Pyrosequencing assay to rapidly detect clarithromycin resistance mutations in Canadian Helicobacter pylori isolates.

BACKGROUND: Mutations at positions 2142 or 2143 in the twocopy 23S ribosomal RNA gene of Helicobacter pylori are highly predictive of in vitro clarithromycin resistance and failure of clarithromycin-containing treatment regimens. OBJECTIVE: To design an assay to rapidly detect these mutations using rapid polymerase chain reaction and pyrosequencing, a novel method of 'sequencing by synthesis', and to test this assay with a collection of Canadian H pylori isolates. METHODS: Forty-two H pylori isolates (24 clarithromycin-resistant, 18 clarithromycin-susceptible) were studied. A target region in the 23S gene was rapidly amplified and sequenced by pyrosequencing. RESULTS: Mutations at one of the two positions studied were present in 20 of the 24 (83%) clarithromycin-resistant isolates; 13 had double- copy A2143G mutations, four had double-copy A2142G mutations and three had single-copy A2143G mutations. There were no mutations in 17 of the 18 (94%) susceptible isolates. A single-copy A2142G mutation was detected in one susceptible isolate. CONCLUSIONS: The pyrosequencing assay developed was able to detect and differentiate mutations at positions 2142 and 2143 in either one or both copies of the H pylori 23S ribosdomal RNA gene. Further study is needed to determine whether this pyrosequencing assay can be used to determine H pylori susceptibility to clarithromycin from clinical specimens such as stools or gastric biopsies.

Acquisition of resistance to extended-spectrum cephalosporins by Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey poult intestinal tract.

Salmonella enterica subsp. enterica serovar Newport resistant to the extended-spectrum cephalosporins (ESCs) and other antimicrobials causes septicemic salmonellosis in humans and animals and is increasingly isolated from humans, animals, foods, and environmental sources. Mechanisms whereby serovar Newport bacteria become resistant to ESCs and other classes of antimicrobials while inhabiting the intestinal tract are not well understood. The present study shows that 25.3% of serovar Newport strains isolated from the turkey poult intestinal tract after the animals were dosed with Escherichia coli harboring a large conjugative plasmid encoding the CMY-2 beta-lactamase and other drug resistance determinants acquired the plasmid and its associated drug resistance genes. The conjugative plasmid containing the cmy-2 gene was transferred not only from the donor E. coli to Salmonella serovar Newport but also to another E. coli serotype present in the intestinal tract. Laboratory studies showed that the plasmid could be readily transferred between serovar Newport and E. coli intestinal isolates. Administration of a single dose of ceftiofur, used to prevent septicemic colibacillosis, to 1-day-old turkeys did not result in the isolation of ceftiofur-resistant E. coli or Salmonella serovar Newport. There was a remarkable association between serotype, drug resistance, and plasmid profile among the E. coli strains isolated from the poults. This study shows that Salmonella serovar Newport can become resistant to ESCs and other antibiotics by acquiring a conjugative drug resistance plasmid from E. coli in the intestines.

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.

Comparison of the Velogene Rapid MRSA Identification Assay, Denka MRSA-Screen Assay, and BBL Crystal MRSA ID System for rapid identification of methicillin-resistant Staphylococcus aureus.

Early detection of methicillin-resistant S.aureus (MRSA) is critical for both the management of infected patients, and the timely institution of appropriate infection control measures. Although detection of the mecA gene by PCR remains the gold standard, this technology is inaccessible for many laboratories. Therefore, we sought to evaluate several new rapid identification systems and compare them to PCR. A total of 71 methicillin-susceptible S. aureus (MSSA), 25 borderline oxacillin-resistant S. aureus (BORSA), and 213 MRSA were selected for study. S.aureus was identified using standard methods. Initial screening was performed on a Mueller-Hinton agar plate with 6 mg/L of oxacillin. MRSA strains were identified using PCR with primers specific for the mecA gene. PCR was used as the reference method. All isolates were tested using the BBL Crystal MRSA ID System (Becton Dickinson Microbiology Systems, Maryland, USA), the MRSA-Screen Assay (Denka Seiken Co., Ltd., Tokyo, Japan), and the Velogene Rapid MRSA Identification Assay (ID Biomedical Corp, Vancouver, BC). With minor modifications, all assays were performed according to manufacturers' instructions. Overall, the 3 commercial assays performed well. The sensitivity and specificity of the BBL, Denka, and Velogene systems were 99%/100%, 99%/100%, and 96%/100% respectively. The advantages of the phenotypic tests-BBL Crystal Kit and Denka MRSA-Screen Assay include lower cost per test, shelf-life, ease of use, and rapid turn-around times. Advantages of the Velogene Rapid MRSA include ability to perform genotypic high-volume testing without the equipment requirements and technical complexity involved with PCR. Turn-around times ranged from 15 min for the Denka MRSA-Screen Assay, 2 h for the Velogene Rapid MRSA, and 4 h for the BBL Crystal. The BBL Crystal, Denka MRSA-Screen, and Velogene Rapid MRSA identification systems are rapid, easy to perform, and provide accurate identification of MRSA. These rapid kits offer an acceptable alternative for smaller, non-reference, laboratories and reduce the dependency on PCR in larger laboratories for routine confirmation.

