Carbapenemase-producing Enterobacterales in hospital drains in Southern Ontario, Canada.

BACKGROUND: Hospital drains may be an important reservoir for carbapenemase-producing Enterobacterales (CPE). AIM: To determine prevalence of CPE in hospital drains exposed to inpatients with CPE, relatedness of drain and patient CPE, and risk factors for drain contamination. METHODS: Sink and shower drains in patient rooms and communal shower rooms exposed to 310 inpatients with CPE colonization/infection were cultured at 10 hospitals. Using short- and long-read whole-genome sequencing, inpatient and corresponding drain CPE were compared. Risk factors for drain contamination were assessed using multi-level modelling. FINDINGS: Of 1209 exposed patient room and communal shower room drains, 53 (4%) yielded 62 CPE isolates in seven (70%) hospitals. Of 49 CPE isolates in patient room drains, four (8%) were linked to prior room occupants. Linked drain/room occupant pairs included Citrobacter freundii ST18 isolates separated by eight single nucleotide variants (SNVs), related blaKPC-containing IncN3-type plasmids (different species), related blaKPC-3-containing IncN-type plasmids (different species), and related blaOXA-48-containing IncL/M-type plasmids (different species). In one hospital, drain isolates from eight rooms on two units were Enterobacter hormaechei separated by 0-6 SNVs. Shower drains were more likely to be CPE-contaminated than hand hygiene (odds ratio: 3.45; 95% confidence interval: 1.66-7.16) or patient-use (13.0; 4.29-39.1) sink drains. Hand hygiene sink drains were more likely to be CPE-contaminated than patient-use sink drains (3.75; 1.17-12.0). CONCLUSION: Drain contamination was uncommon but widely dispersed. Drain CPE unrelated to patient exposure suggests contamination by undetected colonized patients or retrograde (drain-to-drain) contamination. Drain types had different contamination risks.

Multi-modal optimization of bismuth vanadate photoanodes via combinatorial alloying and hydrogen processing.

Alloying transition metals, such as Mo, into BiVO4 has emerged as the primary mechanism for improving carrier transport in this photoanode for solar fuels production. The present work establishes the generality of improving photoelectrochemical performance through co-alloying with a transition metal electron donor and a structure-modulating rare earth. Further improvement for all such alloys is obtained by annealing the oxide materials in H2, ultimately producing photoanodes with above 3 mA cm-2 photocurrent density under AM 1.5G illumination, in the top tier of compact BiVO4 films.

ORMOCHALCs: organically modified chalcogenide polymers for infrared optics.

A novel method combining elemental sulfur and selenium was developed, yielding crystalline sulfur-selenium compounds. The compounds were melted, and an organic comonomer added. Once the organic comonomer was consumed, the viscous compound was vitrified and allowed to cool yielding organic-inorganic hybrid polymers that are termed Organically Modified Chalcogenide (ORMOCHALC) polymers.

Single-step deposition of high-mobility graphene at reduced temperatures.

Current methods of chemical vapour deposition (CVD) of graphene on copper are complicated by multiple processing steps and by high temperatures required in both preparing the copper and inducing subsequent film growth. Here we demonstrate a plasma-enhanced CVD chemistry that enables the entire process to take place in a single step, at reduced temperatures (<420 °C), and in a matter of minutes. Growth on copper foils is found to nucleate from arrays of well-aligned domains, and the ensuing films possess sub-nanometre smoothness, excellent crystalline quality, low strain, few defects and room-temperature electrical mobility up to (6.0±1.0) × 10(4) cm(2) V(-1) s(-1), better than that of large, single-crystalline graphene derived from thermal CVD growth. These results indicate that elevated temperatures and crystalline substrates are not necessary for synthesizing high-quality graphene.

Serratia marcescens harbouring SME-type class A carbapenemases in Canada and the presence of blaSME on a novel genomic island, SmarGI1-1.

