The Canine Vaginal Flora: A Large-Cohort Retrospective Study.

Microbiological examinations are frequently performed as part of breeding management examinations in the bitch, but also in case of (suspected) reproductive tract problems. As most bacteria are opportunistic pathogens, evaluation of bacterial findings is challenging for veterinarians. Besides, breeders might request antimicrobial treatment in breeding bitches, fearing conception failure-even without medical indication. Considering the rising threat of antimicrobial resistance, gaining deeper insights into the bacterial findings from the vagina of healthy and (suspected) reproductive-diseased bitches might contribute to the knowledge of the canine aerobic vaginal flora and consequently improve the responsible use of antibiotics. We analyzed results from bacteriological cultures of 23,254 vaginal swabs sent in to three commercial laboratories in Germany between 2015 and 2021, where standard aerobic microbiological examination was carried out. We found a variety of 319 bacterial species that mostly grew in mixed cultures of two or more bacterial species. Commonly found species were Escherichia coli, beta-hemolytic Streptococci, coagulase-positive Staphylococci, Pasteurellales, and aerobic sporulators, as well as other Streptococcus spp. Our results showed a large diversity of the canine vaginal flora in healthy and (suspected) reproductive-diseased bitches. They largely support earlier findings of small studies on the physiological canine vaginal flora, emphasizing that solely the results of a bacterial evaluation should not be the base for antimicrobial treatment. Instead, bacterial findings should be evaluated with the results of a clinical gynecological examination.

Intervention with impact: Reduced isolation of methicillin-resistant Staphylococcus pseudintermedius from dogs following the introduction of antimicrobial prescribing legislation in Germany.

BACKGROUND: Legislation was introduced in Germany in 2018, requiring bacterial culture and antimicrobial susceptibility testing before the prescription of fluoroquinolones and third-generation cephalosporins to dogs. We hypothesised that, following this intervention, the number of clinical samples testing positive for methicillin-resistant Staphylococcus pseudintermedius (MRSP) would reduce. METHODS: Reports of S. pseudintermedius isolated from canine clinical samples by three German veterinary diagnostic microbiology laboratories during the 38 months before the introduction of the legislation and the 46 months after were compared. Bacterial identification was performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and antimicrobial susceptibility testing followed recognised recommendations but with changes during the study period. RESULTS: Among a total of 120,571 S. pseudintermedius isolates, MRSP accounted for 7.1% overall. Following the legislative intervention, monthly submissions yielding S. pseudintermedius increased at all three laboratories. The MRSP percentage was lower in the period after the intervention in two of the three laboratories (p < 0.001); in the third laboratory, there was no change between periods, but a year-on-year reduction in MRSP percentages occurred after the intervention (p = 0.0004). LIMITATIONS: Changing susceptibility testing methods limited the direct comparison of resistance patterns among laboratories. CONCLUSION: The reduction in MRSP in canine clinical samples following the introduction of this legislation suggests a positive impact of compulsory laboratory testing on reducing antimicrobial resistance.

[Occurrence and resistance of bacteria isolated from the rabbit nares - a retrospective evaluation]. / Vorkommen und Resistenzen von Bakterien in Proben aus Kaninchennasen ­ eine retrospektive Auswertung.

OBJECTIVE: Rabbit snuffles is one of the most common challenges in veterinary practice. The aim of the present study was to evaluate nasal samples of rabbits submitted between 2015-2019, with regard to bacterial distribution and antimicrobial resistance. MATERIAL AND METHODS: Each sample was plated on four different agar plates and enriched in a non-selective broth. Isolates were identified by MALDI Biotyper® (MBT) (Bruker Daltonik GmbH, Bremen, Germany) and antimicrobial susceptibility was performed by broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI, Wayne, PA, USA). RESULTS: A total of 1261 samples were evaluated. Among the samples that tested positive (n=941), one bacterial species was detected in 79.1% of the cases, and more than one bacterial species (mixed culture) was found in 20.9% of the cases. A total of 150 species from 14 families were identified. Isolates belonging to the family Pasteurellaceae were identified most frequently, followed by Enterobacteriaceae, Pseudomonadaceae and Staphylococcaceae.A total of 467 antibiograms of the most common pathogens with possible clinical relevance (Pasteurella multocida [14.6%], Pasteurella species [10.0%], Staphylococcus aureus [5.9%], Pseudomonas aeruginosa [5.4%] and Bordetella bronchiseptica [4.8%]) were evaluated. Quinolones showed the highest efficacy and clindamycin the lowest. Furthermore, among S. aureus, MRSA were most frequently detected in 2016 reaching 23.1% of cases. CONCLUSIONS AND CLINICAL RELEVANCE: Since the causal bacteria for rabbit snuffles are mostly found in the deeper areas of the nose and the nasal vestibule is often contaminated with ubiquitous and coliform bacteria, it would make sense to take samples from the depth of the nasal cavity, ideally via nasal lavage. Due to the demonstrated pathogen diversity and long-term therapy associated with the disease, bacterial culture and sensitivity testing is recommended as part of the management. In the absence of an antibiogram, enrofloxacin is the drug of first choice due to its favorable resistance pattern and tolerability. However, since quinolones are considered as "critically important" antibiotics, their use should be limited to a minimum.

