[Show abstract][Hide abstract] ABSTRACT: The MBEC™ Physiology & Genetics Assay recently became the first approved ASTM standardized biofilm disinfectant efficacy test method. This report summarizes the results of the standardization process using Pseudomonas aeruginosa biofilms. Initial ruggedness testing of the MBEC method suggests that the assay is rugged (i.e., insensitive) to small changes to the protocol with respect to 4 factors: incubation time of the bacteria (when varied from 16-18 hours), treatment temperature (20-24°C), sonication duration (25-35 minutes), and sonication power (130 - 480W). In order to assess the repeatability of MBEC results across multiple tests in the same laboratory and the reproducibility across multiple labs, an 8-lab study was conducted in which 8 concentrations of each of 3 disinfectants (a non-chlorine oxidizer, a phenolic, and a quaternary ammonium compound) were applied to biofilms using the MBEC method. The repeatability and reproducibility of the untreated control biofilms were acceptable, as indicated by small repeatability and reproducibility standard deviations (SD) (0.33 and 0.67 log10(CFU/mm(2)), respectively). The repeatability SDs of the biofilm log reductions after application of the 24 concentration and disinfectant combinations ranged from 0.22 to 1.61, and the reproducibility SDs ranged from 0.27 to 1.70. In addition, for each of the 3 disinfectant types considered, the assay was statistically significantly responsive to the increasing treatment concentrations.
Journal of microbiological methods 05/2014; · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clavibacter michiganensis subsp. michiganensis (Cmm) is an infectious, biofilm-forming bacterium that causes bacterial canker on tomato, a destructive disease of both field and greenhouse-grown tomatoes. Chemical disinfection of tools, equipment and structures is a commonly used method of disease management for bacterial canker. The potential of Cmm biofilm formation on hard surfaces in and around tomato production facilities has not been characterized. Furthermore, the ability of chemical biocides to disinfect or eradicate Cmm biofilms has not been characterized on contaminated surfaces. The BEST™ Assay, a versatile, high throughput, multiwell plate assay, was used to assess the ability of Cmm to form biofilms on six surface materials commonly found in tomato production, handling and storage systems. The BEST™ Assay was also used to compare the performances of eight chemical disinfectants in their abilities to eradicate or reduce Cmm biofilms on five surfaces. The results clearly demonstrated that Cmm forms biofilms on many surface materials, however some surfaces were more effectively colonized than others. For example, plywood surfaces were able to support the highest biofilm populations whereas metals and plastics supported the lowest populations. Biofilms, formed using the BEST™ Assay plate, were much more difficult to disinfect and eradicate than the planktonic cells free-floating in the wells of the plate. Disinfectant efficacy versus Cmm biofilms varied with surface material with porous surfaces being more difficult to disinfect than smooth. The data presented here highlight the importance of biofilm surface testing in the discovery, development and registration of chemical disinfectants and other disease management chemistries.
Science against microbial pathogens: communicating current research and technological advances, Microbiology Book Series - Number 3, Vol 2 edited by A. Mendez-Vilas, 01/2011: chapter A multi-well plate method for rapid growth, characterization and biocide sensitivity testing of microbial biofilms on various surface materials: pages 872-877; Formatex., ISBN: ISBN-13: 978-84-9398943-2-8
[Show abstract][Hide abstract] ABSTRACT: The impact of microbial biofilm formation on survival, tolerance and virulence is now well documented for many pathogenic bacteria and yeast. One of the challenges of biofilm research has been difficulty asssociated with culturing, characterizing and testing biofilms in traditional biofilm reactors. These traditional techniques can be cumbersome when many treatments or replicates are needed as they are not well-suited for rapid, high throughput screening experiments or factorial experiments with many replicates. The MBEC™ Assay is a technique that involves a patented, multi-well plate technology specifically designed for rapid, high throughput culturing, characterzing and testing of microbial biofilms. Furthermore, planktonic cell testing can be performed simultaneously in the same plate. Up to 96 biofilms can be cultured on pegs extending from the plate lid into the wells of the plate bottom. The lid, with biofilms attached, can then be conveniently transferred through a series of solutions or treatments such as rinses, challenges, neutralizations and etc. culminating with the recovery of planktonic cells, or biofilm cells which can be enumerated via standard plate count methods. This assay is now an ASTM standard method for disinfectant and biocide efficacy testing versus microbial biofilms (ASTM E2799-11). We report that this method can be used for many purposes including; simultaneous culture of planktonic cells and biofilms, characterization of biofilm formation and population growth curves, description of biofilm morphology, assessment of biocide efficacy breakpoints, and high througput screening for novel anti-biofilm compounds. This technique is highly versatile and can be used for virtually all culturable species of bacteria. As an example of this, we evaluated the biocidal effect of commercially available disinfectant formulations, approved by the Canadian Food Inspection Agency CFIA to be used in food and feed processing areas, based on MBC (Minimum Bactericidal Concentration) and MBEC (Minimum Biofilm Eradication Concentration) values of Escherichia coli O157:H7 (clinical isolate), and Listeria.