OBJECTIVES: An increasing prevalence since 2010 of Serratia marcescens harbouring the Ambler class A carbapenemase SME prompted us to further characterize these isolates. METHODS: Isolates harbouring bla(SME) were identified by PCR and sequencing. Phenotypic analysis for carbapenemase activity was carried out by a modified Hodge test and a modified Carba NP test. Antimicrobial susceptibilities were determined by Etest and Vitek 2. Typing was by PFGE of macrorestriction digests. Whole-genome sequencing of three isolates was carried out to characterize the genomic region harbouring the bla(SME)-type genes. RESULTS: All S. marcescens harbouring SME-type enzymes could be detected using a modified Carba NP test. Isolates harbouring bla(SME) were resistant to penicillins and carbapenems, but remained susceptible to third-generation cephalosporins, as well as fluoroquinolones and trimethoprim/sulfamethoxazole. Isolates exhibited diverse genetic backgrounds, though 57% of isolates were found in three clusters. Analysis of whole-genome sequence data from three isolates revealed that the bla(SME) gene occurred in a novel cryptic prophage genomic island, SmarGI1-1. CONCLUSIONS: There has been an increasing occurrence of S. marcescens harbouring bla(SME) in Canada since 2010. The bla(SME) gene was found on a genomic island, SmarGI1-1, that can be excised and circularized, which probably contributes to its dissemination amongst S. marcescens.

Complete sequences of a novel blaNDM-1-harbouring plasmid from Providencia rettgeri and an FII-type plasmid from Klebsiella pneumoniae identified in Canada.

OBJECTIVES: Emergence of plasmids harbouring bla(NDM-1) is a major public health concern due to their association with multidrug resistance and their potential mobility. METHODS: PCR was used to detect bla(NDM-1) from clinical isolates of Providencia rettgeri (PR) and Klebsiella pneumoniae (KP). Antimicrobial susceptibilities were determined using Vitek 2. The complete DNA sequence of two bla(NDM-1) plasmids (pPrY2001 and pKp11-42) was obtained using a 454-Genome Sequencer FLX. Contig assembly and gap closures were confirmed by PCR-based sequencing. Comparative analysis was done using BLASTn and BLASTp algorithms. RESULTS: Both clinical isolates were resistant to all ß-lactams, carbapenems, aminoglycosides, ciprofloxacin and trimethoprim/sulfamethoxazole, and susceptible to tigecycline. Plasmid pPrY2001 (113 295 bp) was isolated from PR. It did not show significant homology to any known plasmid backbone and contained a truncated repA and novel repB. Two bla(NDM-1)-harbouring plasmids from Acinetobacter lwoffii (JQ001791 and JQ060896) shared 100% similarity to a 15 kb region that contained bla(NDM-1). pPrY2001 also contained a type II toxin/antitoxin system. pKp11-42 (146 695 bp) was isolated from KP. It contained multiple repA genes. The plasmid backbone had the highest homology to the IncFIIk plasmid type (51% coverage, 100% nucleotide identity). The bla(NDM-1) region was unique in that it was flanked upstream by IS3000 and downstream by a novel transposon designated Tn6229. pKp11-42 also contained a number of mutagenesis and plasmid stability proteins. CONCLUSIONS: pPrY2001 differed from all known plasmids due to its novel backbone and repB. pKp11-42 was similar to IncFIIk plasmids and contained a number of genes that aid in plasmid persistence.

The VanE operon in Enterococcus faecalis N00-410 is found on a putative integrative and conjugative element, Tn6202.

OBJECTIVES: To date no complete genetic structure of acquired DNA harbouring a d-Ala-d-Ser operon in an Enterococcus is known. We wished to characterize the acquired DNA harbouring the vanE operon located in the Enterococcus faecalis N00-410 chromosome. METHODS: Whole genome sequencing of E. faecalis N00-410 was conducted by massively parallel sequencing. Two sequence contigs harbouring the vanE region were linked by PCR and the acquired DNA harbouring the vanE operon was completely characterized. Excision/integration of the region was determined by PCR and transfer attempted by conjugation. RESULTS: The regions flanking the vanE operon were analysed and a total of 42 open reading frames were identified in a region flanked by inverted terminal and direct repeats (Tn6202). Tn6202 could be excised from the chromosome, circularized and the target site rejoined, but transfer could not be demonstrated. CONCLUSIONS: The vanE operon was found on the putative integrative and conjugative element Tn6202 in the E. faecalis N00-410 chromosome. This represents the first characterization of acquired DNA harbouring a D-Ala-D-Ser operon.

