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.

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.

Detection of severe acute respiratory syndrome coronavirus in stool specimens by commercially available real-time reverse transcriptase PCR assays.

Three commercially available real-time reverse transcriptase PCR assays (the Artus RealArt HPA coronavirus LightCycler, the Artus RealArt HPA coronavirus Rotor-Gene, and the EraGen severe acute respiratory syndrome coronavirus POL assay) and three RNA extraction methodologies were evaluated for the detection of severe acute respiratory syndrome coronavirus RNA from 91 stool specimens. The assays' sensitivities were highest (58% to 75%) for specimens obtained 8 to 21 days after symptom onset. The assays were less sensitive when specimens were obtained less than 8 days or more than 21 days after the onset of symptoms. All assays were 100% specific.

Methicillin-resistant Staphylococcus aureus in horses at a veterinary teaching hospital: frequency, characterization, and association with clinical disease.

Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging equine pathogen. To attempt to control nosocomial and zoonotic transmission, an MRSA screening program was established for all horses admitted to the Ontario Veterinary College Veterinary Teaching Hospital, whereby nasal screening swabs were collected at admission, weekly during hospitalization, and at discharge. MRSA was isolated from 120 (5.3%) of 2,283 horses: 61 (50.8%) at the time of admission, 53 (44.2%) during hospitalization, and 6 from which the origin was unclear because an admission swab had not been collected. Clinical infections attributable to MRSA were present or developed in 14 (11.7%) of 120 horses. The overall rate of community-associated colonization was 27 per 1,000 admissions. Horses colonized at admission were more likely to develop clinical MRSA infection than those not colonized at admission (OR 38.9, 95% CI 9.49 160, P < 0.0001). The overall nosocomial MRSA colonization incidence rate was 23 per 1,000 admissions. The incidence rate of nosocomial MRSA infection was at the rate of 1.8 per 1,000 admissions, with an incidence density of 0.88 per 1,000 patient days. Administration of ceftiofur or aminoglycosides during hospitalization was the only risk factor associated with nosocomial MRSA colonization. MRSA screening of horses admitted to a veterinary hospital was useful for identification of community-associated and nosocomial colonization and infection, and for monitoring of infection control practices.

Suspected transmission of methicillin-resistant Staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household.

OBJECTIVE: To describe MRSA infection and colonization in household pets, and transmission of MRSA between animals and humans. METHODS: MRSA infection and colonization in household pets and human contacts were evaluated during investigations initiated after identification of MRSA infection or colonization of a household pet in order to determine if there had been transmission between animals and humans. All MRSA isolates were screened for Panton-Valentine leukocidin (PVL) genes by use of polymerase chain reaction, and isolate relatedness was determined by use of pulsed-field gel electrophoresis (PFGE). RESULTS: Investigations of six situations where MRSA was identified in one or more animals in a household or veterinary facility were performed. MRSA was isolated from 8 animals (5 dogs and 3 cats) with clinical infections, 1 cat that was in contact with 2 infected cats and 14/88 (16%) of household contacts or veterinary personnel. Both animal-to-human and human-to-animal transmission were suspected. An indistinguishable MRSA isolate was recovered from at least one human that was in contact with each animal case. All isolates were classified as Canadian epidemic MRSA-2, the predominant community-associated MRSA clone in humans in Canada. No isolates possessed genes encoding for the PVL. CONCLUSIONS: Transmission of MRSA between humans and animals, in both directions, was suspected. MRSA appears to be an emerging veterinary and zoonotic pathogen.

An outbreak of methicillin-resistant Staphylococcus aureus skin infections resulting from horse to human transmission in a veterinary hospital.

There are increasing reports of methicillin-resistant Staphylococcus aureus (MRSA) infection and colonization in horses and evidence that MRSA can be transmitted between horses and humans. The objective of this study was to investigate reports of skin infection in personnel working with a foal with community-associated MRSA colonization and subsequent infection. Clinical diagnostic specimens were collected from individuals reporting skin lesions following contact with the affected foal. Nasal and groin screening swabs were collected from other veterinary personnel that attended a voluntary screening clinic. MRSA skin infections were identified in three neonatal intensive care unit personnel. Nasal colonization was subsequently identified in 10/103 (9.7%) other veterinary hospital personnel. Isolates were indistinguishable by pulsed field gel electrophoresis, classified as Canadian epidemic MRSA-5, possessed SCCmecIV, were negative for the Panton-Valentine leukocidin and were multidrug resistant. Transmission to veterinary personnel despite short-term contact with standard protective barriers highlights the potential importance of MRSA as an emerging zoonotic pathogen, and indicates that further evaluation of interspecies transmission of MRSA and means to prevent zoonotic infection are required.

