Ron Read

[25] The MBEC assay system: Multiple equivalent biofilms for antibiotic and biocide susceptibility testing

Publisher Summary A number of technologies have been developed to study biofilm growth. Although these technologies produce reproducible biofilms for the study of biofilm growth, structure, and physiology, they have not been amenable for the routine study of biofilm susceptibility to antibiotics and biocides. For this reason, virtually every antibiotic and biocide available has been selected for activity against planktonic organisms. These drugs often have been found to lack activity against microbial biofilms. The MBEC (minimum biofilm eradication concentration) Assay System using the Calgary Biofilm Device provides, for the first time, an assay easily applicable to screening antibiotics and biocides for activity against microbial biofilms. The MBEC Assay System is ideally suited either for screening new putative antibiotics and/or biocides, or for the determination of both the MIC (minimal inhibitory concentration) and MBEC values in clinical situations for the treatment of chronic, recurrent, or device-related infections. The MBEC Assay System produces 96 equivalent biofilms formed under flow conditions, without the need for pumps. Further, as it is based on the standard 96 well platform, it conforms to existing technology available in most laboratories. The MBEC Assay System consists of a two-piece disposable plastic apparatus used for biofilm formation.

Molecular Characterization of Syphilis in Patients in Canada: Azithromycin Resistance and Detection of Treponema pallidum DNA in Whole-Blood Samples versus Ulcerative Swabs

ABSTRACT Although detection of Treponema pallidum DNA in whole-blood specimens of syphilis patients has been reported, it is uncertain at what stage of the disease such specimens are most suitable for the molecular diagnosis of syphilis. Also, few studies have directly compared the different gene targets for routine laboratory diagnostic usage in PCR assays. We examined 87 specimens from 68 patients attending two urban sexually transmitted disease clinics in Alberta, Canada. PCR was used to amplify the T. pallidum tpp47, bmp, and polA genes as well as a specific region of the 23S rRNA gene linked to macrolide antibiotic susceptibility. In primary syphilis cases, PCR was positive exclusively (75% sensitivity rate) in ulcerative swabs but not in blood specimens, while in secondary syphilis cases, 50% of the blood specimens were positive by PCR. Four out of 14 (28.6%) of our PCR-positive syphilis cases were found to be caused by an azithromycin-resistant strain(s). Our results confirmed that swabs from primary ulcers are the specimens of choice for laboratory diagnostic purposes. However, further research is required to determine what specimen(s) would be most appropriate for molecular investigation of syphilis in secondary and latent syphilis.

Evidence for Increased Chlamydia Case Finding After the Introduction of Rectal Screening Among Women Attending 2 Canadian Sexually Transmitted Infection Clinics

BACKGROUND Chlamydia trachomatis is the most common notifiable disease in Canada, and extragenital sites are believed to serve as hidden reservoirs for ongoing transmission of infection. There are no specific Canadian screening guidelines for asymptomatic individuals from extragenital sites. We sought to determine the prevalence and factors associated with rectal C. trachomatis among female sexually transmitted infection (STI) clinic attendees in Alberta, Canada. METHODS Between 20 July and 31 December 2012, all female attendees at 2 Provincial STI clinics receiving a pelvic examination, regardless of a history of anal intercourse, were screened for rectal C. trachomatis using the Gen-Probe Aptima COMBO 2 Assay. Demographic and behavior variables were compared between rectal-only chlamydia cases and genitourinary cases using χ(2) or Fisher exact test, Mann-Whitney test, and logistic regression. RESULTS A total of 3055 women were screened for rectal chlamydia. The prevalence of rectal chlamydia ranged from 11.7% to 13.5%. There were 133 rectal-only cases, increasing case detection by 44.3% from 300 genitourinary cases to 433 total cases, ranging from 21.7% to 88.2% by clinic. Women who were a contact to an STI were less likely to have rectal-only chlamydia for both clinics (P ≤ .001). CONCLUSIONS Our findings add to the growing body of evidence supporting universal rectal screening in high-risk women such as those undergoing pelvic exams at STI clinics.

Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics

BackgroundEnvironmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA.ResultsThe concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments.ConclusionThe abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.

Comparison of CMY-2 plasmids isolated from human, animal, and environmental Escherichia coli and Salmonella spp. from Canada

A total of 244 CMY-2 plasmids from 5 separate studies involving Escherichia coli and Salmonella human clinical cases as well as E. coli from feedlots and water sources were examined. Genetically similar CMY-2 plasmids isolated from either E. coli or Salmonella from human, animal, and environmental sources are widely distributed across Canada and cluster into replicon types I1, A/C, and K/B and an unidentified group.

Genetic characterization and antimicrobial susceptibility of Mannheimia haemolytica isolated from the nasopharynx of feedlot cattle.

A surveillance study was undertaken to examine the population dynamics and antimicrobial resistance of Mannheimia haemolytica isolated from feedlot cattle. A total of 416 isolates were collected from the nasopharynx either upon entry or exit from two feedlots in southern Alberta, Canada. Isolates were serotyped, characterized by pulsed-field gel electrophoresis and tested for susceptibility to ten antimicrobial agents via disk diffusion. Resistant isolates were screened by PCR for select antimicrobial-resistance gene determinants. Isolates were highly diverse, with 335 unique pulsed-field profiles identified among 147 strongly related clusters (similarity ≥ 85%). Clonal spread of isolates throughout the feedlots was limited and no clear association was found between genetic relatedness of M. haemolytica and sampling event (entry or exit). Pulsed-field profiles sharing a common serotype and resistance phenotype tended to cluster together. The majority of isolates were identified as serotype 2 (74.5%) although both serotype 1 (11.9%) and 6 (12.7%) were detected. Only 9.54% of isolates exhibited antimicrobial resistance. Resistance to oxytetracycline was most prevalent (n=16), followed by ampicillin (n=10), and amoxicillin/clavulanic acid (n=7). Multi-drug resistance was observed in five isolates. The tetH gene was detected in all but two oxytetracycline resistant isolates. Other detectable resistance determinates included ermX and bla(ROB-1). In the two feedlots examined, M. haemolytica exhibited considerable genetic diversity and limited resistance to common veterinary antibiotics. Garnering further information on the linkage between genotype and phenotype should contribute toward a better understanding of the pathogenesis and dissemination of M. haemolytica in feedlots.

Similar cefoxitin-resistance plasmids circulating in Escherichia coli from human and animal sources

The aim of this study was to determine the molecular epidemiology of cefoxitin-resistance Escherichia coli identified in cattle entering feedlots and determine if there were any similarities to E. coli causing human infections in Canadian hospitals. A total of 51 E. coli were isolated from a total of 2483 cattle entering four feedlots in southern Alberta, Canada. DNA fingerprinting using pulsed-field gel electrophoresis revealed thirty-two unique patterns with two major clusters observed comprised of Cluster A (11 strains) and Cluster B (7 strains). PCR and sequence analysis revealed 38 isolates (74.5%) harboured bla(CMY-2), whereas the remainder were found to contain mutations in the promoter region of the chromosomal ampC gene, which has been previously associated with cefoxitin resistance. No resistance to nalidixic acid, ciprofloxacin, or amikacin was observed in the clinical isolates. bla(CMY-2) harbouring plasmids were transferred to E. coli DH10B. All of the plasmids carrying bla(CMY-2) contained the A/C replicon and also harboured other resistance genes. Plasmid fingerprinting using BglII revealed 17 unique patterns with all but one clustering within 70% similarity. Comparison of the plasmid fingerprints to those isolated from human clinically significant E. coli in Canada during a similar time period [Mulvey, M.R., Bryce, E., Boyd, D.A., Ofner-Agostini, M., Land, A.M., Simor, A.E, Paton, S., 2005. The Canadian Hospital Epidemiology Committee, and The Canadian Nosocomial Infection Surveillance Program, Health Canada. Molecular characterization of cefoxitin resistant Escherichia coli from Canadian hospitals. Antimicrob. Agents Chemother. 49, 358-365] revealed four strains that harboured bla(CMY-2) A/C replicon type plasmid with fingerprint similarities of greater than 90% to the ones identified in E. coli from the cattle in this study. These findings highlight the potential linkage of multidrug resistant organisms in food producing animals and human infections in Canadian hospitals. The plasmids conferred resistance to multiple antibiotics which could limit options for the treatment of infections caused by these strains.