Emerging importance of multidrug-resistant Acinetobacter species and Stenotrophomonas maltophilia as pathogens in seriously ill patients: geographic patterns, epidemiological features, and trends in the SENTRY Antimicrobial Surveillance Program (1997-1999).

As part of the SENTRY Antimicrobial Surveillance Program, a total of 1078 Acinetobacter species and 842 Stenotrophomonas maltophilia isolates were collected between January 1997 and December 1999 from 5 geographic regions (Canada, the United States, Latin America, Europe, and the Asia-Pacific). The frequency of infections (by geographic region and body site), including those due to imipenem-resistant Acinetobacter species and trimethoprim-sulfamethoxazole (TMP-SMZ)-resistant S. maltophilia, was evaluated. The possibility of seasonal variations in bloodstream infections caused by Acinetobacter species was studied, as was the activity of several therapeutic antimicrobials against all strains. Acinetobacter species and S. maltophilia were most frequently associated with pulmonary infections, independent of the region evaluated. In contrast, patterns of antimicrobial resistance markedly varied among distinct geographic regions, especially for nosocomial isolates. Although the carbapenems were the most active antimicrobials against Acinetobacter species, nearly 11.0% of the nosocomial isolates were resistant to this drug group in both regions. TMP-SMZ, ticarcillin-clavulanic acid, gatifloxacin, and trovafloxacin were the only agents with consistent therapeutic activity against S. maltophilia isolates. Rates of resistance to TMP-SMZ ranged from 2% in Canada and Latin America to 10% in Europe. The geographic differences in resistance patterns among Acinetobacter species and S. maltophilia isolates observed in this study emphasize the importance of local surveillance in determining the most adequate therapy for acinetobacter and S. maltophilia infections and the possible clonal, epidemic nature of occurrence.

Intra and inter technologist variability in the quality assessment of respiratory tract specimens.

The majority of microbiology laboratories have implemented quality improvement procedures such as a Q scoring system to assess the nature of clinical specimens. Our study reviewed the sources and the amount of variation when Q scoring of lower respiratory secretions was performed. In total, 450 slides representing lower respiratory tract secretions were Q scored by three experienced technologists. Total agreement regarding the number of neutrophils, squamous epithelial cells and Q scores was 76%, 57% and 57% respectively. The major factor influencing Q score values was the enumeration of epithelial cells. From our findings, we expect that there is greater variability in Q scoring then is generally acknowledged and there is a substantial degree of subjectivity on part of individual technologists reading gram stains.

Evaluation of four commercial transport media for the survival of Neisseria gonorrhoeae.

We evaluated four commercial transport systems with a standardized inoculum of clinical isolates of N. gonorrhoeae (NG), and assessed survival after holding for up to 48 hours at both ambient and refrigeration temperatures. Suspensions of clinical isolates of NG were standardized and adsorbed onto four transport swab types: Culturette EZ (Becton Dickinson [BD], Cockeysville, MD, USA); Cultureswab (Difco Laboratories, Detroit, MI, USA); Venturi Transystem (Copan Italia, Bovezzo, Italy); and a recently modified Starswab (Starplex Scientific, Etobicoke, ON). Swabs were plated to chocolate agar at 0, 6, 24, and 48 hours, and colonies counted. Each swab type was tested in quadruplicate with each NG strain for all time and temperature variables. There was a marked reduction in NG CFUs after only 6 hours incubation with each of the swabs tested. Survival was best using Venturi Transystem and Cultureswab transports (colony counts were reduced to 15.3% and 13.0%, respectively, at 6 hours) when compared with the Culturette EZ and Starswab (colony counts were reduced to 2.2% and 4.3%, respectively, at 6 hours). After the 24-hour holding period, 94% of the cultures from the Venturi Transystem were positive, 82% from the Cultureswab, 24% from the Starswab; and 17% from the Culturette EZ. After 48 hours, recovery dropped to 72%, 43%, 14%, and 0.04%, respectively. All of the systems tested had at least an 80% decrease in recovered colonies after only 6 hours. Further studies are required to determine how poor transport conditions influence the number of positive cultures and what the public health implications are. Of the swabs tested, Cultureswab and Venturi Transystem were most acceptable.

