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.

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.

BackgroundCastration is one of the most common procedures performed on beef and dairy cattle. The objective of the study was to determine the efficacy of meloxicam oral suspension in reducing pain and inflammation in calves following band or surgical castration.MethodsTwo identical trials with the exception of the method of castration (Band Castration Study 1 and Surgical Castration Study 2) were conducted. Sixty (60) healthy Holstein calves 4 to 5 months of age (138–202 Kg) were used. Animals received either Meloxicam Oral Suspension at a dose of 1 mg/kg BW (n = 15 Study 1 and 15 Study 2) or Saline (n = 15 Study 1 and 15 Study 2) 2 h before castration. Physiological (Heart Rate, Plasma Cortisol and Plasma Substance P) and Behavioral (Visual Analog Scale (VAS), Accelerometers and tail Pedometers) evaluations were conducted before (day -1) and after Castration (Day 0, 1, 2, 3). Inflammation was evaluated daily by providing an individual animal score (Study1) or with a measurement of scrotal thickness (Study 2).ResultsHeart rates were significantly greater in control animals following band and surgical castration. Plasma cortisol and substance P were significantly reduced in animals receiving Meloxicam Oral Suspension. Control animals had significantly greater VAS scores. Accelerometers showed that meloxicam treated animals had a significantly greater motion index and number of steps as well as less % time lying and number of lying bouts. The scrotal inflammation (based on scrotal swelling) was significantly decreased in the meloxicam treated animals compared to the control animals on day 1, day 2 and 3.ConclusionMeloxicam Oral Suspension was able to significantly reduce the display of painful behaviors and physiological responses to pain in band castrated and surgical castrated calves for up to 72 h following a single oral treatment of 1 mg/kg body weight. Meloxicam Oral Suspension was able to significantly reduce scrotal inflammation in band castrated and surgical castrated calves.

Bovine respiratory disease (BRD) is the most important illness of feedlot cattle. Disease management targets the associated bacterial pathogens, Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida, Histophilus somni, and Trueperella pyogenes. We conducted a cross-sectional study to measure the frequencies of antimicrobial-resistant BRD pathogens using a collaborative network of veterinarians, industry, government, and a diagnostic laboratory. Seven private veterinary practices in southern Alberta collected samples from both living and dead BRD-affected animals at commercial feedlots. Susceptibility testing of 745 isolates showed that 100% of the M. haemolytica, M. bovis, P. multocida, and T. pyogenes isolates and 66.7% of the H. somni isolates were resistant to at least one antimicrobial class. Resistance to macrolide antimicrobials (90.2% of all isolates) was notable for their importance to beef production and human medicine. Multidrug resistance (MDR) was high in all target pathogens with 47.2% of the isolates resistant to four or five antimicrobial classes and 24.0% resistance to six to nine classes. We compared the MDR profiles of isolates from two feedlots serviced by different veterinary practices. Differences in the average number of resistant classes were found for M. haemolytica (p < 0.001) and P. multocida (p = 0.002). Compared to previous studies, this study suggests an increasing trend of resistance in BRD pathogens against the antimicrobials used to manage the disease in Alberta. For the veterinary clinician, the results emphasize the importance of ongoing susceptibility testing of BRD pathogens to inform treatment protocols. Surveillance studies that collect additional epidemiological information and manage sampling bias will be necessary to develop strategies to limit the spread of resistance.

Like many other Gram-negative bacteria, Burkholderia cepacia naturally releases membrane vesicles (n-MVs) during normal growth. Through filtration and differential centrifugation, n-MVs from clinical isolates of the IIIa and V genomovars were isolated and their characteristics compared. Electron microscopy revealed that they were spherical, 30-220 nm in diameter, and bilayered. Virulence factors thought to play a role in pathogenicity (e.g., lipase, phospholipase-N, and protease, including a metalloprotease) were found associated with n-MVs, while peptidoglycan zymogram analysis also revealed 26, 28, 36, and 66 kDa peptidoglycan-degrading enzymes. n-MVs were often contaminated with flagella and pili when isolated by traditional methods, and a new strategy using a linear isopycnic sucrose gradient was utilized. For better characterization, this was applied to a representative genomovar IIIa strain (C5424) and showed that n-MVs consisted of a subset of specific outer membrane and periplasmic proteins as well as lipopoly saccharide possessing only a putative minor O-side chain polymer. This finding suggests that certain components are selected by B. cepacia during n-MV formation, and since some are putative virulence factors, this property could help deliver the factors to tissue, thereby aiding infection.