Melatonin improves rate of monospermic fertilization and early embryo development in a bovine IVF system.

The developmental competence of male and female gametes is frequently reduced under in vitro conditions, mainly due to oxidative stress during handling. The amino-acid derived hormone melatonin has emerged as a potent non-enzymatic antioxidant in many biological systems. The goal of the present study was to evaluate the effects of melatonin on post-thaw sperm quality, fertilizing ability, and embryo development and competence in vitro after in vitro fertilization. Frozen-thawed bovine spermatozoa were incubated either in the presence of 10-11 M melatonin (MT), or its solvent (ethanol; Sham-Control), or plain Tyrode's Albumin Lactate Pyruvate medium (TALP, Control). Computer-Assisted Sperm Analysis (CASA) and flow cytometry data after 30 min, 120 min, and 180 min incubation did not reveal any significant effects of melatonin on average motility parameters, sperm subpopulation structure as determined by hierarchical cluster, or on the percentage of viable, acrosome intact sperm, or viable sperm with active mitochondria. Nevertheless, in vitro matured cumulus-oocyte-complexes fertilized with spermatozoa which had been preincubated with 10-11 M melatonin (MT-Sperm) showed higher (P < 0.01) rates of monospermic fertilization, reduced (P < 0.05) polyspermy and enhanced (P < 0.05) embryo development compared to the Control group. Moreover, the relative abundance of MAPK13 in the in vitro-derived blastocysts was greater (P < 0.05) than observed in the Control group. In conclusion, adding melatonin to the sperm-preparation protocol for bovine IVF improved proper fertilization and enhanced embryonic development and competence in vitro.

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.

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.

Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) among employees and in the environment of a small animal hospital.

The aim of the study was to investigate methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) among employees of a small animal hospital and the hospital environment. In total, 96 swabs from employees and 73 swabs from the clinic environment were investigated. Cation-adjusted-Mueller-Hinton broth (CAMHB) + 6.5% NaCl was used for enrichment before plating on Mueller-Hinton (MH) agar with 2% NaCl and 0.25 mg/L oxacillin. The staphylococcal species was determined using MALDI-TOF MS. The isolates were subjected to mecA-PCR, macrorestriction analysis, and antimicrobial susceptibility testing. MRSA were present in five nasal swabs of the 55 employees tested and in six environmental samples, MRSP in two employees (nasal and hand swabs, each) and in three environmental samples. All isolates harboured mecA. Susceptibility testing revealed that all but one of the isolates were multiresistant. All isolates were resistant to ß-lactams and fluoroquinolones. All but one of the isolates were resistant to macrolides and lincosamides. A single MRSA was resistant to gentamicin. All MRSP were resistant to trimethoprim/sulfamethoxazole and non-susceptible to gentamicin. One isolate was also resistant to tetracycline. Macrorestriction analysis revealed three main SmaI patterns for MRSA and two main SmaI patterns for MRSP. All environmental isolates were found in areas of high people and animal traffic, such as dog ward areas, waiting and triage rooms. The finding of indistinguishable MRSA or MRSP among employees and in the environment of the small animal hospital suggests the possibility of transfer of these bacteria between humans, animals, and the hospital environment.