Bovine Tuberculosis in Wild Boar (Sus scrofa) in Slovenia.

Mycobacteria of the Mycobacterium tuberculosis complex (MTC) are capable of infecting a wide variety of animals. Wild boar (Sus scrofa) has been recognized as an important wildlife reservoir for bovine tuberculosis. We screened wild boar in Slovenia for the presence of (1) Mycobacterium bovis in tissues and (2) antibodies to M. bovis in blood samples. In 2016 and 2017, 1284 tissue samples from 676 wild boar were subjected to cultivation. In 2018 and 2019, blood samples from 132 wild boar were examined using an ELISA kit. None of the MTC species were isolated from the tissue samples, and no antibodies to M. bovis were detected in the blood samples. Several nontuberculous mycobacteria (NTM), identified by 16S rRNA sequencing and/or matrix-assisted laser desorption ionization time-of-flight mass spectrometry, were found in the tissues of 9.8% of the wild boar: Mycobacterium nonchromogenicum, Mycobacterium peregrinum/Mycobacterium septicum, Mycobacterium avium, Mycobacterium engbaekii, Mycobacterium arupense, Mycobacterium algericum, Mycobacterium bohemicum, Mycobacterium confluentis, Mycobacterium flavescens, Mycobacterium fortuitum, Mycobacterium thermoresistibile, and Mycobacterium vaccae. Species-level identification was not possible for 21.2% of the isolates. At the time of the study, wild boar in Slovenia were not at risk from bTB; the significance of the presence of NTM in wild boar remains to be clarified and evaluated from a One Health perspective.

Campylobacter magnus sp. nov., isolated from caecal contents of domestic pigs (Sus scrofa domesticus).

During the 2021 European Food Safety Authority coordinated harmonized monitoring of antimicrobial resistance in Campylobacter species in Slovenia, five Campylobacter-like strains were cultured from caeca of a total of 104 domestic pigs that could not be identified using the standard-prescribed biochemical tests or MALDI-TOF MS. The isolates were obtained using the standard ISO 10272 procedure for the isolation of thermotolerant Campylobacter with prolonged cultivation time. Small Campylobacter-like colonies were observed on mCCDA and CASA agar plates after 2-4 days of incubation; dark-field microscopy revealed relatively big spirilli-shaped bacteria exhibiting characteristic Campylobacter-like motility. The cells were 1.5-3 µm long and 0.5-0.7 µm wide, Gram-negative, oxidase-positive and catalase-positive. MALDI-TOF mass spectra were distinctive and consistent, but with low MALDI-TOF MS log scores and the closest matches being those of Campylobacter hyointestinalis and Campylobacter fetus. All five strains underwent whole-genome sequencing. Analysis of 16S rRNA gene sequences revealed that the isolates were most similar (98.3-98.4 % identity) to Campylobacter lanienae. Pairwise average nucleotide identity (ANI) values revealed that the five studied strains shared pairwise ANI of 96.2-96.5 % but were clearly distinct from the previously described Campylobacter species (ANI ≤72.8 %). The core genome-based phylogeny confirmed that the new strains form a distinct and well-supported clade within the genus Campylobacter. The conducted polyphasic taxonomic analysis confirmed that the five strains represent a novel Campylobacter species for which the name Campylobacter magnus sp. nov. is suggested, with strain 46386T (=DSM 115534T=CCUG 76865T) as the type strain.

Helicobacter colisuis sp. nov., isolated from caecal contents of domestic pigs (Sus scrofa domesticus).