Science against microbial pathogens: communicating current research and technological advances, Microbiology Book Series - Number 3, Vol 2 edited by A. Mendez-Vilas, 01/2011: chapter A rapid, high-throughput method for culturing, characterizing and biocide efficacy testing of both planktonic cells and biofilms: pages 864-871; Formatex., ISBN: ISBN-13: 978-84-939843-2-8
[Show abstract][Hide abstract] ABSTRACT: For many pathogenic microbes, biofilm formation is a critical component of the disease cycle and is required for pathogenicity or full virulence. Recent innovation in in vitro biofilm reactors has allowed high throughput evaluation and testing of chemical treatments against microbial biofilms. For example, the BESTTM Assay is a versatile and specialized biofilm reactor capable of culturing bacterial and fungal biofilms in a replicated, high throughput, multi-well format. The microorganism forms a biofilm on the surfaces of pegs extending into liquid media contained within the microtitre plate wells. Additionally, planktonic cells also grow within the media in each well which allows simultaneous growth of biofilms and planktonic cultures within the wells of each reactor. Comparisons and evaluations of microbial biofilms on different surface materials can also be performed. For example, the bacterial ring rot pathogen Clavibacter michiganensis subsp. sepedonicus was studied in the BESTTM Assay to determine the conditions that promote growth in the biofilm form, the sensitivities of biofilm and planktonic cells to biocides, and the effects of biocide concentrations, exposure times and substrate types on efficacy. Technologies such as the BESTTM Assay excel at efficacy testing of chemical and biological agents being screened or developed for control of plant diseases, including postharvest diseases. Plant pathology research, phenotype screening, evaluating and formulating biological control products and protocols often ignore the critical aspect of biofilm potential. As a result, biofilm biology remains uncharacterized or unconfirmed for many plant-pathogen interactions and for most biocontrol agents. Biofilm reactors, such as the BESTTM Assay, provide technology for characterizing and evaluating these important issues.
The Americas Journal of Plant Science and Biotechnology. 12/2010; 4(SI2):43-47.
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium andersoni has not been previously reported in feedlot beef cattle in Western Australia. Faecal samples were collected from 10 groups of cattle ranging in age from 11 to 36 months in five different feedlots in Western Australia. The incidence of C. andersoni ranged from 0% to 26%. There were no clinical signs associated with C. andersoni infection, but there was a significant reduction in rate of gain of 0.44 kg in infected animals compared with negative pen mates. Cryptosporidium andersoni is characterised by large oocysts (7.4 x 5.5 μm) and was confirmed by 18S sequencing.