Carbapenem-resistant Gram-negative bacilli in Canada 2009-10: results from the Canadian Nosocomial Infection Surveillance Program (CNISP).

OBJECTIVES: To investigate the occurrence and molecular mechanisms associated with carbapenemases in carbapenem-resistant Gram-negative isolates from Canadian cases. METHODS: Twenty hospital sites across Canada submitted isolates for a 1 year period starting 1 September 2009. All Enterobacteriaceae with MICs ≥ 2 mg/L and Acinetobacter baumannii and Pseudomonas aeruginosa with MICs ≥ 16 mg/L of carbapenems were submitted to the National Microbiology Laboratory (NML) where carbapenem MICs were confirmed by Etest and isolates were characterized by PCR for carbapenemase genes, antimicrobial susceptibilities, PFGE and plasmid isolation. RESULTS: A total of 444 isolates (298 P. aeruginosa, 134 Enterobacteriaceae and 12 A. baumannii) were submitted to the NML of which 274 (61.7%; 206 P. aeruginosa, 59 Enterobacteriaceae and 9 A. baumannii) met the inclusion criteria as determined by Etest. Carbapenemase genes were identified in 30 isolates: bla(GES-5) (n = 3; P. aeruginosa), bla(KPC-3) (n = 7; Enterobacteriaceae), bla(NDM-1) (n = 2; Enterobacteriaceae), bla(VIM-2) and bla(VIM-4) (n = 8; P. aeruginosa) bla(SME-2) (n = 1; Enterobacteriaceae) and bla(OXA-23) (n = m9; A. baumannii). PFGE identified a cluster in each of Enterobacteriaceae, P. aeruginosa and A. baumannii corresponding to isolates harbouring carbapenemase genes. Three KPC plasmid patterns (IncN and FllA) were identified where indistinguishable plasmid patterns were identified in unrelated clinical isolates. CONCLUSIONS: Carbapenemases were rare at the time of this study. Dissemination of carbapenemases was due to both dominant clones and common plasmid backbones.

Plasmid comparison and molecular analysis of Klebsiella pneumoniae harbouring bla(KPC) from New York City and Toronto.

OBJECTIVES: This study examined Klebsiella pneumoniae clinical isolates and their bla(KPC) plasmids to determine potential relatedness of the isolates and their plasmids harbouring carbapenem resistance mechanisms. METHODS: K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae from New York City (NYC) (n = 19) and Toronto (n = 2) were typed by PFGE and multilocus sequence typing (MLST). bla(KPC)-harbouring plasmids were transformed into Escherichia coli DH10B(TM), restricted using EcoRI and analysed for bla content and replicon (rep) type. Susceptibility profiles for clinical and transformed strains were determined by automated microbroth dilution using CLSI breakpoints. Outer membrane protein (OMP) genes were analysed by sequencing of ompk35 and ompk36. RESULTS: PFGE analysis identified 17 related strains (≥ 80% similarity; 11 KPC-2, 6 KPC-3) where ST258 was the dominant clonal type. All clinical isolates contained both bla(SHV) and bla(TEM-1) and, with the exception of one isolate, were multidrug resistant (MDR). Transformed KPC plasmids (n = 21) carried TEM-1 (n = 18) and were MDR (n = 5). Three plasmid clusters, repFIIA (n = 10), repR (n = 3) and an unknown type (n = 3), were observed. repFllA plasmids were observed from both NYC and Toronto strains. OMP gene analysis revealed premature stop codons in ompk35 and numerous deletions and insertions in ompk36. CONCLUSIONS: The dissemination of bla(KPC) is due both to carriage of similar KPC-harbouring plasmids within genetically distinct K. pneumoniae and to clonal spread of K. pneumoniae with unrelated KPC plasmids.

