report here optimization of this technique and show how varying selection conditions impact the characteristics of resultant aptamer pools, including the binding affinity, selectivity, and the secondary structures. We found that the use of larger starting library sequence diversity, gel purification of the subsequent pools, and the introduction of counter-selection resulted in a more efficient SELEX process and more selective aptamers. A SELEX protocol with lower starting sequence diversity, the use of heat denaturation, and the absence of counter-selection still resulted in high-affinity aptamer sequences specific to the target cell types; however, the SELEX process was inefficient, requiring 20 rounds, and the aptamers were not specific to the strain of the bacterial cells. Strikingly, two different SELEX methodologies yielded the same sequence that bound strongly to the target S. pyogenes cells, suggesting the robustness of the bacterial cell-SELEX technique.

First Report of Wohlfahrtiimonas chitiniclastica Isolation from a Patient with Cellulitis in the United States.

We report the first documented isolation of Wohlfahrtiimonas chitiniclastica from a human in the United States. Initially misidentified as Acinetobacter lwoffii by Vitek-2, the isolate was subsequently identified as W. chitiniclastica by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing. While the clinical significance of the isolate in this case is unclear, it highlights the superior performance of MALDI-TOF MS for bacterial identification.

DNA aptamers binding to multiple prevalent M-types of Streptococcus pyogenes.

This paper describes the selection of high affinity DNA aptamers binding to multiple M-types of the pathogenic species Streptococcus pyogenes (Group A Streptococcus or GAS). Unlike common aptamer selection techniques that use purified molecules of a monoclonal cell population as targets, this work has achieved the selection of aptamers against the various M-types of S. pyogenes. Cell mixtures containing equal numbers of the 10 most prevalent S. pyogenes M-types were incubated with 80-nucleotide DNA libraries, centrifuged, and washed to separate cell-bound from unbound DNA sequences. The DNA bound to the cells was amplified using the polymerase chain reaction, and the amplicons were tested for their binding to the target cells. The amplicons were also used as new DNA libraries for subsequent rounds of selection. Cloning, sequencing, and subsequent analysis of selected aptamers showed that they bind preferentially to GAS over other common and related bacteria. Resultant DNA aptamers showed strong and preferential binding to GAS, including the 10 most prevalent GAS M-types and another 10 minor M-types tested. Estimated K(d) values were in the range of 4 to 86 nM. Two aptamers, 20A24P and 15A3P (with estimated binding dissociation constants of 9 and 10 nM, respectively), are particularly promising. These aptamers could potentially be used to improve the detection of GAS, a pathogen that is the causative agent of many infectious diseases, most notably strep throat.

Selection and analytical applications of aptamers binding microbial pathogens.

DNA aptamers specifically recognizing microbial cells and viruses have a range of analytical and therapeutic applications. This article describes recent advances in the development of aptamers targeting specific pathogens (e.g., live bacteria, whole viral particles, and virally-infected mammalian cells). Specific aptamers against pathogens have been used as affinity reagents to develop sandwich assays, to label and to image cells, to bind with cells for flow-cytometry analysis, and to act as probes for development of whole-cell biosensors. Future applications of aptamers to pathogens will benefit from recent advances in improved selection and new aptamers containing modified nucleotides, particularly slow off-rate modified aptamers (SOMAmers).

Selection of aptamers against live bacterial cells.

Single-stranded DNA or RNA aptamer molecules have usually been selected against purified target molecules. To eliminate the need of purifying target molecules on the cell surface, we have developed a selection technique using live bacterial cells in suspension as targets, to select for ssDNA aptamers specific to cell surface molecules. Lactobacillus acidophilus cells were chosen to demonstrate proof of principle based on their high abundance of surface molecules (potential targets). Aptamer pools obtained after 6-8 rounds of selection demonstrated high affinity for and selective binding with L. acidophilus cells when tested via flow cytometry, microscopy, and fluorescence measurements. Out of 27 aptamers that were cloned and sequenced, one sequence, hemag1P, was found to bind to L. acidophilus much more strongly and specifically than other cells tested. This aptamer was predicted to have a tight hairpin secondary structure. On average, an estimated 164 +/- 47 aptamer molecules were bound to a target cell with an apparent K d of 13 +/- 3 nM. A likely putative molecular target of hemag1P is the S-layer protein on the cell surface.

The impact of blood culture identification by MALDI-TOF MS on the antimicrobial management of pediatric patients.

OBJECTIVE: To assess the impact of MALDI-TOF MS coupled with antimicrobial stewardship on clinical outcomes for pediatric inpatients with bloodstream infections. METHODS: Outcomes of pediatric inpatients were compared before and after MALDI-TOF MS implementation. Outcomes measured included time until organism identification and susceptibility, duration of antibiotics, patient length of stay (LOS), mortality and hospital costs. RESULTS: 210 and 135 patient events were compared pre- and post-intervention. Average time to organism identification decreased from 41 to 11 hours (P = <0.0001). Time to i) susceptibilities decreased from 50.8 to 37.7 hours (P = <0.0001), ii) de-escalation of antibiotics decreased from 58 to 23 hours (P = <0.0001), iii) discontinuation of unnecessary antibiotics decreased from 49 to 20 hours (P = <0.0001). Infection-related LOS decreased from 10.5 to 8.37 days (P = 0.006). No significant differences were seen for other outcomes. CONCLUSIONS: MALDI-TOF MS identification of bacteria from blood culture broth improves time to appropriate antibiotic treatment for pediatric inpatients.