Australian Veterinary Journal 11/2010; 88(11):458-60. · 1.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peritoneal dialysis (PD)-related peritonitis is a common and morbid complication of PD. Bacteria are able to create a biofilm on the PD catheter, which can be a source of recurrent infection. Biofilms undergo a phenotypic change resulting in increased antibiotic resistance. ♢
21 clinical isolates of different patients with PD peritonitis secondary to Staphylococcus aureus were collected. They were analyzed for their antibiotic susceptibility in the planktonic form using the standard minimum inhibitory concentration (MIC) and in a biofilm using minimum biofilm eradication concentration (MBEC). Chi-square was used to compare the sensitivity results. ♢
The isolates were susceptible to all the antibiotics tested using MIC. Every antibiotic except gentamicin lost its efficacy when the bacteria were grown in a biofilm (p > 0.05). The change in susceptibility was statistically significant to a level of p < 0.001 for all antibiotics tested. ♢ Discussion: In PD peritonitis that is long standing, recurrent, or not responsive to therapy, MBEC testing should be considered as a biofilm may be present. Gentamicin should be strongly considered over other agents for empiric gram-negative coverage as it may be providing synergy in the setting of Staphylococcus aureus. Also, the newer anti-staphylococcal drugs should be tested for their performance in a biofilm using the MBEC method.
Peritoneal dialysis international : journal of the International Society for Peritoneal Dialysis. 11/2010; 30(6):652-6.
[Show abstract][Hide abstract] ABSTRACT: Giardia and Cryptosporidium are protozoan parasites known to cause enteric disease in terrestrial wildlife species (mammals, reptiles and birds). Few surveys for Giardia and Cryptosporidium in marine wildlife species, such as pinnipeds, have been reported. The objective of this study was to determine the prevalence and genotype of Giardia and Cryptosporidium in two species of pinnipeds, harp seal (Phoca groenlandica) and hooded seal (Cystophora cristata), from the Gulf of St. Lawrence, Canada. Faecal samples were collected from pup and adult seals and examined for the presence of cysts of Giardia and oocysts of Cryptosporidium using microscopy and immunofluorescent staining. Tissues from the small intestine of adult seals were also collected and examined for infections using the polymerase chain reaction (PCR) technique. Giardia cysts were found in the faeces of 42% (16/38) of adult harp seals, but in none of the harp seal pups (0/20). Although Giardia cysts were not detected in faeces of adult hooded seals (0/10) using microscopy, 80% tested positive for Giardia using PCR of intestinal tissue indicative of a true replicating infection. Both harp and hooded seals harboured infections with the zoonotic strain, Giardia duodenalis Assemblage A, as determined using a nested-PCR technique to amplify a small subunit ribosomal (SSU-rRNA) gene of Giardia. Cryptosporidium was not detected by microscopy, nor using the PCR technique on intestinal tissues from any of the 68 seals examined.
[Show abstract][Hide abstract] ABSTRACT: Batch culture of biofilms on peg lids is a versatile method that can be used for microtiter determinations of biofilm antimicrobial susceptibility. In this paper, we describe a core protocol and a set of parameters (surface composition, the rate of rocking or orbital motion, temperature, cultivation time, inoculum size, atmospheric gases and nutritional medium) that can be adjusted to grow single- or multispecies biofilms on peg surfaces. Mature biofilms formed on peg lids can then be fitted into microtiter plates containing test agents. After a suitable exposure time, biofilm cells are disrupted into a recovery medium using sonication. Microbicidal endpoints can be determined qualitatively using optical density measurements or quantitatively using viable cell counting. Once equipment is calibrated and growth conditions are at an optimum, the procedure requires approximately 5 h of work over 4-6 d. This efficient method allows antimicrobial agents and exposure conditions to be tested against biofilms on a high-throughput scale.
[Show abstract][Hide abstract] ABSTRACT: While antibiotic resistance has grabbed the headlines and the attention of the pharmaceutical industry, the lack of susceptibility of biofilms formed both on animate and inanimate surfaces deserve greater attention from the industry, medical practitioners and regulators. The current literature tells us that the inherent tolerance to antibiotics demonstrated by antibiotic-sensitive organisms when grown as a biofilm clearly identifies a major disconnect between our current practices in antimicrobial development, diagnostics and efficacy in patient treatment. A paradigm shift is required in the way we utilize conventional antimicrobials and in the way we screen for next-generation antibiotics with efficacy to treat biofilms associated with chronic, recurrent and device related infections. This paradigm shift must not only take place in industry but also in how drugs are brought to the marketplace for acceptance.