Seven Helicobacter-like isolates were cultured from caecal contents of 100 domestic pigs (Sus scrofa domesticus) sampled as part of the EFSA-coordinated harmonized monitoring of antimicrobial resistance in Campylobacter sp. in 2015. The bacteria were isolated using the standard ISO 10272 procedure for the isolation of thermotolerant Campylobacter with extended incubation time and formed small, grey, moist and flat colonies with a metallic sheen (small Campylobacter-like colonies) on modified Charcoal-Cefoperazone-Deoxycholate Agar (mCCDA) and Skirow agar plates. Morphologically, the bacterial cells were spirilli-shaped and highly motile, 1-2 µm long and ≤0.5 µm wide, Gram-negative, oxidase-positive and catalase-positive. They could not be identified using the standard-prescribed biochemical tests and had uniform, unique and reproducible MALDI-TOF mass spectra that most closely matched those of Helicobacter pullorum. Three strains (11154-15T, 14348-15 and 16470-15) underwent whole-genome sequencing. Analysis of 16S rRNA gene sequences revealed a high similarity (≥99.8 % identity) to Helicobacter canadensis. Pairwise average nucleotide identity (ANI) values revealed that the three studied strains were closely related (ANI ≥98.9 %), but distinct from the previously described Helicobacter species (ANI ≤90.6 %). The core genome-based phylogeny confirmed that the new strains form a distinct clade most closely related to H. canadensis. The conducted polyphasic taxonomic analysis confirmed that the three strains represent a novel Helicobacter species for which the name Helicobacter colisuis sp. nov. is suggested, with strain 11154-15T (= DSM 113688T = CCUG 76053T) as the type strain.

Corrigendum: Evaluation of Human Milk Microbiota by 16S rRNA Gene Next-Generation Sequencing (NGS) and Cultivation/MALDI-TOF Mass Spectrometry Identification.

[This corrects the article DOI: 10.3389/fmicb.2019.02612.].

Evaluation of Human Milk Microbiota by 16S rRNA Gene Next-Generation Sequencing (NGS) and Cultivation/MALDI-TOF Mass Spectrometry Identification.

The aim of the present study was to characterize human milk microbiota (HMM) with 16S rRNA gene amplicon next-generation sequencing and cultivation/matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) identification approaches. We analyzed 31 human milk samples from healthy Slovenian mothers. To check the accuracy of MALDI-TOF MS identification, several colonies representing most abundant genera and those, which could not be reliably identified by MALDI-TOF, were subjected to Sanger sequencing of their 16S rRNA gene. We showed that cultivation/MALDI-TOF MS was a suitable tool for culture-dependent determination of HMM. With both approaches, Staphylococcus and Streptococcus were found as predominant genera in HMM and the abundance of Staphylococcus was associated with decreased microbial diversity. In addition, we characterized factors that might influence HMM. The use of a breast pump was significantly associated with composition of HMM, lower microbial load, and higher abundance of cultivable staphylococci. Moreover, our study suggests that administration of probiotics to the suckling infant might influence HMM by increased abundance of lactobacilli and the presence of viable probiotic bacteria in human milk. However, since our study was observational with relatively small sample size, more targeted studies are needed to study possible transfer of probiotics to the mammary gland via an external route and the physiological relevance of these events.

Microbiological Aspects of Naturally Occurring Primary Endodontic Infections in Dogs.

Dental fractures are common in dogs, but data on microbiology of naturally occurring primary endodontic infections, and their relation to clinical and radiographic signs, are lacking. Samples were obtained from root canals of 32 periodontally healthy fractured teeth under aseptic conditions and immediately cultured for aerobic and anaerobic bacteria. Cultures were further identified by matrix-assisted laser desorption/ionization, time of flight technology. Sixty-one bacteria (30 bacterial species) were isolated from root canals; 54% were Gram-negative bacteria, 53% were facultative anaerobic, and 42% were anaerobic bacteria. Number of bacterial species in the root canals declined with the duration of fractures over 12 months. No statistically significant association was found between the number of bacterial species involved in the root canal infection and any of the clinical or radiographic signs of endodontic disease. Naturally occurring primary endodontic infections in dogs appear to be polymicrobial and involve only a selected number of opportunistic pathogen species.

Tear production, intraocular pressure and conjunctival bacterial flora in selected captive wild ruminants.