Characterization of HLA-A2-restricted HPV-16 E7-specific CD8(+) T-cell immune responses induced by DNA vaccines in HLA-A2 transgenic mice.

We have recently demonstrated that linkage of DNA-encoding calreticulin to DNA-encoding human papillomavirus-16 E7 antigen strongly enhances the efficacy of DNA vaccines against E7-expressing tumors in animal models. In this study, as a prelude to clinical translation, we characterized the ability of DNA-encoding calreticulin linked to DNA-encoding E7 antigen to generate HLA-A2-restricted E7-specific CD8(+) T-cell responses in HLA-A2 (AAD) transgenic mice, as well as antitumor effects against an E7(+) HLA-A2(+) tumor cell line, TC-1/A2. Our results show that while vaccination with CRT/E7 DNA generates strong H-2D(b)-restricted E7 (amino acid (aa)49-57)-specific CD8(+) T-cell immune responses in both C57BL/6 and HLA-A2 (AAD) transgenic mice, no such responses were generated to HLA-A2-restricted epitopes in either type of mouse. In contrast, vaccination with DNA-encoding calreticulin linked to DNA encoding a mutant version of E7 with a deleted aa49-57 epitope leads to the generation of an HLA-A2-restricted E7 (aa11-20)-specific CTL response in HLA-A2 (AAD) transgenic mice. More importantly, vaccination with CRT/mtE7 (del aa49-57) DNA protects against a lethal challenge with TC-1/A2 tumor cells in HLA-A2 (AAD) transgenic mice. Furthermore, our in vitro studies demonstrate that the presence of the E7 (aa49-57) epitope does not suppress presentation of the HLA-A2-restricted E7 (aa11-20) epitope through MHC class I molecules. Thus, the predominant E7 aa49-57-specific CD8+ T-cell immune response in HLA-A2 transgenic mice vaccinated with CRT/E7 is likely due to preferred expansion of E7 aa49-57-specific CD8(+) T cells in vaccinated mice. These results highlight the importance of epitope immunodominance in the evaluation of immune responses in HLA-A2 (AAD) transgenic mice.

Cancer immunotherapy using a DNA vaccine encoding a single-chain trimer of MHC class I linked to an HPV-16 E6 immunodominant CTL epitope.

The potency of DNA vaccines may be affected by the efficiency of intracellular processing and MHC class I presentation of encoded antigens. Since a single-chain trimer (SCT) composed of peptide, beta2-microglobulin (beta2m), and MHC class I heavy chain has been shown to bypass antigen processing and lead to stable presentation of peptides, we investigated the efficacy of a DNA vaccine encoding a SCT composed of an immunodominant CTL epitope of human papillomavirus type 16 (HPV-16) E6 antigen, beta2m, and H-2Kb MHC class I heavy chain (pIRES-E6-beta2m-Kb). Transfection of 293 cells with pIRES-E6-beta2m-Kb can bypass antigen processing and lead to stable presentation of E6 peptide. Furthermore, C57BL/6 mice vaccinated with pIRES-E6-beta2m-Kb exhibited significantly increased E6 peptide-specific CD8+ T-cell immune responses compared to mice vaccinated with DNA encoding wild-type E6. Most importantly, 100% of mice vaccinated with pIRES-E6-beta2m-Kb DNA were protected against a lethal challenge of E6-expressing TC-1 tumor cells. In contrast, all mice vaccinated with wild-type E6 DNA or control plasmid DNA grew tumors. Our data indicate that a DNA vaccine encoding a SCT can lead to stable enhanced MHC class I presentation of encoded antigenic peptide and may be useful for improving DNA vaccine potency to control tumors or infectious diseases.

Comparison of HPV DNA vaccines employing intracellular targeting strategies.