Four-day incubation for detection of bacteremia using the BACTEC 9240.

Records of 29,356 blood cultures performed between April 1994 and April 1997, using the BACTEC 9240 continuous monitoring blood culture system, were reviewed retrospectively. From these, 3,127 blood culture vials became positive. Of 95 blood culture isolates detected after three days of incubation, 63 were recovered on day four and 32 on day five. Twenty-six contaminants were recovered on day four, and 21 on day five. Chart review was performed for all day four and five isolates that did not meet our definition of a contaminant. Of the 40 isolates that were clinically insignificant, 31 were recovered on day four, and nine on day five. Of eight clinically significant isolates, six were recovered on day four, and two on day five. Our data support a four-day incubation protocol for the recovery of all clinically significant bacteria with overall sensitivity reduced by only 0.06% when compared with a five-day protocol.

The epidemiology of penicillin resistance in Streptococcus pneumoniae.

Pneumococci are not intrinsically resistant penicillin or other commonly used antibiotics. Penicillin-resistant strains were not encountered until 1965 when two strains were identified in Boston. At that time, the resistance was of a minor degree and its significance was not recognized by the authors. In 1971, resistant strains were encountered in New Guinea and, by the late 1970s, penicillin-resistant pneumococci had spread worldwide. By the early 1980s, areas where more than 10% of isolates were found to be penicillin resistant included Israel, Poland, Spain, South Africa, New Guinea, and the United States from New Mexico to Alaska. In this decade a number of countries including South Korea, Hungary, and Spain have reported dramatic increases in penicillin resistance. Penicillin resistance, once acquired by a virulent pneumococcal clone, has the ability to spread from country to country and to other continents in a relatively short time. Coincident with the increasing penicillin resistance has been the development of resistance to a wide variety of other antibiotics, including other cephalosporins, macrolides, trimethoprim-sulfamethoxazole, tetracycline, and chloramphenicol. Some strains are so highly resistant as to significantly impair our ability to treat patients with meningitis and to choose an appropriate oral agent for the treatment of pneumococcal otitis media.

Cross-Canada survey of resistance of 2747 aerobic blood culture isolates to piperacillin/tazobactam and other antibiotics.

OBJECTIVE: To compare the activity of piperacillin/tazobactam with that of other broad parenteral antibiotics against aerobic and facultative anaerobic blood culture isolates in a Canada-wide survey. DESIGN: Fifty-eight laboratories in nine provinces each contributed up to 50 consecutive clinically significant aerobic and facultative anaerobic isolates for susceptibility testing. SETTING: Participating hospitals included both tertiary care and community hospitals. MATERIALS AND METHODS: Testing was performed in five regional centres by using the same microbroth dilution method, and results were interpreted according to National Commitee for Clinical Laboratory Standards M7-A3 and M100-S5 guidelines. RESULTS: Piperacillin/tazobactam and imipenem were both active against more than 99% of the 1616 strains of Enterobacteriaceae species tested. The minimum inhibitory concentration of 90% of isolates (MIC(90)) of all Enterobacteriaceae species was 2 mg/L for piperacillin/tazobactam compared with 64 mg/L for piperacillin alone. Seventeen per cent of strains of Enterobacteriaceae species were susceptible to piperacillin/tazobactam but resistant to piperacillin. Piperacillin/tazobactam was highly active against Pseudomonas aeruginosa, inhibiting 99.1% of strains. MIC(90) was 8 mg/L. Nine per cent of P aeruginosa strains were not susceptible to imipenem. Most of these strains had a MIC of 8 mg/L, which falls in the intermediate category. Ninety-seven per cent of P aeruginosa were susceptible to ciprofloxacin and 97.3% to tobramycin. Ninety-six per cent of strains of Actinobacter species were susceptible to piperacillin/tazobactam, whereas only 76% of strains were susceptible to piperacillin alone. Overall, piperacillin/tazobactam was the most active agent tested; 98% of all strains were susceptible, followed closely by imipenem, to which 97.8% of strains were susceptible. CONCLUSIONS: Aerobic blood culture isolates from Canadian centres continue to be highly susceptible to a variety of antibiotics. The broad spectrum of activity of piperacillin/tazobactam suggests that this combination should be considered for empirical treatment of sepsis while awaiting results of cultures and susceptibility testing.

