Household Transmission of Carbapenemase-producing Enterobacterales in Ontario, Canada.

BACKGROUND: Data on household transmission of carbapenemase-producing Enterobacterales (CPE) remain limited. We studied risk of CPE household co-colonization and transmission in Ontario, Canada. METHODS: We enrolled CPE index cases (identified via population-based surveillance from January 2015 to October 2018) and their household contacts. At months 0, 3, 6, 9, and 12, participants provided rectal and groin swabs. Swabs were cultured for CPE until September 2017, when direct polymerase chain reaction (PCR; with culture of specimens if a carbapenemase gene was detected) replaced culture. CPE risk factor data were collected by interview and combined with isolate whole-genome sequencing to determine likelihood of household transmission. Risk factors for household contact colonization were explored using a multivariable logistic regression model with generalized estimating equations. RESULTS: Ninety-five households with 177 household contacts participated. Sixteen (9%) household contacts in 16 (17%) households were CPE-colonized. Household transmission was confirmed in 3/177 (2%) cases, probable in 2/177 (1%), possible in 9/177 (5%), and unlikely in 2/177 (1%). Household contacts were more likely to be colonized if they were the index case's spouse (odds ratio [OR], 6.17; 95% confidence interval [CI], 1.05-36.35), if their index case remained CPE-colonized at household enrollment (OR, 7.00; 95% CI, 1.92-25.49), or if they had at least 1 set of specimens processed after direct PCR was introduced (OR, 6.46; 95% CI, 1.52-27.40). CONCLUSIONS: Nine percent of household contacts were CPE-colonized; 3% were a result of household transmission. Hospitals may consider admission screening for patients known to have CPE-colonized household contacts.

Enterobacter cloacae Complex Isolates Harboring blaNMC-A or blaIMI-Type Class A Carbapenemase Genes on Novel Chromosomal Integrative Elements and Plasmids.

Carbapenem-resistant Enterobacter cloacae complex isolates submitted to a reference laboratory from 2010 to 2015 were screened by PCR for seven common carbapenemase gene groups, namely, KPC, NDM, OXA-48, VIM, IMP, GES, and NMC-A/IMI. Nineteen of the submitted isolates (1.7%) were found to harbor Ambler class A blaNMC-A or blaIMI-type carbapenemases. All 19 isolates were resistant to at least one carbapenem but susceptible to aminoglycosides, trimethoprim-sulfamethoxazole, tigecycline, and ciprofloxacin. Most isolates (17/19) gave positive results with the Carba-NP test for phenotypic carbapenemase detection. Isolates were genetically diverse by pulsed-field gel electrophoresis macrorestriction analysis, multilocus sequence typing, and hsp60 gene analysis. The genes were found in various Enterobacter cloacae complex species; however, blaNMC-A was highly associated with Enterobacter ludwigii Whole-genome sequencing and bioinformatics analysis revealed that all NMC-A (n = 10), IMI-1 (n = 5), and IMI-9 (n = 2) producers harbored the carbapenemase gene on EludIMEX-1-like integrative mobile elements (EcloIMEXs) located in the identical chromosomal locus. Two novel genes, blaIMI-5 and blaIMI-6, were harbored on different IncFII-type plasmids. Enterobacter cloacae complex isolates harboring blaNMC-A/IMI-type carbapenemases are relatively rare in Canada. Though mostly found integrated into the chromosome, some variants are located on plasmids that may enhance their mobility potential.

Vancomycin-variable Enterococcus faecium: in vivo emergence of vancomycin resistance in a vancomycin-susceptible isolate.

We report the emergence of vancomycin resistance in a patient colonized with a vanA-containing, vanRS-negative isolate of Enterococcus faecium which was initially vancomycin susceptible. This is a previously undescribed mechanism of drug resistance with diagnostic and therapeutic implications.

Distinct signatures of diversifying selection revealed by genome analysis of respiratory tract and invasive bacterial populations.