Intradermal vaccination via gene gun efficiently delivers DNA vaccines into dendritic cells (DCs) of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. In the context of DNA vaccines, we previously used the gene gun approach to test several intracellular targeting strategies that are able to route a model antigen, such as the human papillomavirus type-16 (HPV-16) E7, to desired subcellular compartments in order to enhance antigen processing and presentation to T cells. These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat-shock protein 70 (HSP70), calreticulin (CRT) and the translocation domain (dII) of Pseudomonas aeruginosa exotoxin A (ETA). Vaccination with DNA vaccines encoding E7 antigen linked to any of these molecules all led to a significant enhancement of E7-specific CD8(+) T-cell immune responses and strong antitumor effects against an E7-expressing tumor, TC-1. However, we were interested in identifying the most potent DNA vaccine for our future clinical trials. Thus, we performed a series of experiments to directly compare the potency of the various DNA vaccines. Among the DNA vaccines we tested, we found that vaccination with pcDNA3-CRT/E7 generated the highest number of E7-specific CD8(+) T cells and potent long-term protection and treatment effects against E7-expressing tumors in mice. Interestingly, we observed that pcDNA3-CRT/E7 is also capable of protecting against an E7-expressing tumor with downregulated MHC class I expression, a common feature associated with most HPV-associated cervical cancers. Our data suggest that the DNA vaccine linking CRT to E7 (CRT/E7) may be a suitable candidate for human trials for the control of HPV infections and HPV-associated lesions.

Molecular characterization of the vanE gene cluster in vancomycin-resistant Enterococcus faecalis N00-410 isolated in Canada.

The vanE operon was characterized from Enterococcus faecalis N00-410 (MIC of vancomycin = 24 microg/ml). The organization of the vanE operon was identical to that of the vanC1 operon from Enterococcus gallinarum, with protein identities ranging from 46 to 63%. An open reading frame located downstream of the vanE operon showed significant homology to a number of integrase genes, all of which are located downstream of the chromosomal GMP synthase gene guaA.

Defects in D-alanyl-lipoteichoic acid synthesis in Streptococcus mutans results in acid sensitivity.

In the cariogenic organism, Streptococcus mutans, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:ISS1 was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in dltB of the dlt operon encoding four proteins (DltA-DltD) involved in the synthesis of D-alanyl-lipoteichoic acid. Inactivation of the dltC gene, encoding the D-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a similar average number of glycerolphosphate residues, permeabilized cells of BH97LC did not incorporate D-[(14)C]alanine into this amphiphile. This defect was correlated with the deficiency of Dcp. Chemical analysis of the LTA purified from the mutant confirmed the absence of D-alanine-esters. Electron micrographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild-type cells. Proton permeability assays revealed that the mutant was more permeable to protons than the wild type. This observation suggests a mechanism for the loss of the characteristic acid tolerance response in S. mutans.

Partial characterization of a genomic island associated with the multidrug resistance region of Salmonella enterica Typhymurium DT104.

This study describes the identification of the insertion site and partial characterization of a 43-kb region harboring the genes associated with the penta-resistant phenotype of a Canadian isolate of Salmonella enterica Typhymurium DT104 labelled 96-5227. The 43-kb fragment, here referred to as Salmonella genomic island I (SgiI), was found in the genome of S. enterica Typhymurium between the thdf and a prophage CP-4-like integrase (int2) gene and is flanked by an imperfect 18-bp direct repeat. A region downstream of sulI in the right end of SgiI contained four open reading frames which includes an IS6100 element, and a 2-kb region from the left end contained two open reading frames which showed homology to an integrase and an excisionase. Furthermore, a 1.9-kb retron sequence located between int2 and yidY was identified which may be unique to the S. enterica Typhymurium genome. The int-retron sequence is flanked by a 27-bp imperfect direct repeat.

Molecular characterization of the vanD gene cluster and a novel insertion element in a vancomycin-resistant enterococcus isolated in Canada.