Methicillin-resistant Staphylococcus aureus in horses and horse personnel, 2000-2002.

Methicillin-resistant Staphylococcus aureus (MRSA) infection was identified in 2 horses treated at a veterinary hospital in 2000, prompting a study of colonization rates of horses and associated persons. Seventy-nine horses and 27 persons colonized or infected with MRSA were identified from October 2000 to November 2002; most isolations occurred in a 3-month period in 2002. Twenty-seven (34%) of the equine isolates were from the veterinary hospital, while 41 (51%) were from 1 thoroughbred farm in Ontario. Seventeen (63%) of 27 human isolates were from the veterinary hospital, and 8 (30%) were from the thoroughbred farm. Thirteen (16%) horses and 1 (4%) person were clinically infected. Ninety-six percent of equine and 93% of human isolates were subtypes of Canadian epidemic MRSA-5, spa type 7 and possessed SCCmecIV. All tested isolates from clinical infections were negative for the Panton-Valentine leukocidin genes. Equine MRSA infection may be an important emerging zoonotic and veterinary disease.

Laboratory contamination of specimens with quality control strains of vancomycin-resistant enterococci in Ontario.

Cross-contamination with laboratory control strains of vancomycin-resistant Enterococcus faecalis was documented in 15 clinical specimens from nine clinical microbiology laboratories in Ontario, Canada. Laboratories should be alert to the possibility of contamination of specimens with vancomycin-resistant enterococci from the laboratory environment. Molecular typing of strains may assist in elucidating the source of such contamination.

Activity of BMS-284756, a novel des-fluoro(6) quinolone, against Staphylococcus aureus, including contributions of mutations to quinolone resistance.

The in vitro activity of BMS-284756 against 602 Staphylococcus aureus isolates, including 152 that were both methicillin and ciprofloxacin resistant (MIC > or = 4 microg/ml), was determined. For ciprofloxacin-susceptible and nonsusceptible isolates, the MICs at which 50% of organisms were inhibited were 0.015 and 2 microg/ml and the MICs at which 90% of organisms were inhibited were 0.03 and 4 microg/ml, respectively.

Molecular mechanisms of cefoxitin resistance in Escherichia coli from the Toronto area hospitals.

Escherichia coli may become resistant to cephamycines and oxyimino cephalosporins by virtue of promotor and attenuator mutations or because they have acquired mobilized beta-lactamases from other gram-negative bacilli. This study examined Canadian strains to determine how often promotor and/or attenuator mutations account for this mechanism of resistance and the extent to which clonal spread of these organisms has occurred. We sequenced the promotor and attenuator region of 30 strains resistant to cefoxitin. Twenty-two strains had promotor mutations, 26 had attenuator mutations. Most promotor mutations resulted either in a change in the -35 promotor region towards the E. coli sigma 70 consensus sequence or in the creation of a new consensus hexamer upstream. Eight strains had mutations that increased the typical ampC 16-nucleotide spacer region to the consensus 17- or an 18-nucleotide sequence. Of the attenuator mutations, most did not substantially affect the attenuator loop. Several of the mutations have previously been described in South Africa, Scandinavia, and France. There was evidence that strains bearing certain mutations were clonally disseminated; however, the 11 strains bearing a complex set of attenuator mutations were not. The majority of cephamycin resistant E. coli strains in Toronto have attenuator and/or promotor mutations upstream of the chromosomal ampC gene.

Streptococcal meningitis resulting from contact with an infected horse.

We report a case of group C streptococcal meningitis in a woman with a history of close animal contact as well as head trauma as a result of a kick by a horse. Blood and cerebrospinal fluid cultures grew Streptococcus equi subsp. zooepidemicus, as did a throat culture taken from the colt that had kicked her 2 weeks prior to admission.

Evaluation of D-xylose and 1% methyl-alpha-D-glucopyranoside fermentation tests for distinguishing Enterococcus gallinarum from Enterococcus faecium.