Characteristics of individuals with male-to-male and heterosexually acquired infectious syphilis during an outbreak in Calgary, Alberta, Canada.

Background Eliminating syphilis is important not only to prevent the sequelae of infection but also to control the spread of HIV. Current prevention and control efforts in Canada have been ineffective in eliminating this disease. Goal The goal of the study was to determine the characteristics of individuals with infectious syphilis due to male-to-male and heterosexual contact, diagnosed during an outbreak in Calgary, Alberta, Canada. Study Design This was a prospective study of individuals with infectious syphilis diagnosed at the STD clinic in Calgary between January 2000 and April 2002. Results The outbreak reported here (September 2000 to April 2002) involves 32 cases of infectious syphilis, corresponding to rates of 0.9/100,000 population during 2000 and 1.8/100,000 population during 2001. Between September 2000 and June 2001, the cases diagnosed were among men who have sex with men (MSM); between May 2001 and April 2002, they were due to locally acquired infections among heterosexuals, including one case of congenital syphilis. Compared to the heterosexuals, MSM tended to be older, be coinfected with HIV, and report excessive alcohol use (versus injection drug use) and had infectious syphilis diagnosed earlier. MSM used the Internet and bars or bathhouses to initiate sexual contact, whereas heterosexually acquired infections were largely among sex workers and their clients. Contact tracing was more successful among the heterosexuals than among MSM. The public health staff at the STD clinic initiated a series of multifaceted interventions in response to the outbreak. These interventions were moderately successful, as measured by the increased numbers of individuals seeking counseling and testing services at the clinic. Conclusion The results highlight key differences in the risk factor-specific characteristics of the outbreak that should be taken into account when designing prevention and control strategies.

Molecular typing of Treponema pallidum strains in western Canada: predominance of 14d subtypes.

Background: Resurgence of syphilis in Canada and worldwide requires laboratories to update their methods for molecular epidemiology investigation and surveillance. This study utilizes polymerase chain reaction diagnostic tests for syphilis, identifies macrolide resistance, and uses a molecular typing system to characterize Treponema pallidum clinical strains causing syphilis in Alberta and Northwest Territories, Canada. Methods: In total 449 specimens including genital swabs, whole blood, sera, and cerebrospinal fluid were obtained from 374 patients with suspect syphilis in Alberta and Northwest Territories. Molecular subtyping was based on genetic characterization of treponemal repeat genes, arp and tpr. Detection of macrolide resistance was accomplished by identification of the 23S rRNA gene mutation associated with the resistance pattern. Results: Forty-nine specimens obtained from 43 patients were found to be positive for T. pallidum DNA using bmp, tpp47 and polA polymerase chain reaction assays. Four molecular subtypes were identified, with one type, 14d, accounting for 70% of all cases and 83% of typeable strains. Seven patients (16%) were found to be infected by macrolide-resistant strains, of which 6 were men who have sex with men and 1 whose infection was acquired in China. Conclusions: A single molecular type of T. pallidum, characterized as 14d, caused the majority of the syphilis cases identified in this study. A more discriminatory typing method would be required to determine if these strains are clonal. Treatment of infectious syphilis with macrolide antibiotics should be restricted to patient populations where resistance is rare and clinical and serological follow up of patients is possible.

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis.

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.