Many pathogens colonize different anatomical sites, but the selective pressures contributing to survival in the diverse niches are poorly understood. Group A Streptococcus (GAS) is a human-adapted bacterium that causes a range of infections. Much effort has been expended to dissect the molecular basis of invasive (sterile-site) infections, but little is known about the genomes of strains causing pharyngitis (streptococcal "sore throat"). Additionally, there is essentially nothing known about the genetic relationships between populations of invasive and pharyngitis strains. In particular, it is unclear if invasive strains represent a distinct genetic subpopulation of strains that cause pharyngitis. We compared the genomes of 86 serotype M3 GAS pharyngitis strains with those of 215 invasive M3 strains from the same geographical location. The pharyngitis and invasive groups were highly related to each other and had virtually identical phylogenetic structures, indicating they belong to the same genetic pool. Despite the overall high degree of genetic similarity, we discovered that strains from different host environments (i.e., throat, normally sterile sites) have distinct patterns of diversifying selection at the nucleotide level. In particular, the pattern of polymorphisms in the hyaluronic acid capsule synthesis operon was especially different between the two strain populations. This finding was mirrored by data obtained from full-genome analysis of strains sequentially cultured from nonhuman primates. Our results answer the long-standing question of the genetic relationship between GAS pharyngitis and invasive strains. The data provide previously undescribed information about the evolutionary history of pathogenic microbes that cause disease in different anatomical sites.

Molecular complexity of successive bacterial epidemics deconvoluted by comparative pathogenomics.

Understanding the fine-structure molecular architecture of bacterial epidemics has been a long-sought goal of infectious disease research. We used short-read-length DNA sequencing coupled with mass spectroscopy analysis of SNPs to study the molecular pathogenomics of three successive epidemics of invasive infections involving 344 serotype M3 group A Streptococcus in Ontario, Canada. Sequencing the genome of 95 strains from the three epidemics, coupled with analysis of 280 biallelic SNPs in all 344 strains, revealed an unexpectedly complex population structure composed of a dynamic mixture of distinct clonally related complexes. We discovered that each epidemic is dominated by micro- and macrobursts of multiple emergent clones, some with distinct strain genotype-patient phenotype relationships. On average, strains were differentiated from one another by only 49 SNPs and 11 insertion-deletion events (indels) in the core genome. Ten percent of SNPs are strain specific; that is, each strain has a unique genome sequence. We identified nonrandom temporal-spatial patterns of strain distribution within and between the epidemic peaks. The extensive full-genome data permitted us to identify genes with significantly increased rates of nonsynonymous (amino acid-altering) nucleotide polymorphisms, thereby providing clues about selective forces operative in the host. Comparative expression microarray analysis revealed that closely related strains differentiated by seemingly modest genetic changes can have significantly divergent transcriptomes. We conclude that enhanced understanding of bacterial epidemics requires a deep-sequencing, geographically centric, comparative pathogenomics strategy.

Group A Streptococcus emm gene types in pharyngeal isolates, Ontario, Canada, 2002-2010.

Group A Streptococcus (GAS) is a human-adapted pathogen that causes a variety of diseases, including pharyngitis and invasive infections. GAS strains are categorized by variation in the nucleotide sequence of the gene (emm) that encodes the M protein. To identify the emm types of GAS strains causing pharyngitis in Ontario, Canada, we sequenced the hypervariable region of the emm gene in 4,635 pharyngeal GAS isolates collected during 2002-2010. The most prevalent emm types varied little from year to year. In contrast, fine-scale geographic analysis identified inter-site variability in the most common emm types. Additionally, we observed fluctuations in yearly frequency of emm3 strains from pharyngitis patients that coincided with peaks of emm3 invasive infections. We also discovered a striking increase in frequency of emm89 strains among isolates from patients with pharyngitis and invasive disease. These findings about the epidemiology of GAS are potentially useful for vaccine research.