To determine the validity of the rapid xylose and methyl-alpha-D-glucopyranoside (MDG) fermentation tests in distinguishing Enterococcus gallinarum from Enterococcus faecium, 156 well-characterized clinical isolates of enterococci (55 E. gallinarum, 91 E. faecium, and 10 Enterococcus faecalis isolates) known to be of different clones were examined in a blinded fashion. Species identification was confirmed by PCR of the ddl ligase genes of E. faecium and E. faecalis and the vanC1 gene of E. gallinarum. Xylose tests were performed with D-xylose tablets by using a heavy bacterial suspension and were interpreted after 2 h of incubation. Standard MDG fermentation tests were read after 24 h of incubation. The xylose fermentation test had a sensitivity of 98% (54 of 55) and a specificity of 99% (100 of 101) in distinguishing E. gallinarum from E. faecium and E. faecalis. The standard MDG test had a sensitivity of 100% (55 of 55) and a specificity of 95% (96 of 101) after 24 h. The xylose fermentation test is a simple method, easily incorporated into laboratory protocols, that distinguishes E. gallinarum from E. faecium with high sensitivity and specificity in 2 h. The standard MDG test has high sensitivity and can be useful in ruling out the presence of E. gallinarum but requires overnight incubation.

Practical approach to the identification of clinically relevant Enterococcus species.

Enterococci have become important nosocomial pathogens, with Enterococcus faecalis and then Enterococcus faecium predominating. Because of the emergence of glycopeptide (vancomycin and teicoplanin) resistance in enterococci, laboratories have been required to screen for resistant strains and to identify them to the species level. This has resulted in the need for accurate identification of species less commonly associated with clinical infections, such as Enterococcus casseliflavus and Enterococcus gallinarum, which are inherently resistant to the glycopeptides. Studies evaluating commonly used commercial identification systems, have found error rates for enterococcal species identification of 2-21% for E. faecalis, 5-9% for E. faecium, and 14-79% for other species. Reporting errors may have adverse effects on the management of clinical infections, as well as in the control of multidrug-resistant strain outbreaks. The purpose of this document is to present a simplified approach to the identification of Enterococcus species that uses a combination of rapid, readily available, and inexpensive tests.

Molecular characterization of multidrug resistance in Streptococcus mitis.

Antimicrobial resistance was characterized for 14 strains of Streptococcus mitis. HinfI restriction fragment length mapping of gyrA PCR amplicons from three ciprofloxacin-resistant isolates correlated with mutations associated with such resistance in other organisms. By using PCR, seven erythromycin-resistant strains were found to possess either the mef or ermB gene. Hybridization revealed tet(M) in seven tetracycline-resistant isolates.

Streptococcus iniae, a human and animal pathogen: specific identification by the chaperonin 60 gene identification method.

It was recently reported that Streptococcus iniae, a bacterial pathogen of aquatic animals, can cause serious disease in humans. Using the chaperonin 60 (Cpn60) gene identification method with reverse checkerboard hybridization and chemiluminescent detection, we identified correctly each of 12 S. iniae samples among 34 aerobic gram-positive isolates from animal and clinical human sources.

Invasive infections due to a fish pathogen, Streptococcus iniae. S. iniae Study Group.

BACKGROUND: Streptococcus iniae is a pathogen in fish, capable of causing invasive disease and outbreaks in aquaculture farms. During the winter of 1995-1996 in the greater Toronto area there was a cluster of four cases of invasive S. iniae infection in people who had recently handled fresh, whole fish from such farms. METHODS: We conducted a prospective and retrospective community-based surveillance for cases of S. iniae infection in humans. To obtain a large sample of isolates, we studied cultures obtained from the surface of fish from aquaculture farms. Additional isolates were obtained from the brains of infected tilapia (oreochromis species). All the isolates were characterized by pulsed-field gel electrophoresis (PFGE). RESULTS: During one year, our surveillance identified a total of nine patients with invasive S. iniae infection (cellulitis of the hand in eight and endocarditis in one). All the patients had handled live or freshly killed fish, and eight had percutaneous injuries. Six of the nine fish were tilapia, which are commonly used in Asian cooking. Thirteen additional S. iniae isolates (2 from humans and 11 from infected tilapia) were obtained from normally sterile sites. The isolates from the nine patients were indistinguishable by PFGE and were highly related to the other clinical isolates. There was substantial genetic diversity among the 42 surveillance isolates from the surface of fish, but in 10 isolates the PFGE patterns were identical to those from the patients with S. iniae infection. CONCLUSIONS: S. iniae can produce invasive infection after skin injuries during the handling of fresh fish grown by aquaculture. We identified a clone of S. iniae that causes invasive disease in both humans and fish.

Experience with a hospital-wide outbreak of vancomycin-resistant enterococci.