Retrospective report of antimicrobial susceptibility observed in bacterial pathogens isolated from ocular samples at Mount Sinai Hospital, 2010 to 2015.

BACKGROUND: Antimicrobial resistance has emerged as a major threat to global public health. Thus, the surveillance of changes in antimicrobial resistance in local and global settings is a paramount necessity. While many studies have tracked antimicrobial resistance, only a small percentage surveyed ocular isolates. The purpose of this study was to report the in vitro susceptibility of bacterial pathogens isolated from ocular samples in New York, NY from 2010 to 2015. METHODS: A retrospective review of ocular isolates was conducted. All organisms were collected by 25 separate inpatient wards and outpatient clinics, and were analyzed by the clinical microbiology laboratory at Mount Sinai Hospital. Clinical Laboratory and Standards Institute (CLSI) guidelines were followed for susceptibility testing and breakpoint interpretations. RESULTS: A total of 549 bacterial organisms were isolated from 1664 cultures (33%) during the 6-year study period. Of these, 358 isolates (65.2%) underwent susceptibility testing. 182 (50.8%) isolates were Gram-positive. The most common Gram-positive bacterium was Staphylococcus aureus (62.1%). Methicillin-resistance decreased in S. aureus isolates (31.3% in 2010, 14.1% in 2015) but was without significant change (p = 0.25). When analyzing all S. aureus isolates recovered during the study period, there were significantly more methicillin-resistant S. aureus (MRSA) isolates resistant to fluoroquinolones (p <0.0001), erythromycin (p <0.0001), and trimethoprim/sulfamethoxazole (TMP/SMZ; p <0.05). Overall, Streptococcus pneumoniae isolates showed reduced susceptibility to erythromycin, but were otherwise susceptible to the other antimicrobials tested. Haemophilus influenzae (26.1%) and Pseudomonas aeruginosa (23.9%) were the most common Gram-negative bacteria isolated. Resistance to ampicillin and TMP/SMZ was observed in several of the H. influenzae isolates. P. aeruginosa isolates did not show high resistance overall, however, it was noted that isolates resistant to meropenem were also resistant to other antimicrobials (p < 0.01). CONCLUSION: Overall, antimicrobial resistance was infrequent for the Gram-negative and Gram-positive bacteria analyzed. While the MRSA isolates demonstrated increased resistance to multiple antimicrobial classes, this is expected for this pathogen. Due to the continued use of broad-spectrum oral and systemic antimicrobials to treat ocular infections, findings of this study and other surveillance studies specific to ocular isolates should be used as resources in effective decision making in the treatment of ocular disease.

Report of two paediatric cases of central line infections caused by species of the genus Kocuria.

INTRODUCTION: Species of the genus Kocuria are Gram-positive cocci of the family Micrococcacceae that are ubiquitous in the environment and part of the normal skin and oral flora in humans. A paucity of cases have been reported of Kocuria as human pathogens and there are currently no evidence-based guidelines for managing these uncommon infections. CASE PRESENTATION: We present two paediatric cases of central line infections with species of the genus Kocuria that required line removal despite antimicrobial therapy. CONCLUSION: Species of the genus Kocuria are uncommon human pathogens that have rarely been reported to cause opportunistic infections in both adult and paediatric populations. The cases presented here add to the growing body of literature documenting the pathogenicity of these organisms and the possible need for line removal to achieve clinical cure in central line-associated bacteraemia caused by species of the genus Kocuria.

T2Candida Provides Rapid and Accurate Species Identification in Pediatric Cases of Candidemia.

OBJECTIVES: The goal of this study is to assess the ability of the T2Candida platform (T2 Biosystems, Lexington, MA) to accurately identify Candida species from pediatric blood specimens with low volumes. METHODS: Whole blood from 15 children with candidemia was collected immediately following blood culture draw. The amount of blood required by the system was reduced by pipetting whole blood directly onto the T2Candida cartridge. Specimens were subsequently run on the T2Dx Instrument (T2 Biosystems). RESULTS: The T2Candida panel provided the appropriate result for each specimen compared with blood culture-based species identification and correctly identified 15 positive and nine negative results in 3 to 5 hours. While the time to species identification for blood culture was not reported, the T2Candida results include species data. CONCLUSIONS: T2Candida can be used to efficiently diagnose or rule out candidemia using low-volume blood specimens from pediatric patients. This could result in improved time to appropriate antifungal therapy or reduction in unnecessary empirical antifungal therapy.

High-risk HPV detection and genotyping by APTIMA HPV using cervical samples.

High-risk human papillomavirus (HPV) detection and genotyping is critical for cervical cancer screening. Testing of 967 cervical cytology specimens in PreservCyt preservative revealed similar positivity rates for HC2 (13.8%) and APTIMA HPV (AHPV) tests (13.5%, p=0.89) and high overall agreement (94.6%, κ=0.77). A trend towards higher HPV16 positivity rates by the Cobas HPV test (23.0%, 26/113) compared to the AHPV genotyping assay (19.5%, 22/113; p=0.125) was noted. No cross-contamination was detected with AHPV in a challenge experiment.

Assays for cytokines using aptamers.

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.