New Delhi metallo-ß-lactamase-1: local acquisition in Ontario, Canada, and challenges in detection.

New Delhi metallo-ß-lactamase-1 (NDM-1) is a recently identified metallo-ß-lactamase that confers resistance to carbapenems and all other ß-lactam antibiotics, with the exception of aztreonam. NDM-1 is also associated with resistance to many other classes of antibiotics. The enzyme was first identified in organisms isolated from a patient in Sweden who had previously received medical treatment in India, but it is now recognized as endemic throughout India and Pakistan and has spread worldwide. The gene encoding NDM-1 has been found predominantly in Escherichia coli and Klebsiella pneumoniae. We describe the isolation NDM-1-producing organisms from two patients in Toronto, Ontario. To the best of our knowledge, this is the first report of an organism producing NDM-1 that was locally acquired in Canada. We also discuss the evidence that NDM-1 can affect bacterial species other than E. coli and K. pneumoniae, the limited options for treatment and the difficulty laboratories face in detecting organisms that produce NDM-1.

Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses.

Emerging infectious diseases have caused a global effort for development of fast and accurate detection techniques. The rapidly mutating nature of viruses presents a major difficulty, highlighting the need for specific detection of genetically diverse strains. One such infectious agent is SARS-associated coronavirus (SARS-CoV), which emerged in 2003. This study aimed to develop a real-time RT-PCR detection assay specific for SARS-CoV, taking into account its intrinsic polymorphic nature due to genetic drift and recombination and the possibility of continuous and multiple introductions of genetically non-identical strains into the human population, by using mismatch-tolerant molecular beacons designed to specifically detect the SARS-CoV S, E, M and N genes. These were applied in simple, reproducible duplex and multiplex real-time PCR assays on 25 post-mortem samples and constructed RNA controls, and they demonstrated high target detection ability and specificity. This assay can readily be adapted for detection of other emerging and rapidly mutating pathogens.

Multiplex real-time PCR for rapid Staphylococcal cassette chromosome mec typing.

Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets (mec class, ccr) for each SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the DeltamecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the DeltamecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital- and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCCmec typing of MRSA isolates.

Molecular characterization of Enterococcus faecalis N06-0364 with low-level vancomycin resistance harboring a novel D-Ala-D-Ser gene cluster, vanL.

Enterococcus faecalis N06-0364, exhibiting a vancomycin MIC of 8 microg/ml, was found to harbor a novel D-Ala-D-Ser gene cluster, designated vanL. The vanL gene cluster was similar in organization to the vanC operon, but the VanT serine racemase was encoded by two separate genes, vanTm(L) (membrane binding) and vanTr(L) (racemase).

Prevalence of vancomycin-variable Enterococcus faecium (VVE) among vanA-positive sterile site isolates and patient factors associated with VVE bacteremia.

Vancomycin-variable enterococci (VVE) are vanA-positive, vancomycin-susceptible enterococci with the ability to revert to a vancomycin-resistant phenotype on exposure to vancomycin. We sought to assess the prevalence of VVE and to determine clinical characteristics of patients infected with VVE. We prospectively collected Enterococcus faecium sterile site isolates from Toronto Invasive Bacterial Diseases Network hospitals from January 2015 to June 2016 and calculated VVE (defined as vanA-positive, vancomycin-susceptible isolates) prevalence among vanA-containing isolates. We performed chart reviews of VVE and vancomycin-resistant E. faecium (VRE) bacteremias identified from January 2012 to June 2016, and on a random sample of patients with bacteremia due to vanA/vanB-negative, vancomycin-susceptible enterococci (VSE) from January 2015 to June 2016. Clinical characteristics were compared and factors associated with mortality assessed. Because of the potential reversion from VVE to VRE, pulsed-field gel electrophoresis (PFGE) was performed for strains causing breakthrough bacteremia in order to identify relatedness among strains with different phenotypic resistance within the same patient. VVE comprised 47% (18/38) of vanA-positive isolates. The charts of 36 VRE, 25 VVE, and 79 VSE patients were reviewed. Central venous catheter associated bacteremia was more common in VVE (44%) and VRE patients (57%) than in VSE patients (28%) (P = 0.01). The Pitt bacteremia (OR 1.3, P = 0.002) and the Charlson score (OR 1.2, P = 0.008) were the only independent mortality predictors. PFGE of strains causing breakthrough bacteremia showed high within-patient clonality, irrespective of vanA-positivity or vancomycin-susceptibility. A substantial proportion of vanA-positive isolates are VVE and are therefore not detected with conventional selective culture methods. Bacteremia sources of patients with VVE are similar to those infected with VRE. We detected no association between VVE and 30-day mortality or breakthrough bacteremia.