Expert Opinion on Pharmacotherapy 04/2010; 11(8):1233-7. · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The discovery of biofilm formation in bacteria and yeasts has led to a better understanding of microbial ecology and to new insights into the mechanisms of virulence and persistence of pathogenic microorganisms. However, it is generally assumed that filamentous fungi, some of which have a significant impact on our health or our economy, do not form biofilms. In contrast to this assumption, here we discuss recent findings supporting the hypothesis that surface-associated filamentous fungi can form biofilms. Based on these findings and on previous models for bacterial and yeast systems, we propose preliminary criteria and a model for biofilm formation by filamentous fungi.
Trends in Microbiology 11/2009; 17(11):475-80. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The autochthonous microbial flora of the gastrointestinal and biliary system were studied in ten cats using standard bacteriological cultures and electron microscopic techniques. The most common organisms isolated from the gastrointestinal tract were streptococci, anaerobes and coliform bacteria. A spiral organism was identified in the feline stomach and duodenum. The bile and the biliary system were entirely sterile except in one cat. Bacteria were isolated from the sphincter of Oddi. Electron microscopy confirmed that bacteria had colonised in the thick mucous layer in the gastrointestinal tract and the sphincter of Oddi. No bacteria could be found above the sphincter by electron microscopy. The sphincter of Oddi is hence the natural boundary and probably serves as a physical barrier to the upward migration of bacteria from the colonised gastrointestinal tract to the uncolonised biliary system. The cat may be a suitable animal model for bacteriological studies of the biliary system in the human.
Microbial Ecology in Health and Disease 07/2009; 3(4):199-207.
[Show abstract][Hide abstract] ABSTRACT: Coyotes from southern Alberta and Saskatchewan, Canada, were examined for the presence of Giardia and Cryptosporidium and cohabiting helminths. Toxascaris was present in over 90% of the 70 animals examined, and Taenia sp. in 6.5-25% of the two groups of animals studied. Giardia (12.5-21.7%) and Cryptosporidium (0-17.4%) were also common and molecular characterisation revealed both zoonotic and host-adapted genotypes of Giardia, whereas the Cryptosporidium proved to be a variant of the canine species C. canis. The seasonal variation observed in the occurrence of Cryptosporidium may be related to stress-induced shedding of the parasite.
[Show abstract][Hide abstract] ABSTRACT: Biofilms are slimy aggregates of microbes that are likely responsible for many chronic infections as well as for contamination of clinical and industrial environments. Pseudomonas aeruginosa is a prevalent hospital pathogen that is well known for its ability to form biofilms that are recalcitrant to many different antimicrobial treatments. We have devised a high-throughput method for testing combinations of antimicrobials for synergistic activity against biofilms, including those formed by P. aeruginosa. This approach was used to look for changes in biofilm susceptibility to various biocides when these agents were combined with metal ions. This process identified that Cu(2+) works synergistically with quaternary ammonium compounds (QACs; specifically benzalkonium chloride, cetalkonium chloride, cetylpyridinium chloride, myristalkonium chloride, and Polycide) to kill P. aeruginosa biofilms. In some cases, adding Cu(2+) to QACs resulted in a 128-fold decrease in the biofilm minimum bactericidal concentration compared to that for single-agent treatments. In combination, these agents retained broad-spectrum antimicrobial activity that also eradicated biofilms of Escherichia coli, Staphylococcus aureus, Salmonella enterica serovar Cholerasuis, and Pseudomonas fluorescens. To investigate the mechanism of action, isothermal titration calorimetry was used to show that Cu(2+) and QACs do not interact in aqueous solutions, suggesting that each agent exerts microbiological toxicity through independent biochemical routes. Additionally, Cu(2+) and QACs, both alone and in combination, reduced the activity of nitrate reductases, which are enzymes that are important for normal biofilm growth. Collectively, the results of this study indicate that Cu(2+) and QACs are effective combinations of antimicrobials that may be used to kill bacterial biofilms.