[The importance of wildlife as reservoir of antibiotic-resistant bacteria in Bavaria--first results]. / Die Bedeutung von Wildtieren als Reservoir für antibiotikaresistente Erreger in Bayern--erste Ergebnisse.

The use of antimicrobial agents is responsible for the emergence and spread of antibiotic resistant bacteria. Nevertheless, multiresistant bacteria have been found in animals that have never been exposed to antimicrobial agents. Wild animals that are carriers of methicillin-resistant organisms represent a hazard since they can transmit their bacteria to other animals and to humans. In the hunting season 2009/2010 nasal swabs of 98 red deer and 109 wild boars were examined for the presence of methicillin-sensitive and methicillin-resistant staphylococci. From each wild boar methicillin-susceptible staphylococci (Staphylococcus aureus in 28% and Staphylococcus spp. in 72% of the animals) were isolated. In red deer the detection rate of Staphylococcus (S.) aureus and methicillin-susceptible staphylococci was 49% and 17%, respectively. The occurrence of S. aureus was significantly higher (p < 0.05) in red deer than in wild boars. Methicillin-resistant staphylococci were not found. However, in one third of the red deer, methicillin-resistant bacteria of the genus Enterococcus spp. and Bacillus spp. were isolated. The results of the present study indicate that wildlife, especially red deer are an important reservoir for S. aureus and that the upper respiratory tract of red deer is regularly colonised with methicillin-resistant bacteria such as Bacillus spp. and Enterococcus spp. Primarily, commensal bacteria are harmless to human health, however, red deer may be a reservoir for antibiotic-resistant bacteria.

High frequency of multiresistant coagulase-positive Staphylococcus aureus found in slaughter pigs in Uruguay.

Staphylococcus aureus are a hazard to human health since they can cause infections and food poisoning. Antimicrobial resistant strains render the treatment of infections problematic and contribute to the spread of antimicrobial resistance. They are therefore of great public concern. This study determined the resistance pattern of coagulase-positive S. aureus (CPSA) isolated from nasal swabs of 100 slaughter pigs from one farm in Uruguay. Out of 69 animals, 71 CPSA were collected. Minimum inhibitory concentrations of 20 antimicrobials were determined using the broth microdilution method in accordance with CLSI recommendations. No methicillin-resistant S. aureus were detected. All CPSA were resistant to three or more classes of antimicrobials (i.e., multiresistant), whereby all CPSA were resistant to spectinomycin. Most of the isolates (46%) were resistant to six classes of antimicrobials. Almost all isolates were resistant to penicillin (99%), ampicillin (99%), gentamicin (96%), tetracycline (90%), and tilmicosin (87%). Very high resistance rates were observed against erythromycin (77%) and clindamycin (70%). High resistance was observed against tiamulin (40%), enrofloxacin (31%), and florfenicol (23%) and low resistance was observed against amoxicillin/clavulanic acid (4%). All CPSA isolates were mecA negative. The results of the present study could be related to an overuse of antimicrobials in pig production and should encourage veterinarians and pig holders to practice a controlled administration of chemotherapeutics in pig husbandry.

Antimicrobial susceptibility and distribution of ß-lactamase A (blaA) and ß-lactamase B (blaB) genes in enteropathogenic Yersinia species.