Emerging antimicrobial resistance among Escherichia coli strains in bloodstream infections in Toronto, 2006-2016: a retrospective cohort study.

BACKGROUND: Enterobacteriaceae that produce extended-spectrum ß-lactamase (ESBL) have emerged as a serious threat, with variable rates depending on geographic region. We determined the prevalence of ESBL-producing Escherichia coli, Klebsiella pneumoniae, K. oxytoca and Proteus mirabilis in bloodstream infections in Toronto from 2006 through 2016. METHODS: All patients with E. coli, K. pneumoniae, K. oxytoca and P. mirabilis isolated from blood in a tertiary care microbiology laboratory in Toronto between 2006 and 2016 (1 isolate per species per patient per year) were included in this retrospective cohort study. Organisms were identified by conventional methods, and susceptibility testing was performed according to Clinical and Laboratory Standards Institute standards. Screening for ESBL and phenotypic confirmatory testing were done with a modified Clinical and Laboratory Standards Institute method. ST131 clonal type was determined by means of an established protocol. RESULTS: The proportion of ESBL-producing E. coli isolates increased significantly between 2006 and 2016, from 6.4% (19/296) to 17.3% (89/513) (p < 0.001). This trend was seen in both intensive care units and emergency departments. Concurrently, the proportion of ST131 among ESBL-producing E. coli also increased significantly, from 31.6% (6/19) in 2006 to 73.0% (65/89) in 2016 (p = 0.03). Among ESBL-producing E. coli, significant resistance was noted to multiple antimicrobial classes. Comparable increases in the proportion of ESBL-producing K. pneumoniae, K. oxytoca and P. mirabilis were not noted. INTERPRETATION: We observed a significant increase in the proportion of ESBL-producing E. coli in bloodstream infections in Toronto temporally correlated with an increase in the ST131 clonal type. Recognition of this dramatic rise is important to inform empiric antibiotic treatment.

A nosocomial outbreak of community-associated methicillin-resistant Staphylococcus aureus among healthy newborns and postpartum mothers.

BACKGROUND: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has increasingly been isolated from individuals with no predisposing risk factors; however, such strains have rarely been linked to outbreaks in the hospital setting. The present study describes the investigation of an outbreak of CA-MRSA that occurred in the maternal-newborn unit of a large community teaching hospital in Toronto, Ontario. METHODS: Screening and clinical specimens collected from mothers and newborns delivered during the outbreak period, as well as from staff on the affected unit, were submitted for microbiological testing. Computerized delivery logs and nursing notes were reviewed, and a case control study was conducted. RESULTS: Analysis by pulsed-field gel electrophoresis revealed 38 babies and seven mothers with MRSA colonization and/or infection by the same unique strain (Canadian MRSA-10-related) from September to December 2004. Isolates were characterized as having the staphylococcal chromosome cassette mec type IVa and were positive for the Panton-Valentine leukocidin gene. No one health care worker was associated with all cases; however, mothers and newborns exposed to one particular nurse (Nurse A) were almost 23 times (odds ratio 22.7, 95% CI 3.3 to 195.9) more likely to acquire MRSA than those with no such contact. MRSA was successfully isolated from Nurse A and from an environmental swab of a telephone recently used by Nurse A; both isolates matched the pulsed-field gel electrophoresis pattern of the outbreak strain. CONCLUSION: The first nosocomial outbreak of CA-MRSA among healthy newborns and postpartum mothers in Canada is described. Effective control of sustained MRSA transmission within an institution may require prompt identification, treatment and monitoring of colonized and/or infected staff.

