Antibiotic prophylaxis and hospitalization of horses subjected to median laparotomy: gut microbiota trajectories and abundance increase of Escherichia.

Introduction: Horse clinics are hotspots for the accumulation and spread of clinically relevant and zoonotic multidrug-resistant bacteria, including extended-spectrum ß-lactamase producing (ESBL) Enterobacterales. Although median laparotomy in cases of acute equine colic is a frequently performed surgical intervention, knowledge about the effects of peri-operative antibiotic prophylaxis (PAP) based on a combination of penicillin and gentamicin on the gut microbiota is limited. Methods: We collected fecal samples of horses from a non-hospitalized control group (CG) and from horses receiving either a pre-surgical single-shot (SSG) or a peri-operative 5-day (5DG) course of PAP. To assess differences between the two PAP regimens and the CG, all samples obtained at hospital admission (t0), on days three (t1) and 10 (t2) after surgery, were screened for ESBL-producing Enterobacterales and subjected to 16S rRNA V1-V2 gene sequencing. Results: We included 48 samples in the SSG (n = 16 horses), 45 in the 5DG (n = 15), and 20 in the CG (for t0 and t1, n = 10). Two samples of equine patients receiving antibiotic prophylaxis (6.5%) were positive for ESBL-producing Enterobacterales at t0, while this rate increased to 67% at t1 and decreased only slightly at t2 (61%). Shannon diversity index (SDI) was used to evaluate alpha-diversity changes, revealing there was no significant difference between horses suffering from acute colic (5DG, SDImean of 5.90, SSG, SDImean of 6.17) when compared to the CG (SDImean of 6.53) at t0. Alpha-diversity decreased significantly in both PAP groups at t1, while at t2 the onset of microbiome recovery was noticed. Although we did not identify a significant SDImean difference with respect to PAP duration, the community structure (beta-diversity) was considerably restricted in samples of the 5DG at t1, most likely due to the ongoing administration of antibiotics. An increased abundance of Enterobacteriaceae, especially Escherichia, was noted for both study groups at t1. Conclusion: Colic surgery and PAP drive the equine gut microbiome towards dysbiosis and reduced biodiversity that is accompanied by an increase of samples positive for ESBL-producing Enterobacterales. Further studies are needed to reveal important factors promoting the increase and residency of ESBL-producing Enterobacterales among hospitalized horses.

New variant strain of Streptococcus canis with Lancefield group C isolated from canine otitis externa.

Every basic course in microbiology teaches us, Streptococcus canis always tests positive for Lancefield group G. Surprisingly, we identified a strain of S. canis with Lancefield group C, cultured from a dog with otitis externa after lateral ear canal resection. Whole genome sequencing data and analysis points towards a horizontal gene transfer event between S. canis and S. dysgalactiae. Although these species are closely related, gene transfer in this region of the genome of S. canis has not been described before. The value of technologies as MALDI-TOF MS and sequencing in microbiological diagnostics will grow as more diverse streptococci arise that do not always conform anymore to the classical Lancefield group typing.

How to survive pig farming: Mechanism of SCCmec element deletion and metabolic stress adaptation in livestock-associated MRSA.

Previous research on methicillin susceptible Staphylococcus aureus (MSSA) belonging to livestock-associated (LA-) sequence type (ST) 398, isolated from pigs and their local surroundings, indicated that differences between these MSSA and their methicillin resistant predecessors (MRSA) are often limited to the absence of the staphylococcal cassette chromosome mec (SCCmec) and few single nucleotide polymorphisms. So far, our understanding on how LA-MRSA endure the environmental conditions associated with pig-farming as well as the putative impact of this particular environment on the mobilisation of SCCmec elements is limited. Thus, we performed in-depth genomic and transcriptomic analyses using the LA-MRSA ST398 strain IMT38951 and its methicillin susceptible descendant. We identified a mosaic-structured SCCmec region including a putative replicative SCCmecVc which is absent from the MSSA chromosome through homologous recombination. Based on our data, such events occur between short repetitive sequences identified within and adjacent to two distinct alleles of the large cassette recombinase genes C (ccrC). We further evaluated the global transcriptomic response of MRSA ST398 to particular pig-farm associated conditions, i.e., contact with host proteins (porcine serum) and a high ammonia concentration. Differential expression of global regulators involved in stress response control were identified, i.e., ammonia-induced alternative sigma factor B-depending activation of genes for the alkaline shock protein 23, the heat shock response and the accessory gene regulator (agr)-controlled transcription of virulence factors. Exposure to serum transiently induced the transcription of distinct virulence factor encoding genes. Transcription of genes reported for mediating the loss of methicillin resistance, especially ccrC, was not significantly different compared to the unchallenged controls. We concluded that, from an evolutionary perspective, bacteria may save energy by incidentally dismissing a fully replicative SCCmec element in contrast to the induction of ccr genes on a population scale. Since the genomic SCCmec integration site is a hot-spot of recombination, occasional losses of elements of 16 kb size may restore capacities for the uptake of foreign genetic material. Subsequent spread of resistance, on the other hand, might depend on the autonomous replication machinery of the deleted SCCmec elements that probably enhance chances for reintegration of SCCmec into susceptible genomes by mere multiplication.

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.

Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from zoo and wild animals.

Antimicrobial resistance of Staphylococcus aureus is a major problem in human and veterinary medicine. The aim of this study was to characterise S. aureus isolates from wild and zoo animals mainly associated with bacterial infections. In total, 23 S. aureus isolates, including nine from wild animals and 14 from zoo animals, were obtained during routine diagnostics. All isolates were subjected to multilocus sequence typing (MLST), spa typing, macrorestriction analysis with subsequent SmaI pulsed-field gelelectrophoresis (PFGE), antimicrobial susceptibility testing and S. aureus-specific DNA-microarray analysis. Resistant isolates were also tested for their respective resistance genes by PCR. Isolates from zoo animals and wildlife showed a high diversity of MLST types, spa types and PFGE patterns. Nineteen different spa types were identified, including three novel types and 16 main macrorestriction patterns. Only few isolates were resistant to members of four classes of antimicrobial agents and harboured the respective resistance genes (ß-lactams [blaZ, mecA, mecC], tetracyclines [tet(K), tet(L)] and chloramphenicol [catpC221]) or mutations (fluoroquinolones). The DNA microarray analysis identified one isolate from a zoo animal harbouring the toxic shock syndrome toxin gene tst1. Moreover, several enterotoxin genes were detected in five S. aureus isolates. All isolates were negative for Panton-Valentine leukocidin (PVL) genes, but the animal-associated leukocidin genes lukM/lukF-P83 were found in three isolates from two animals.