One hundred eighty-six strains of enteropathogenic Yersinia (Y.) enterocolitica of bioserotypes 2/O:5,27, 2/O:9, 3/O:3, and 4/O:3 and 12 strains of Yersinia pseudotuberculosis of bioserotypes 1/O:1, 1/O:2, and 2/O:1 from different human (feces) and nonhuman (pig, pork, wild boar, monkey, chinchilla, mara, capybara, salad) sources collected in the years 1995-2009 were examined. Antimicrobial resistance patterns for 12 antimicrobial agents were generated using broth microdilution. The presence and characterization of the ß-lactamase genes blaA and blaB were studied using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP), respectively. The expression of ß-lactamase BlaA and BlaB was detected using double-disc diffusion. Y. enterocolitica strains showed resistance to ampicillin (92%), streptomycin (13%), and sulfamethoxazole (2%). Intermediate susceptibility to tetracycline was shown by two Y. enterocolitica strains. All Y. pseudotuberculosis strains were susceptible to all tested antimicrobial agents. Most (99%) of the Y. enterocolitica strains carried both ß-lactamase genes. One strain of bioserotype 3/O:3 lacked both genes. In contrast, all Y. pseudotuberculosis strains carried neither of the ß-lactamase genes. Homogeneity was detected in all blaA and blaB genes of Y. enterocolitica using PCR-RFLP. The majority (89%) of Y. enterocolitica strains expressed both ß-lactamase enzymes, whereas none of the Y. pseudotuberculosis strains showed expression of either enzyme. Also, it seems that the resistance of Y. enterocolitica has not changed during the last years.

Comparison of broth microdilution and disk diffusion test for antimicrobial resistance testing in Yersinia enterocolitica 4/O:3 strains.

The aim of this study was to compare the results obtained from two methods for the determination of antimicrobial resistance in 110 Yersinia enterocolitica 4/O:3 strains. Ten antimicrobial agents were tested using broth microdilution and disk diffusion. Similar results were determined for six antimicrobials. Very major errors (false-susceptible by disk diffusion test) were detected for ampicillin (at a rate of 1.8%). Major errors (false-resistant by disk diffusion test) were found for streptomycin (0.9%) and sulfamethoxazole (1.8%). Minor errors (intermediate susceptible by disk diffusion and resistant or susceptible by microdilution) were obtained for ampicillin (2.7%) and sulfamethoxazole (13.6%). All Y. enterocolitica were resistant to at least one antimicrobial agent. Resistances to three classes of antimicrobial agents were obtained by 3% of the strains included in the study. A slightly higher frequency of multiresistance was obtained by disk diffusion (3%) compared with broth microdilution (1%). Resistance to streptomycin was found frequently (13%); in contrast, resistance to tetracycline was rare (1%). The disk diffusion test produced unacceptably high rates of very major errors for ampicillin and a high frequency of minor errors for sulfamethoxazole. Susceptibility tests should thus be carried out by the more reliable method of microdilution. Most of the antimicrobials that can be used for therapy were very effective when tested against Y. enterocolitica. In order to identify changes in susceptibilities as early as possible, antimicrobial resistance in Y. enterocolitica should be regularly surveyed.

Detection of Listeria monocytogenes in pork and beef using the VIDAS® LMO2 automated enzyme linked immunoassay method.

Listeria (L.) monocytogenes, a foodborne pathogen, is known to be a possible contaminant of foods during production and processing. Samples (n=985) of raw meat and by-products obtained from beef and pork were first screened by the VIDAS system for the presence of Listeria spp., followed by testing for the presence of L. monocytogenes. Positive L. monocytogenes results were confirmed by plating on selective agars: 14% of the samples were positive for Listeria and 4% tested positive for L. monocytogenes, of which 3% were confirmed on selective agars. In by-products (17%) the contamination with listeriae was higher than in meat cuts (10%). Only samples strongly positive for Listeria spp. by VIDAS were positive for L. monocytogenes. Overall, the prevalence of L. monocytogenes in beef and pork samples was rather low in comparison to most previous studies. The VIDAS system was shown to be a suitable method for screening out Listeria-negative samples; the main advantage being a markedly reduced assay time.

The degree of mycoheterotrophic carbon gain in green, variegated and vegetative albino individuals of Cephalanthera damasonium is related to leaf chlorophyll concentrations.