Antimicrobial Agents and Chemotherapy 08/2008; 52(8):2870-81. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antibiotic-resistant Escherichia coli in 300 feedlot steers receiving subtherapeutic levels of antibiotics was investigated through the collection of 3,300 fecal samples over a 314-day period. Antibiotics were selected based on the commonality of use in the industry and included chlortetracycline plus sulfamethazine (TET-SUL), chlortetracycline (TET), virginiamycin, monensin, tylosin, or no antibiotic supplementation (control). Steers were initially fed a barley silage-based diet, followed by transition to a barley grain-based diet. Despite not being administered antibiotics prior to arrival at the feedlot, the prevalences of steers shedding TET- and ampicillin (AMP)-resistant E. coli were >40 and <30%, respectively. Inclusion of TET-SUL in the diet increased the prevalence of steers shedding TET- and AMP-resistant E. coli and the percentage of TET- and AMP-resistant E. coli in the total generic E. coli population. Irrespective of treatment, the prevalence of steers shedding TET-resistant E. coli was higher in animals fed grain-based compared to silage-based diets. All steers shed TET-resistant E. coli at least once during the experiment. A total of 7,184 isolates were analyzed for MIC of antibiotics. Across antibiotic treatments, 1,009 (13.9%), 7 (0.1%), and 3,413 (47.1%) E. coli isolates were resistant to AMP, gentamicin, or TET, respectively. In addition, 131 (1.8%) and 143 (2.0%) isolates exhibited potential resistance to extended-spectrum beta-lactamases, as indicated by either ceftazidime or cefpodoxime resistance. No isolates were resistant to ciprofloxacin. The findings of the present study indicated that subtherapeutic administration of tetracycline in combination with sulfamethazine increased the prevalence of tetracycline- and AMP-resistant E. coli in cattle. However, resistance to antibiotics may be related to additional environmental factors such as diet.
Applied and Environmental Microbiology 07/2008; 74(14):4405-16. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The GacS/GacA two-component regulatory system in pseudomonads regulates genes involved in virulence, secondary metabolism and biofilm formation. Despite these regulatory functions, some Pseudomonas species are prone to spontaneous inactivating mutations in gacA and gacS. A gacS(-) strain of Pseudomonas aeruginosa PA14 was constructed to study the physiological role of this sensor histidine kinase. This loss-of-function mutation was associated with hypermotility, reduced production of acylhomoserine lactones, impaired biofilm maturation, and decreased antimicrobial resistance. Biofilms of the gacS(-) mutant gave rise to phenotypically stable small colony variants (SCVs) with increasing frequency when exposed to silver cations, hydrogen peroxide, human serum, or certain antibiotics (tobramicin, amikacin, azetronam, ceftrioxone, oxacilin, piperacillin or rifampicin). When cultured, the SCV produced thicker biofilms with greater cell density and greater antimicrobial resistance than did the wild-type or parental gacS(-) strains. Similar to other colony morphology variants described in the literature, this SCV was less motile than the wild-type strain and autoaggregated in broth culture. Complementation with gacS in trans restored the ability of the SCV to revert to a normal colony morphotype. These findings indicate that mutation of gacS is associated with the occurrence of stress-resistant SCV cells in P. aeruginosa biofilms and suggests that in some instances GacS may be necessary for reversion of these variants to a wild-type state.
[Show abstract][Hide abstract] ABSTRACT: Although they differ considerably with respect to their biology, both Giardia duodenalis and Cryptosporidium parvum are common in ruminants, whereas Cryptosporidium andersoni is not. G. duodenalis infections are acquired during the first few months of life, tend to be chronic, and may be a production-limiting disease of ruminants. C. parvum infections remain an important cause of diarrhea in neonatal ruminants. Abomasal cryptosporidiosis, caused by C. andersoni, is an emerging disease of cattle that may affect both beef and dairy herds. This article reviews the life cycles, production impacts, treatments, controls, and zoonotic potentials of these important ruminant parasites.