A single vanD-containing Enterococcus faecium strain (N97-330) was isolated in Canada. The vanD-containing region was cloned and sequenced. Although the proteins have more than 96% identity to a previously described vanD region in BM4339, the vanS(D) gene contains a frameshift mutation that leads to a predicted truncated protein. Furthermore, sequence analysis of the ddl gene revealed the presence of an IS982-like element (ISEfm1) which interrupted the D-Ala-D-Ala ligase. This suggested the constitutive expression of the vanD operon, which was confirmed. Pulsed-field gel electrophoresis fingerprinting demonstrated that BM4339 was not related to N97-330 (>15 band differences). Both strains contained multiple copies of the IS982-like element.

Identification of the operon for the sorbitol (Glucitol) Phosphoenolpyruvate:Sugar phosphotransferase system in Streptococcus mutans.

Transposon mutagenesis and marker rescue were used to isolate and identify an 8.5-kb contiguous region containing six open reading frames constituting the operon for the sorbitol P-enolpyruvate phosphotransferase transport system (PTS) of Streptococcus mutans LT11. The first gene, srlD, codes for sorbitol-6-phosphate dehydrogenase, followed downstream by srlR, coding for a transcriptional regulator; srlM, coding for a putative activator; and the srlA, srlE, and srlB genes, coding for the EIIC, EIIBC, and EIIA components of the sorbitol PTS, respectively. Among all sorbitol PTS operons characterized to date, the srlD gene is found after the genes coding for the EII components; thus, the location of the gene in S. mutans is unique. The SrlR protein is similar to several transcriptional regulators found in Bacillus spp. that contain PTS regulator domains (J. Stülke, M. Arnaud, G. Rapoport, and I. Martin-Verstraete, Mol. Microbiol. 28:865-874, 1998), and its gene overlaps the srlM gene by 1 bp. The arrangement of these two regulatory genes is unique, having not been reported for other bacteria.

Regulation of sugar transport via the multiple sugar metabolism operon of Streptococcus mutans by the phosphoenolpyruvate phosphotransferase system.

In this report, we provide evidence that the transport of sugars in Streptococcus mutans via the multiple sugar metabolism system is regulated by the phosphoenolpyruvate phosphotransferase system. A ptsI-defective mutant (DC10), when grown on the multiple sugar metabolism system substrate raffinose, exhibited reduced growth, transport, and glycolytic activity with raffinose relative to the parent strain BM71. Inhibition of [3H]raffinose uptake was also observed in both BM71 and DC10 with increasing concentrations of glucose and the glucose analogs alpha-methyl glucoside and 2-deoxyglucose.

Glucose transport by a mutant of Streptococcus mutans unable to accumulate sugars via the phosphoenolpyruvate phosphotransferase system.

Streptococcus mutans transports glucose via the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS). Earlier studies indicated that an alternate glucose transport system functions in this organism under conditions of high growth rates, low pH, or excess glucose. To identify this system, S. mutans BM71 was transformed with integration vector pDC-5 to generate a mutant, DC10, defective in the general PTS protein enzyme I (EI). This mutant expressed a defective EI that had been truncated by approximately 150 amino acids at the carboxyl terminus as revealed by Western blot (immunoblot) analysis with anti-EI antibody and Southern hybridizations with a fragment of the wild-type EI gene as a probe. Phosphotransfer assays utilizing 32P-PEP indicated that DC10 was incapable of phosphorylating HPr and EIIAMan, indicating a nonfunctional PTS. This was confirmed by the fact that DC10 was able to ferment glucose but not a variety of other PTS substrates and phosphorylated glucose with ATP and not PEP. Kinetic assays indicated that the non-PTS system exhibited an apparent Ks of 125 microM for glucose and a Vmax of 0.87 nmol mg (dry weight) of cells-1 min-1. Sugar competition experiments with DC10 indicated that the non-PTS transport system had high specificity for glucose since glucose transport was not significantly by a 100-fold molar excess of several competing sugar substrates, including 2-deoxyglucose and alpha-methylglucoside. These results demonstrate that S. mutans possesses a glucose transport system that can function independently of the PEP PTS.