• Achlorophyllous variants of some forest orchids are known to reach almost the same size as their green forms. These vegetative albino forms cover their entire carbon (C) demand through fungi that simultaneously form ectomycorrhizae with trees, while green variants partially draw on C from photosynthesis and C from fungal hosts. Here, we investigate whether the amount of C derived from either source is proportional to leaf chlorophyll concentration. The discovery of two Cephalanthera damasonium populations with variegated leaves enabled a continuous bridging of leaf chlorophyll concentrations between green and albino forms. • Leaves of 27 green, variegated and albino individuals of C. damasonium were compared for chlorophyll concentrations, C sources (as characterized by (13)C abundances) and total C and nitrogen (N) concentrations. • We found a linear relationship between leaf chlorophyll concentrations and the proportional reliance on fungi as a C source. Furthermore, we show that the shift in C gain through mycoheterotrophic means significantly changes leaf total C and N concentrations. • Our results document that partial mycoheterotrophy in C. damasonium is not a static nutritional mode but a flexible mechanism related inter alia to leaf chlorophyll concentrations. The change in proportional reliance on fungi as a C source affects leaf chemical composition.

Salmonella in raw meat and by-products from pork and beef.

After campylobacteriosis, salmonellosis is the second main cause of human bacterial enteritis in Germany. Salmonella is known to colonize the gastrointestinal tract of animals without producing any clinical signs. Therefore, carcasses can become contaminated with Salmonella at the time of slaughter. During an 11-month period, a total of 4,170 raw meat samples and by-products from beef and pork, obtained from seven different slaughterhouses in Southern Germany, were screened by the VIDAS system for Salmonella in this study. Positive results were confirmed by isolation of the pathogen on selective agars. The overall percentage of Salmonella-positive samples was 1.4% by the VIDAS system and 0.7% by culture confirmation. Salmonella was detected in 1.8% of pork samples by the VIDAS system and in 1.1% of samples by culture. In beef samples the presence of Salmonella was verified in 0.6% of samples by the VIDAS system and in 0.1% by culture on selective agars. The highest contamination rates were found in porcine and bovine tongue samples. Salmonella was detected in porcine samples throughout the year, except in samples collected in July, and a slight increase was observed in the colder months. The VIDAS system was shown to be an efficient screening method for the detection of Salmonella, with the advantage of a reduced analysis time.

Role of DNA protection and repair in resistance of Bacillus subtilis spores to ultrahigh shock pressures simulating hypervelocity impacts.

Impact-induced ejections of rocks from planetary surfaces are frequent events in the early history of the terrestrial planets and have been considered as a possible first step in the potential interplanetary transfer of microorganisms. Spores of Bacillus subtilis were used as a model system to study the effects of a simulated impact-caused ejection on rock-colonizing microorganisms using a high-explosive plane wave setup. Embedded in different types of rock material, spores were subjected to extremely high shock pressures (5 to 50 GPa) lasting for fractions of microseconds to seconds. Nearly exponential pressure response curves were obtained for spore survival and linear dependency for the induction of sporulation-defective mutants. Spores of strains defective in major small, acid-soluble spore proteins (SASP) (alpha/beta-type SASP) that largely protect the spore DNA and spores of strains deficient in nonhomologous-end-joining DNA repair were significantly more sensitive to the applied shock pressure than were wild-type spores. These results indicate that DNA may be the sensitive target of spores exposed to ultrahigh shock pressures. To assess the nature of the critical physical parameter responsible for spore inactivation by ultrahigh shock pressures, the resulting peak temperature was varied by lowering the preshock temperature, changing the rock composition and porosity, or increasing the water content of the samples. Increased peak temperatures led to increased spore inactivation and reduced mutation rates. The data suggested that besides the potential mechanical stress exerted by the shock pressure, the accompanying high peak temperatures were a critical stress parameter that spores had to cope with.

Epidemiological data on pathogenic Yersinia enterocolitica in Southern Germany during 2000-2006.

