Antimicrobial and Biocide Resistance among Feline and Canine Staphylococcus aureus and Staphylococcus pseudintermedius Isolates from Diagnostic Submissions.

A total of 114 Staphylococcus isolates from various infections of companion animals, including 43 feline Staphylococcus aureus, 19 canine S. aureus, 11 feline Staphylococcus pseudintermedius and 41 canine S. pseudintermedius were investigated for (i) their susceptibility to 24 antimicrobial agents and three combinations of antimicrobial agents by broth microdilution following CLSI recommendations and (ii) the corresponding resistance genes. In addition, the isolates were tested for their susceptibility to the four biocides benzalkonium chloride, chlorhexidine, polyhexanide and octenidine by a recently developed biocide susceptibility testing protocol. Penicillin resistance via blaZ was the dominant resistance property in all four groups of isolates ranging between 76.7 and 90.9%. About one quarter of the isolates (25.4%) proved to be methicillin-resistant and carried the genes mecA or mecC. Macrolide resistance was the second most prevalent resistance property (27.2%) and all isolates harbored the resistance genes erm(A), erm(B), erm(C), erm(T) or msr(A), alone or in combinations. Fluoroquinolone resistance was detected in 21.1% of all isolates tested, whereas tetracycline resistance via tet(K) and/or tet(M) occurred in 19.3% of the isolates. Resistance to last resort antimicrobial agents in human medicine was seen only in single isolates, if at all. The minimal inhibitory concentrations (MICs) of the four biocides showed unimodal distributions and were very similar for the four groups of staphylococci. Because of the large number of (multi)resistant isolates, antimicrobial susceptibility testing of feline and canine S. aureus and S. pseudintermedius isolates is highly recommended before the start of an antimicrobial chemotherapy. Moreover, no hints towards the development of biocide resistance were detected.

Antimicrobial and Biocide Resistance among Canine and Feline Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii Isolates from Diagnostic Submissions.

A total of 215 isolates from infections of dogs and cats, including 49 Enterococcus faecalis, 37 Enterococcus faecium, 59 Escherichia coli, 56 Pseudomonas aeruginosa, and 14 Acinetobacter baumannii, were investigated for their susceptibility to 27 (Gram-positive bacteria) or 20 (Gram-negative bacteria) antimicrobial agents/combinations of antimicrobial agents by broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute. Moreover, all isolates were analysed for their susceptibility to the biocides benzalkonium chloride, chlorhexidine, polyhexanide, and octenidine by a recently published broth microdilution biocide susceptibility testing method. While the E. faecalis isolates did not show expanded resistances, considerable numbers of the E. faecium isolates were resistant to penicillins, macrolides, tetracyclines, and fluoroquinolones. Even a single vancomycin-resistant isolate that carried the vanA gene cluster was detected. Expanded multiresistance phenotypes were also detected among the E. coli isolates, including a single carbapenem-resistant, blaOXA-48-positive isolate. In addition, multiresistant A. baumannii isolates were detected. The minimal inhibitory concentrations of the biocides showed unimodal distributions but differed with respect to the biocide and the bacterial species investigated. Although there were no indications of a development of biocide resistance, some P. aeruginosa isolates exhibited benzalkonium MICs higher than the highest test concentration.

Development of Quality Control Ranges for Biocide Susceptibility Testing.

Every laboratory test needs validation by quality controls. For biocide susceptibility testing (BST), neither quality control (QC) strains nor QC ranges applicable to these strains are currently available. As QC strains, four well-defined laboratory reference strains (Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442), which have been used previously for biocide efficacy testing, were selected. In an interlaboratory trial with eleven participating laboratories, BST QC ranges should be developed for the aforementioned four strains and the four biocides benzalkonium chloride, chlorhexidine, octenidine and polyhexanide. The performance of three different lots of tryptic soy broth was explored using the broth microdilution method and the data were subsequently evaluated using the RangeFinder software. As a result, QC ranges were defined for all reference strain-biocide combinations, except for P. aeruginosa ATCC® 15442 with the two biocides chlorhexidine and polyhexanide. The development of the latter two QC ranges was not possible, due to the limited solubility of the biocides in the test range required for P. aeruginosa ATCC® 15442. The newly developed QC ranges comprise three to five dilution steps. The establishment of QC ranges will contribute to the validation of BST in the future.

Comparison of two methods for cell count determination in the course of biocide susceptibility testing.

The inoculum density is an important parameter for numerous experimental approaches in bacteriology, including antimicrobial susceptibility testing (AST), biocide susceptibility testing (BST) and biocide efficacy testing (BET). Methods to determine the inoculum density commonly refer to cell counts and have been described for BET according to the German Medical Veterinary Society (Deutsche Veterinärmedizinische Gesellschaft, DVG) and for AST according to the Clinical and Laboratory Standards Institute (CLSI). In this study, the DVG method using 1000 µL volumes of two different dilution steps and the AST method according to CLSI using a 100 µL volume of a single dilution step from the inoculum suspension were compared. For this, each of the four reference strains, Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442, was comparatively tested 28 times using the inoculum preparation according to DVG. The results were statistically analysed using Bland-Altman plots and 95 % limits of agreement (AL). Moreover, cell counts were correlated with the optical density of the bacterial suspensions used. In comparison, the CLSI method measured lower values for colony-forming units (CFU) of -0.12 log10 compared to the DVG method. Overall, both methods returned an AL of -0.52 to 0.27 log10. Since the variations observed between the two methods were within one log10 step and the measured CFUs did not differ systematically, both methods proved to be suitable for cell count determination. Therefore, the CLSI method, which is less complex and less time-consuming, is recommended.