Community- and Healthcare-Associated Methicillin-Resistant Staphylococcus aureus Strains: An Investigation Into Household Transmission, Risk Factors, and Environmental Contamination.

OBJECTIVE To measure transmission frequencies and risk factors for household acquisition of community-associated and healthcare-associated (HA-) methicillin-resistant Staphylococcus aureus (MRSA). DESIGN Prospective cohort study from October 4, 2008, through December 3, 2012. SETTING Seven acute care hospitals in or near Toronto, Canada. PARTICIPANTS Total of 99 MRSA-colonized or MRSA-infected case patients and 183 household contacts. METHODS Baseline interviews were conducted, and surveillance cultures were collected monthly for 3 months from household members, pets, and 8 prespecified high-use environmental locations. Isolates underwent pulsed-field gel electrophoresis and staphylococcal cassette chromosome mec typing. RESULTS Overall, of 183 household contacts 89 (49%) were MRSA colonized, with 56 (31%) detected at baseline. MRSA transmission from index case to contacts negative at baseline occurred in 27 (40%) of 68 followed-up households. Strains were identical within households. The transmission risk for HA-MRSA was 39% compared with 40% (P=.95) for community-associated MRSA. HA-MRSA index cases were more likely to be older and not practice infection control measures (P=.002-.03). Household acquisition risk factors included requiring assistance and sharing bath towels (P=.001-.03). Environmental contamination was identified in 78 (79%) of 99 households and was more common in HA-MRSA households. CONCLUSION Household transmission of community-associated and HA-MRSA strains was common and the difference in transmission risk was not statistically significant. Infect Control Hosp Epidemiol 2016;1-7.

New Delhi metallo-ß-lactamase in Jamaica.

INTRODUCTION: The global dissemination of the New Delhi metallo-beta-lactamase (NDM) gene among certain strains of bacteria has serious implications since the infections caused by such organisms pose a therapeutic challenge. Although the NDM gene has been detected in various parts of the world, this is the first report of its detection in the English-speaking Caribbean. The NDM producing Klebsiella pneumoniae was isolated from an Indian patient who had recently relocated to Jamaica. METHODOLOGY: Identification and susceptibility testing of the K. pneumoniae isolate was performed using the Vitek 2 automated system) in keeping with Clinical and Laboratory Standards Institute (CLSI) standards. It was identified as a metallobetalactamase producer using the Rosco KPC+MBL kit. Genotypic screening for common betalactamase (including carbapenemase) genes, was carried out  using two multiplex PCRs: one for SHV-, TEM-, CTX-M-, OXA-1-, and CMY-2-types, and one for VIM-, KPC-, IMP-, OXA-48, GES-, and NDM-types. Strain typing was conducted by pulsed-field gel electrophoresis (PFGE) using XbaI and multi-locus sequencing (MLS). Plasmid isolation and analysis was also performed. RESULTS: K. pneumoniae (N11-02395), not previously associated with the dissemination of the NDM in India, Sweden or the UK, was found to harbor the NDM-1 gene on plasmid pNDM112395. CONCLUSION: The identification of the NDM-1 gene underscores the need for effective surveillance and infection control measures to identify and prevent spread of multidrug resistant Gram negative bacilli. Strict infection control measures implemented for this patient helped to prevent the spread of this organism to other patients.

PCR ribotyping of Clostridium difficile isolates originating from human and animal sources.