Veterinary Clinics of North America Food Animal Practice 12/2006; 22(3):623-43. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial resistance (AMR) was temporally assessed in campylobacters isolated from beef cattle (7,738 fecal samples from 2,622 animals) in four commercial feedlots in Alberta. All calves were administered chlortetracycline and oxytetracycline in feed, and a majority of the animals (93%) were injected with long-acting oxytetracycline upon arrival at the feedlot. Fecal samples from individual animals were collected upon arrival (i.e., entry sample), 69 days (standard deviation [SD] = 3 days) after arrival (i.e., interim sample), and 189 days (SD = 33 days) after arrival (i.e., exit sample) at the feedlot. In total, 1,586 Campylobacter isolates consisting of Campylobacter coli (n = 154), Campylobacter fetus (n = 994), Campylobacter jejuni (n = 431), Campylobacter hyointestinalis (n = 4), and Campylobacter lanienae (n = 3) were recovered and characterized. The administration of antimicrobials did not decrease carriage rates of campylobacters, and minimal resistance (< or =4%) to azithromycin, ciprofloxacin, enrofloxacin, gentamicin, and meropenem was observed. In contrast, substantive increases in the prevalence of isolates resistant to tetracycline and doxycycline (56 to 89%) for C. coli, C. fetus, and C. jejuni, as well as in the number of animals (7 to 42%) from which resistant isolates were recovered, were observed during the feedlot period. Increased resistance to erythromycin (total isolates and carriages rates) was also observed in isolates of C. coli over the three isolation times. The majority of C. fetus isolates recovered were resistant to nalidixic acid, but this was independent of when they were isolated. A relatively limited number of multidrug-resistant isolates were recovered and consisted primarily of C. coli resistant to tetracyclines and erythromycin (10% of isolates). Over the course of the feedlot period, considerable increases in antimicrobial resistance were observed in C. coli, C. fetus, and C. jejuni, but with the exception of erythromycin resistance in C. coli, the administration of antimicrobial agents to beef cattle was found to have a minimal impact on resistance to macrolides and fluoroquinolones, the two classes of antimicrobials used to treat campylobacteriosis in humans. However, the widespread use of antimicrobial agents in beef production and the possible horizontal transfer of mobile genetic elements with antimicrobial resistance determinants among Campylobacter and other bacterial taxa emphasize the need to monitor AMR development in bacteria from beef cattle.
Applied and Environmental Microbiology 06/2006; 72(6):4088-95. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fibroproliferative scars in humans often demonstrate familial inheritance patterns, and genetics may contribute to healing and scarring. Genetic factors may also influence the scarring phenotype in a porcine model. Healing of full thickness excisional skin wounds in Yorkshire pigs closely resembles normal healing in humans, while identical wounds in red Duroc pigs form hypercontracted, hyperpigmented scars. The present study has evaluated the healing process in the first generation cross (F1) of red Duroc and Yorkshire pigs. Gross and histologic analysis revealed that the F1 animals exhibit an intermediate healing phenotype, with some features of each parent breed. F1 full thickness wounds were significantly hypercontracted and fibrotic, but apigmented. Analysis of mRNA expression patterns for a panel of relevant molecules (N=32) in the F1 animals revealed some similarities to each parent breed, as well as unique patterns for other molecules. Furthermore, a depth dependency to the healing response was observed at the gross, histologic, and molecular levels, with deep dermal wounds healing similar to Yorkshire wounds. These findings suggest that the genetic contribution to scar phenotype in this animal model is complex. However, the results indicate that further understanding in this model may provide insights into risk factors for hypertrophic scarring in human burn patients.
[Show abstract][Hide abstract] ABSTRACT: Many issues concerning the taxonomy of Echinococcus have been resolved in recent years with the application of molecular tools. However, the status of Echinococcus maintained in transmission cycles involving cervid intermediate hosts remains to be determined. The recent characterization of the parasite from cervids in Finland has highlighted the paucity of data available, particularly that from North America. In this study, we have characterized a large number of Echinococcus isolates from cervids from Western Canada on the basis of morphology and molecular genetic techniques. Our results support earlier studies suggesting that Echinococcus of cervid origin is phenotypically and genetically distinct to Echinococcus maintained in domestic host assemblages, and also confirms that Echinococcus of cervid origin does not constitute a genetically homogeneous group. However, our data do not support the existence of 2 distinct genotypes (strains/subspecies) with separate geographical distributions. Our data appear to support the existence of only 1 species in cervids, but additional isolates from cervids and wolves in other endemic regions should be characterized before a final decision is made on the taxonomic status of Echinococcus in cervids.