The welfare impacts of mechanical and manual broiler catching and of circumstances at loading under field conditions.

Loading of broilers for transport to the processing plant poses a notable injury risk for broilers. Therefore, the poultry industry has developed mechanical methods as alternatives to manual loading methods. Our objective in the present study was to compare manual loading (MAN) of broilers with the mechanical loading (MECH). We assessed the injuries of broilers of 12 MAN and 12 MECH flocks on-farm before and immediately after loading, documented the numbers of broilers dead on arrival reported by the processing plant, and assessed the circumstances at loading. A smaller number of broilers with a hematoma (≥0.5 cm in diameter) on the wing were observed after MAN compared with MECH using the examined harvester (MAN vs. MECH odds ratio: 0.16; 95% confidence interval: 0.10, 0.28). The number of broilers with severe wing injuries did not differ between the loading methods. The number of broilers dead on arrival was greater in mechanically loaded flocks (MAN vs. MECH odds ratio: 0.26; 95% confidence interval: 0.10, 0.68), but lower than in comparable studies. We observed a lower average stocking rate than targeted in the drawers of MECH containers, most likely because the used harvester can adapt to short-term changes in weight and adjust the stocking rate during the loading process. A longer total loading duration in MAN was associated with an increase of wing hematomas, and the involvement of more working people per 10,000 broilers during MAN was associated with a lower occurrence of hematomas. The total loading duration in MECH had no notable influence on the occurrence of injuries. Physical conditions of the involved personnel might play a larger role in MAN than in MECH. The harvester that was examined should be further developed to reduce the occurrence of hematomas. Our results indicate that the choice of loading method alone does not determine the injury risk, and multiple factors are associated with broiler welfare during loading. It is important that the chosen method is performed under the most adequate conditions.

Biocide susceptibility testing of bacteria: Development of a broth microdilution method.

Biocide susceptibility testing (BST) of bacteria lacks standardised methods. Based on a recently established broth macrodilution BST method, a broth microdilution method for BST was developed. To establish the respective protocol, four reference strains Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442 were investigated for their minimal inhibitory concentrations (MICs) towards quaternary ammonium compounds (benzalkonium chloride), cationic compounds (chlorhexidine), aldehydes (glutardialdehyde) and alcohols (isopropanol) using tryptic soy broth. All combinations of (i) inoculum preparation according to the German Veterinary Medical Society (DVG) or the Clinical and Laboratory Standards Institute (CLSI) with some modifications, (ii) use of 1st subculture (SC) and 2nd SC, (iii) direct colony suspension (DCS) with/without glass beads, and (iv) incubation at 37 °C for 24 h, 48 h, and 72 h were compared using seven independent replications. Overall, the reproducibility was high for all abovementioned strain/biocide/parameter combinations. In total, 86.9 % - 100 % of the results were within ± one dilution step of the mode value. The proposed method for a standardised BST protocol comprises (i) two different inoculum densities, (ii) the use of a fresh overnight culture (1st SC or 2nd SC), (iii) the preparation of the inoculum suspension by either of the two methods using DCS with or without glass beads, and (iv) the incubation at 37 °C for 24 h. This broth microdilution method will help to harmonize BST of bacterial pathogens in routine diagnostics.

Development and evaluation of a broth macrodilution method to determine the biocide susceptibility of bacteria.

In comparison to biocide efficacy testing, biocide susceptibility testing of bacteria so far lacks standardized methods for routine use. The aims of the present study were to develop a broth macrodilution method to test bacterial pathogens for their biocide susceptibility and to evaluate this method in an interlaboratory trial. Staphylococcus aureus ATCC®6538 was tested for its susceptibility to benzalkonium chloride, chlorhexidine and isopropanol comparing test strain suspension preparations, test volumes and incubation times. The use of 2 mL volumes for the testing and an incubation time of 24 h were proposed. Ten German laboratories participated in the interlaboratory trial. Four reference strains (S. aureus ATCC®6538, Enterococcus hirae ATCC®10541, Escherichia coli ATCC®10536 and Pseudomonas aeruginosa ATCC®15442) commonly used for biocide activity testing, were included. Strains were tested three times at independent occasions for their susceptibility to benzalkonium chloride, glutardialdehyde and isopropanol. In total, 360 data points were obtained (30 per strain/biocide combination). The modal minimal inhibitory concentration ± one dilution step was defined as acceptable range. For the four reference strains and the three biocides 80-100% of the values were considered as acceptable. The deviations within the laboratories for a strain/biocide combination were rather consistent. In general, the testing was performed without difficulties by the laboratories. Although inoculum plate counts of four laboratories were outside the acceptable range, this did not have a large impact on the results. The proposed method was stable and easy to perform. It may contribute to a harmonization and standardization of biocide susceptibility testing.