OBJECTIVE: Evaluation of tear production (Schirmer's tear test, STT) and measurement of intraocular pressure (IOP) were performed in a population of captive wild ungulates in a Slovenian ZOO during routine annual health check. ANIMALS STUDIED: In total, 10 fallow deer (Dama dama), 25 mouflons (Ovis aries musimon), 20 alpine ibexes (Capra ibex), and three alpine chamois (Rupicapra rupicapra) were included in the study. METHODS: Tear production was performed by Schirmer's tear test, IOP was measured with an applanation tonometer, and ophthalmological examination was conducted with slit-lamp biomicroscopy and indirect ophthalmoscopy. Conjunctival swabs were taken and submitted for aerobic bacteriology and for detection of Chlamydia spp. and Mycoplasma spp. tested by PCR. RESULTS: Average tear production (in mm/min) was 17.8 ± 3.16 for fallow deer, 17.9 ± 3.87 for mouflons, and 11.7 ± 3.87 for ibexes. Mean intraocular pressure (IOP, in mm Hg) was 14.1 ± 2.48 for fallow deer, 14.9 ± 2.20 for mouflons, and 13.1 ± 2.43 for ibexes. For chamois, average tear production and IOP were 14.5 ± 3.0 and 10.2 ± 2.5, respectively; this is the first record of STT I and IOP in chamois. Bacteriological swabs were positive for bacteria in 100% of the fallow deer, 56% of mouflons, 35% of ibexes, and 100% of chamois. Gram-positive bacteria were predominant. Moraxella spp., Chlamydia spp., and Mycoplasma spp. were not detected. CONCLUSION: The reported values were obtained in animals under manual restraint only to be applicative in similar conditions.

Mycobacterium spp. in wild game in Slovenia.

Wildlife species are an important reservoir of mycobacterial infections that may jeopardise efforts to control and eradicate bovine tuberculosis (bTB), caused by Mycobacterium bovis. Slovenia is officially free of bTB, but no data on the presence of mycobacteria in wild animals has been reported. In this study, samples of liver and lymph nodes were examined from 306 apparently healthy free-range wild animals of 13 species in Slovenia belonging to the families Cervidae, Suidae, Canidae, Mustelidae and Bovidae. Mycobacteria were isolated from 36/306 (11.8%) animals (red deer, roe deer, fallow deer, wild boar and jackal) and identified by PCR, commercial diagnostic kits and sequencing. Non-tuberculous mycobacteria identified in five species were Mycobacterium peregrinum, M. avium subsp. hominissuis, M. intracellulare, M. confluentis, M. fortuitum, M. terrae, M. avium subsp. avium, M. celatum, M. engbaekii, M. neoaurum, M. nonchromogenicum and M. vaccae.

Clostridium difficile in goats and sheep in Slovenia: characterisation of strains and evidence of age-related shedding.

Diversity of Clostridium difficile in different age groups of goats (n = 109) and sheep (n = 105) was investigated. C. difficile was detected in 9.2% of goats and 5.7% of sheep. None of the adult animals were positive. Isolates belonged to four toxinotypes (0, V, XIa, XII), six PCR-ribotypes (010, 014/020, 045, 056, SLO 061, SLO 151) and six pulsotypes. PCR-ribotypes 010, 014/020, 045 and 056 were found previously in other animal species and humans in Slovenia. Additionally, three pulsotypes were indistinguishable from restriction patterns in our PFGE database of animal isolates. All strains were susceptible to metronidazol, vancomycin, moxifloxacin, and with the exception of a single non-toxigenic strain also to clindamycin and erythromycin. While all strains were resistant to ciprofloxacin and levofloxacin, oxacillin-resistance was observed only in strains of PCR-ribotype 045. This first study on C. difficile in small ruminants in Slovenia revealed the evidence of age-related shedding as the highest was demonstrated in neonatal goats and sheep aged up to 16 days.

Antimicrobial susceptibility of animal and human isolates of Clostridium difficile by broth microdilution.

A total of 188 human (n = 92) and animal (n = 96) isolates of Clostridium difficile of different PCR ribotypes were screened for susceptibility to 30 antimicrobials using broth microdilution. When comparing the prevalence of antimicrobial resistance, the isolates of animal origin were significantly more often resistant to oxacillin, gentamicin and trimethoprim/sulfamethoxazole (P<0.01). The most significant difference between the animal and human populations (P = 0.0006) was found in the level of imipenem resistance, with a prevalence of 53.3 % in isolates of human origin and 28.1 % in isolates of animal origin. Overall, the results show similar MICs for the majority of tested antimicrobials for isolates from human and animal sources, which were collected from the same geographical region and in the same time interval. This supports the hypothesis that C. difficile could be transmissible between human and animal hosts. Resistant isolates have been found in all animal species tested, including food and companion animals, and also among non-toxigenic isolates. The isolates of the most prevalent PCR ribotype 014/020 had low resistance rates for moxifloxacin, erythromycin, rifampicin and daptomycin, but a high resistance rate for imipenem. Multiresistant strains were found in animals and humans, belonging to PCR ribotypes 012, 017, 027, 045, 046, 078 and 150, and also to non-toxigenic strains of PCR ribotypes 010 and SLO 080.