Molecular typing of Clostridium difficile isolates from animals and humans may be useful for evaluation of the possibility for interspecies transmission. The objective of this study was to evaluate C. difficile isolates from domestic animals and humans using PCR ribotyping. Isolates were also tested using PCR for the presence of genes encoding toxins A and B. One hundred and thirty-three isolates of C. difficile from dogs (n = 92), horses (n = 21) and humans (n = 20), plus one each from a cat and a calf, were evaluated. Overall, 23 ribotypes were identified. Of these, nine were identified from dogs, 12 from horses, seven from humans and one each from the cat and calf. In dogs, humans and horses, one or two different ribotypes predominated. Overall, 25 % of isolates from humans were indistinguishable from isolates from one or more animal species. Genes encoding C. difficile toxins A and B were detected in all human, equine and bovine isolates, and in 69 % of canine isolates. While different ribotypes appear to predominate in different mammalian species, several indistinguishable strains may be found in multiple species. This suggests that there is a potential for interspecies transmission of C. difficile and epidemiological studies are warranted.

Community-associated methicillin-resistant Staphylococcus aureus in horses and humans who work with horses.

OBJECTIVE: To evaluate the prevalence of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) in horses and horse personnel. DESIGN: Prospective prevalence study. SAMPLE POPULATION: 972 horses and 107 personnel from equine farms in Ontario, Canada and New York state. PROCEDURE: Nasal swab specimens were collected from horses and humans on farms with (targeted surveillance) and without (nontargeted surveillance) a history of MRSA colonization or infection in horses during the preceding year. Selective culture for MRSA was performed. Isolates were typed via pulsed-field gel electrophoresis, and antibiograms were determined. RESULTS: MRSA was isolated from 46 of 972 (4.7%) horses (0/581 via nontargeted surveillance and 46/391 [12%] via targeted surveillance). Similarly, MRSA was isolated from 14 of 107 (13%) humans (2/41 [5%] from nontargeted surveillance and 12/66 [18%] from targeted surveillance). All isolates were subtypes of Canadian epidemic MRSA-5, an uncommon strain in humans. All isolates were resistant to at least 1 antimicrobial class in addition to beta-lactams. On all farms with colonized horses, at least 1 human was colonized with an indistinguishable subtype. For horses, residing on a farm that housed > 20 horses was the only factor significantly associated with MRSA colonization. For humans, regular contact with > 20 horses was the only identified risk factor. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirm a reservoir of colonized horses on a variety of farms in Ontario and New York and provide evidence that 1 MRSA strain is predominantly involved in MRSA colonization in horses and humans that work with horses.

Emergence of levofloxacin-resistant pneumococci in immunocompromised adults after therapy for community-acquired pneumonia.

We describe 4 patients infected with levofloxacin-resistant pneumococci after therapy for community-acquired pneumonia (CAP). The 4 patients had 15 episodes of CAP; Streptococcus pneumoniae was isolated from blood or sputum samples obtained during 14 of the episodes. The underlying medical condition was Bruton agammaglobulinemia in 3 patients and chronic lymphoid leukemia in the other. The initial episode of CAP in each patient was due to a levofloxacin-susceptible strain. One of 4 reinfections and 5 of 6 relapses were due to levofloxacin-resistant strains. All of these strains had amino acid substitutions in the quinolone-resistance-determining region of the genes parC and gyrA. The time between episodes of pneumonia varied from 1 to 4 months. In immunocompromised patients with suspected or proven pneumococcal infection, it may be prudent not to use fluoroquinolone monotherapy empirically when the patient has a history of fluoroquinolone therapy in at least the past 4 months.

Activity of GAR-936 and other antimicrobial agents against North American isolates of Staphylococcus aureus.

The comparative in vitro activity of GAR-936 and 12 other drugs against 602 North American isolates of methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus was determined. The GAR-936 MICs ranged from 0.06 to 1.0 mg/l. The MIC(50)s and MIC(90)s were 0.12 and 0.25 mg/l for MSSA and 0.25 and 0.5 mg/l for MRSA.