Cumulative yield from patient surveillance cultures for methicillin-resistant Staphylococcus aureus during a hospital outbreak.

The cumulative yield from cultures of separate sites was determined during the investigation of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak. Surveillance cultures were submitted from clinical sites, nose, groin, and axilla of 421 patients on two different occasions. MRSA was recovered most often from various clinical sites, including lower respiratory tract, surgical wounds, urinary tract, and decubitus ulcers (total, 13 patients). Four additional patients were identified as positive from the first nasal swab, one patient from the second nasal swab, and two others from swabs of the groin. The submission of axillary swabs or a second groin swab did not identify additional MRSA-colonized patients and resulted in additional costs of $4,525.

Use of selenite enrichment broth for the detection of Salmonella from stool: a report of one year experience at a provincial public health laboratory.

Several references recommend that selenite enrichment be used only in stool cultures from suspect carriers, during outbreaks, and in other special circumstances. To determine the impact of such an approach, we examined results from 3977 specimens cultured by our laboratory. Epidemiological information was collected from physicians and the public health department. Salmonella spp. were identified in 74 specimens from 54 patients. Four Shigella spp. were recovered from four patients. Forty-seven of the 74 Salmonella spp. were recovered on both the primary xylose-lysine-deoxycholate (XLD) and after enrichment. No Salmonella or Shigella grew on the primary XLD only. Twenty-six Salmonella spp. were recovered only after selenite enrichment. Of these, 17 were from newly identified patients. The elimination of selenite enrichment would have significantly reduced our yield, whereas the elimination of the primary XLD would not have resulted in any fewer isolates and would have resulted in savings of approximately $4000 yearly.

Study of the comparative activity of piperacillin/tazobactam with currently available antibiotics against 8206 aerobic isolates.

OBJECTIVES: To compare the activity of piperacillin-tazobactam with piperacillin and other parenterally administered antibiotics against aerobic Gram-negative bacilli and Gram-positive cocci isolated from across Canada, and to determine the prevalence of resistance mediated by extended-spectrum cephalosporinases. METHODS: Sixty-one laboratories participated. Disk diffusion testing was performed in accordance with methods outlined by the National Committee for Clinical Laboratory Standards. Susceptibilities were performed on 8206 strains. Escherichia coli and Klebsiella pneumoniae with reduced susceptibilities to third-generation cephalosporins were screened for extended-spectrum beta-lactamases (ESBLs). RESULTS: Piperacillin-tazobactam was active against 92% of the strains, piperacillin against 81% and ticarcillin-clavulanic acid against 88%. Few differences were observed in the relative susceptibility of strains from teaching or community hospitals, from different anatomic sites or from different regions of the country. Aerobic Gram-negative bacilli tested tended to be more susceptible to all the agents than was recently reported in a similar American study. Only 43% of Enterococcus faecium were susceptible to ampicillin and 42% to piperacillin piperacillin with and without tazobactam. Only two enterococcal strains were resistant to vancomycin, and 19 had intermediate zone sizes. Of the 10 strains of E coli and eight strains of K pneumoniae with reduced susceptibility to extended spectrum cephalosporins, only one demonstrated typical ESBL activity. CONCLUSIONS: Canadian aerobic Gram-positive cocci and Gram-negative bacilli remain highly susceptible to many currently available antibiotics. The findings confirm a broad spectrum of activity of piperacillin-tazobactam and indicate that the pattern of susceptibility is quite uniform from different body sites, in both teaching and community hospitals, and across the country.

First reported case of bacterial endocarditis attributable to Actinomyces meyeri.

A 48-year-old man presented to the Victoria General Hospital, Halifax, Nova Scotia in severe congestive heart failure. Echocardiographic studies revealed significant aortic valve insufficiency. Two anaerobic blood cultures performed two weeks apart were both positive for Actinomyces meyeri. The patient was treated with high dose intravenous penicillin. Three weeks after antibiotics were begun, he underwent aortic valve replacement. Intraoperative cultures were negative. Histopathological examination revealed findings in keeping with subacute bacterial endocarditis. The patient completed a six-week course of penicillin and was doing well three months after surgery. This is the first case of endocarditis attributable to A meyeri reported in the literature.

Molecular epidemiology of enterovirus outbreaks in Canada during 1991-1992: identification of echovirus 30 and coxsackievirus B1 strains by amplicon sequencing.