Zero prevalence of Clostridium difficile in wild passerine birds in Europe.

Clostridium difficile is an important bacterial pathogen of humans and a variety of animal species, where it can cause significant medical problems. The major public health concern is the possibility of inapparent animal reservoirs of C. difficile and shedding of bacteria to noninfected individuals or populations, as well as being a source of food contamination. Migrating birds can be a key epizootiological factor for transmission and distribution of pathogens over a wide geographic range. Therefore, the purpose of this study was to investigate whether migrating passerine birds can be a source of spread of C. difficile along their migration routes. Cloacal samples were taken from 465 passerine birds during their migration south over the Alps. Selective enrichment was used for detection of C. difficile. Clostridium difficile was not isolated from any of the samples, which indicates that migrating passerine birds are unlikely to serve as a reservoir and a carrier of C. difficile.

Diversity of Clostridium difficile in pigs and other animals in Slovenia.

A study of Clostridium difficile diversity in pigs, calves and horses in Slovenia was conducted. A total of 547 samples were collected and C. difficile was isolated from 247/485 (50.9%) piglet samples, from 4/42 (9.5%) calf samples, and 1/20 (5%) horse samples. The isolates were characterized by toxinotyping, PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) using restriction endonuclease SmaI. Piglet isolates belonged to two toxinotypes (V and 0), four PCR-ribotypes (066, 029, SI 011, SI 010), and six pulsotypes. Bovine isolates were grouped into two toxinotypes (XIa and 0), three PCR-ribotypes (077, 002, 033), and three pulsotypes. The only equine isolate was indistinguishable from one calf isolate (XIa/033) in toxinotype, PCR-ribotype, and pulsotype. None of detected genotypes was present in all three animal hosts.

Isolation of Clostridium difficile from food animals in Slovenia.

A total of 313 faecal samples from three pig farms and two cattle farms was collected, and Clostridium difficile was isolated from 133/257 piglet samples (51.8%) and from 1/56 calf samples (1.8%). Toxins were tested only in calf samples and were positive in 44.6% (25/56). The only bovine isolate belonged to toxinotype XIa (A(-)B(-)CDT(+)). Porcine isolates belonged to toxinotype 0 (A(+)B(+)CDT(-)) and toxinotype V (A(+)B(+)CDT(+)). Of the two ribotypes usually found in toxinotype V, the strains isolated in this study showed a greater similarity to ribotype 066 than to ribotype 078.

Isolation and characterisation of Mycobacterium avium and Rhodococcus equi from granulomatous lesions of swine lymph nodes in Slovenia.

Granulomatous lesions in bovine and especially swine lymph nodes are still frequently observed during routine veterinary meat inspections even though Mycobacterium bovis infections are no longer detected in domestic animals in Slovenia. Different lymph nodes of pigs (n = 260) were investigated using classical bacteriological and molecular methods. Mycobacterium avium alone was isolated in 47.3% of pigs and in mixed infection with Rhodococcus equi in 3.9% of pigs. R. equi alone was isolated in 27.3% and in mixed infection with mycobacteria other than M. avium in 1.5% of pigs. A total of 133 M. avium isolates were typed using the IS1245, IS901 and FR300 PCR. Almost two thirds (60.9%) of isolates belonged to M. avium hominissuis (IS901-, IS1245+ genotype), 33.8% of isolates belonged to M. avium avium (IS901+, IS1245+ genotype) and 5.3% of isolates remained non-typed. Fifty out of 85 R. equi isolates were tested for the virulence-associated antigens (VapA and VapB). Nearly two thirds (60.0%) were positive for VapB while all the other isolates were VapA- and VapB-negative.