BACKGROUND: Vancomycin-resistant enterococci (VRE) were first detected in our institution in 1991. An outbreak was recognized in late 1992 when there was a sudden rise in the number of patients per month with VRE. Little information exists concerning the natural history of infection with these pathogens, and the effect of antimicrobial therapy is unclear. Recent guidelines emphasize prudent use of vancomycin and prompt institution of barrier precautions to limit the spread of vancomycin resistance. METHODS: Data were obtained by review of microbiologic and clinical records. Patients were categorized according to site of infection, and outcome of therapy was assessed. Hospital antibiotic usage was analyzed to determine any correlation with the outbreak. Infection control measures instituted in 1993 included patient isolation, environmental cleaning, and a reemphasis of barrier precautions. Surveillance cultures were performed to assess the extent of the outbreak in January 1995. RESULTS: VRE were detected in clinical cultures from 159 patients from 1991 through 1994. Mortality rate was 48%, but in most cases death could not be attributed to enterococcal infection. Patients with wound infections healed without specific therapy. Many patients with bacteremia had resolution with ampicillin or without specific therapy. Patients were widely scattered throughout the hospital from the beginning of the outbreak. Hospital usage of cefotaxime correlated with the number of cases. Infection control measures were not successful. Surveillance culture results in January 1995 revealed that 53% of all medical and surgical inpatients had fecal colonization with VRE. Genetic analysis of selected isolates revealed that one strain predominated, but at least seven distinct strains were identified. CONCLUSIONS: Our data suggest that many infections with VRE resolve without specific therapy. The infection control measures we used were ineffective, possibly because of the multiple strains present in our hospital. Isolation of all patients with VRE is impractical when there is widespread fecal carriage.

Treatment of experimental endocarditis caused by multidrug resistant Enterococcus faecium with ramoplanin and penicillin.

Antibiotic resistant strains of enterococci are being isolated with increasing frequency. Effective treatment of infections caused by Enterococcus faecium resistant to ampicillin, vancomycin and aminoglycosides has not been established. We studied the activity of ramoplanin, a new lipoglycopeptide antibiotic, against two strains of multidrug resistant E. faecium. In time kill studies, ramoplanin was bactericidal against both strains, but not in the presence of 50% serum. The combination of ramoplanin and penicillin was bactericidal even in the presence of serum. In rabbits with experimental endocarditis neither penicillin nor ramoplanin significantly reduced vegetation colony counts when given alone, although ramoplanin significantly reduced spleen and kidney bacterial counts of both strains. The combination of ramoplanin plus penicillin resulted in a significant reduction of vegetation bacterial counts (-3.2 and -3.7 log10 cfu/g for strains VA3 and MMC3, respectively, P < 0.01). All spleen cultures and 9 out of 10 kidney cultures from each strain were sterile following combination therapy. While ramoplanin will not be available for parenteral therapy, further research into the development of other lipoglycopeptide antibiotics is warranted.

In vitro activities of antimicrobial combinations against Stenotrophomonas (Xanthomonas) maltophilia.

Stenotrophomonas (Xanthomonas) maltophilia is inherently resistant to multiple antimicrobial agents. In order to investigate the in vitro potential of combinations of antimicrobial agents, we obtained 230 epidemiologically unrelated clinical isolates from seven hospitals across Canada and from Northwestern Memorial Hospital in Chicago. Ticarcillin-clavulanate combined with ciprofloxacin or trimethoprim-sulfamethoxazole were assayed for synergy against 31 ticarcillin-resistant strains of S. maltophilia by using microtiter checkerboard panels and against 20 strains by using time-kill methodology. The combination of ciprofloxacin with ceftazidime was also evaluated by time-kill studies. Ticarcillin-clavulanate plus trimethoprim-sulfamethoxazole demonstrated synergy by checkerboard panels, with fractional inhibitory concentration indices ranging from 0.033 to 0.49, and by time-kill studies for all 20 strains tested. Synergy between ticarcillin-clavulanate plus ciprofloxacin was found by the checkerboard method for 24 of 31 strains (77%), with fractional inhibitory concentration indices ranging from 0.188 to 0.75. A correlation between synergy by the checkerboard method and the reference time-kill study method was not observed for ticarcillin-clavulanate plus ciprofloxacin, with results for 3 of 10 strains being nonconcordant. Synergy with both ticarcillin-clavulanate plus ciprofloxacin and ceftazidime plus ciprofloxacin by the time-kill method was found to correlate with ciprofloxacin MICs of <32 micrograms/ml and zone diameters of >15 mm on Mueller-Hinton agar. Evaluation of these combinations in vivo may be warranted.