The relatedness of enteroviral isolates associated with two recent outbreaks in Canada was assessed using direct sequencing of amplicons derived from a large portion of the 5' nontranslated region (NTR) of the viral genome. The amplicons of 60 echovirus 30 isolates originating from seven different provinces in 1991 were found to share 99% or greater sequence identity. Recent coxsackievirus B1 isolates characterised in the same manner were identical to each other. When the 5' NTR sequence of these isolates was compared to prototype strains a difference of 11-15% in nucleotide composition was observed. These results indicate that the variability of nucleotide sequence found in 5' NTRs can be utilized to identify rapidly enteroviral strains associated with particular outbreaks and distinguish them from other strains and serotypes.

Comparison of TechLab Clostridium difficile Tox-A enzyme immunoassay and Bartels Prima system toxin-A EIA.

We evaluated the Bartles Clostridium difficile toxin A test and the TechLab Tox-A test to detect C. difficile toxin A in stool. The results were compared with C. difficile cytotoxicity assays. Of the 463 specimens tested 82 (17.7%) tested positive by cytotoxicity assay. The sensitivity, specificity, and positive and negative predictive values of the TechLab EIA were 86.6%, 93.7%, 74.7%, and 97.0%, respectively. For the Bartels Prima EIA, sensitivity, specificity, and positive and negative predictive values were 95.1%, 95.5%, 82.1%, and 98.9%, respectively. The differences in sensitivity were statistically significant. Indeterminate results requiring repeat testing were more common with the TechLab EIA than with the Bartels Prima EIA. Of the two kits, the Bartels EIA is preferable, primarily because of its increased sensitivity.

An assessment of the anaerobic vial of paired NR 6A and NR 7N Bactec blood cultures.

OBJECTIVE: To determine the number of obligate anaerobes recovered in anaerobic blood culture vials and to determine if their recovery had a significant impact on patient care. DESIGN: Retrospective review. SETTING: Tertiary care teaching hospital. MAIN RESULTS: Six thousand nine hundred and five pairs of Bactec blood cultures were submitted (each set consisted of one 6A and one 7N vial). Of these, 690 sets were culture-positive in at least one of the vials (10% of pairs). Both vials were positive in 406 (58.8%). The aerobic vial alone was positive in 176 (25.5%) and the anaerobic vial alone was positive in 107 (15.5%). Of these, most were facultative anaerobes; however, 20 blood culture sets from 18 patients were positive for obligate anaerobes. In five of the 18, the isolate was judged to be a contaminant. In 11 of 13 patients, the clinically significant obligate anaerobic bacteremia might have been predicted on clinical grounds, and in eight patients, empirical antianaerobic antibiotics had been started before the results of blood cultures were known. CONCLUSIONS: Clinical laboratories should carefully examine the use of the routine anaerobic blood culture and consider its replacement with larger volume aerobic blood culture vials.

Hepatitis B prenatal screening survey, Nova Scotia, 1990-1991.

We conducted a study in which 5,754 pregnant women who delivered at the Grace Maternity Hospital in Halifax were screened for HBsAg. There were five who were found to be seropositive for the first time (a screening yield for seropositivity of 8.7/10,000). Overall six were seropositive for a prevalence rate of 10.4/10,000. These rates are above the 6.0/10,000 level at which routine prenatal screening is considered to be cost-effective. Screening based upon risk factors would have identified only two of the five women who were found for the first time to be HBsAg seropositive. Based upon the results of this study, we recommend that routine screening for HBsAg be performed on all prenatal women in Nova Scotia.

A comparison of the API Uriscreen with the Vitek Urine Identification-3 and the leukocyte esterase or nitrite strip as a screening test for bacteriuria.

The API Uriscreen is a rapid urine-screening test based on the detection of catalase activity present in somatic cells and in many of the bacteria commonly causing urinary tract infections. Of 487 routine, outpatient urine specimens processed by conventional quantitative culture, API Uriscreen, Vitek UID-3 panel, and a leukocyte esterase-nitrite strip, 142 had no growth. Of 336 urine specimens with > or = 10(3) colony-forming units (CFU)/ml, 79 were considered to be indicative of possible or probable urinary tract infection (Cumitech 2A). The sensitivity and specificity of the API Uriscreen for the detection of bacteriuria at > or = 10(5) CFU/ml were 62% and 85%, those of the leukocyte esterase-nitrite strip was 61% and 82%, those of the Vitek UID-3 panel were 91% and 66%. When bacteriurias were classified into possibly or probably indicative of urinary tract infection, the sensitivity and specificity of the API Uriscreen at > or = 10(5) CFU/ml were 87% and 78%, those of the leukocyte esterase-nitrite were 84% and 76%, those of the Vitek UID-3 were 93% and 55%. In this study, we consider the API Uriscreen did not have significant advantages over the leukocyte esterase-nitrite strip.