Yersinia enterocolitica is the most common species causing enteric yersiniosis, which is still the third most frequently reported foodborne gastroenteritis in Europe. Y. enterocolitica generally causes sporadic human infections, and outbreaks are rare. The most important infection source of yersiniosis is believed to be contaminated pork and pork products. Data on the prevalence of pathogenic Y. enterocolitica in animals and foodstuffs are very limited and old; thus, more information on the extent and range of the prevalence of this enteropathogen in nonhuman sources is needed. In this work, prevalence of pathogenic Y. enterocolitica in different sources in Bavaria is presented. Further, the antimicrobial resistance of human and nonhuman strains is reported. The highest isolation rate of pathogenic Y. enterocolitica (67%) was found in tonsils of slaughter pigs. No pathogenic strains were isolated from cattle, sheep, turkey, and horses. ail-Positive Y. enterocolitica was detected in dogs (5%), cats (3%), and rodents (3%) by real-time PCR. Pathogenic Y. enterocolitica was isolated only from raw pork, especially from edible offal (51%). Surprisingly, 38% of game was contaminated with this pathogen when the samples were studied with PCR. Additionally, some raw pork sausages and one poultry sample were PCR positive. All pathogenic Y. enterocolitica isolates from nonhuman sources were belonging to bioserotype 4/O:3. Antimicrobial resistance of 60 human and 140 porcine strains of bioserotype 4/O:3 was tested by the agar disc diffusion method to 15 different antimicrobial agents. All Y. enterocolitica 4/O:3 strains were susceptible to most of the tested antibacterial agents.

Microbial rock inhabitants survive hypervelocity impacts on Mars-like host planets: first phase of lithopanspermia experimentally tested.

The scenario of lithopanspermia describes the viable transport of microorganisms via meteorites. To test the first step of lithopanspermia, i.e., the impact ejection from a planet, systematic shock recovery experiments within a pressure range observed in martian meteorites (5-50 GPa) were performed with dry layers of microorganisms (spores of Bacillus subtilis, cells of the endolithic cyanobacterium Chroococcidiopsis, and thalli and ascocarps of the lichen Xanthoria elegans) sandwiched between gabbro discs (martian analogue rock). Actual shock pressures were determined by refractive index measurements and Raman spectroscopy, and shock temperature profiles were calculated. Pressure-effect curves were constructed for survival of B. subtilis spores and Chroococcidiopsis cells from the number of colony-forming units, and for vitality of the photobiont and mycobiont of Xanthoria elegans from confocal laser scanning microscopy after live/dead staining (FUN-I). A vital launch window for the transport of rock-colonizing microorganisms from a Mars-like planet was inferred, which encompasses shock pressures in the range of 5 to about 40 GPa for the bacterial endospores and the lichens, and a more limited shock pressure range for the cyanobacterium (from 5-10 GPa). The results support concepts of viable impact ejections from Mars-like planets and the possibility of reseeding early Earth after asteroid cataclysms.

The ectomycorrhizal specialist orchid Corallorhiza trifida is a partial myco-heterotroph.

The leafless, circumboreal orchid Corallorhiza trifida is often assumed to be fully myco-heterotrophic despite contrary evidence concerning its ability to photosynthesize. Here, its level of myco-heterotrophy is assessed by analysing the natural abundance of the stable nitrogen and carbon isotopes (15)N and (13)C, respectively. The mycorrhizal associates and chlorophyll contents of C. trifida were investigated and the C and N isotope signatures of nine C. trifida individuals from Central Europe were compared with those of neighbouring obligate autotrophic and myco-heterotrophic reference plants. The results show that C. trifida only gains c. 52 +/- 5% of its total nitrogen and 77 +/- 10% of the carbon derived from fungi even though it has been shown to specialize on one specific complex of ectomycorrhizal fungi similar to fully myco-heterotrophic orchids. Concurrently, compared with other Corallorhiza species, C. trifida contains a remarkable amount of chlorophyll. Since C. trifida is able to supply significant proportions of its nitrogen and carbon demands through the same processes as autotrophic plants, this species should be referred to as partially myco-heterotrophic.

Control of antimicrobial peptide synthesis by the agr quorum sensing system in Staphylococcus epidermidis: activity of the lantibiotic epidermin is regulated at the level of precursor peptide processing.

The accessory gene regulator (agr) quorum sensing system in staphylococci is responsible for the regulation of surface proteins and exoproteins, including many virulence factors in the pathogenic species Staphylococcus aureus and S. epidermidis. Strain S. epidermidis Tü3298 produces the lantibiotic epidermin. An isogenic agr deletion mutant of this strain showed a strong reduction of epidermin production. Detailed analysis of the impact of agr on epidermin biosynthesis revealed that agr does not interfere with the transcription of epidermin biosynthetic genes, but controls the extracellular processing of the N-terminal leader peptide by the EpiP protease.