Campylobacter devanensis sp. nov., Campylobacter porcelli sp. nov., and Campylobacter vicugnae sp. nov., three novel Campylobacter lanienae-like species recovered from swine, small ruminants, and camelids.

In a previous study characterizing Campylobacter strains deficient in selenium metabolism, 50 strains were found to be similar to, but distinct from, the selenonegative species Campylobacter lanienae. Initial characterization based on multilocus sequence typing and the phylogeny of a set of 20 core genes determined that these strains form three putative taxa within the selenonegative cluster. A polyphasic study was undertaken here to further clarify their taxonomic position within the genus. The 50 selenonegative strains underwent phylogenetic analyses based on the sequences of the 16S rRNA gene and an expanded set of 330 core genes. Standard phenotypic testing was also performed. All strains were microaerobic and anaerobic, Gram-negative, spiral or curved cells with some displaying coccoid morphologies. Strains were motile, oxidase, catalase, and alkaline phosphatase positive, urease negative, and reduced nitrate. Strains within each clade had unique phenotypic profiles that distinguished them from other members of the genus. Core genome phylogeny clearly placed the 50 strains into three clades. Pairwise average nucleotide identity and digital DNA-DNA hybridization values were all below the recommended cut-offs for species delineation with respect to C. lanienae and other related Campylobacter species. The data presented here clearly show that these strains represent three novel species within the genus, for which the names Campylobacter devanensis sp. nov. (type strain RM3662T=LMG 33097T=NCTC 15074T), Campylobacter porcelli sp. nov. (type strain RM6137T=LMG 33098T=CCUG 77054T=NCTC 15075T) and Campylobacter vicugnae sp. nov. (type strain RM12175T=LMG 33099T=CCUG 77055T=NCTC 15076T) are proposed.

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.

Identification and Characterisation of Pseudomonas savastanoi pv. savastanoi as the Causal Agent of Olive Knot Disease in Croatian, Slovenian and Portuguese Olive (Olea europaea L.) Orchards.

Strains of Pseudomonas savastanoi pv. savastanoi (Pss), isolated from infected olive trees (Olea europaea L.) in three European countries (Croatia, Slovenia and Portugal) were identified and characterised according to their colony morphology, physiological and biochemical features. According to the LOPAT scheme, 38.6% of Pss isolates were grouped in the Ib cluster. The Portuguese Pss strains were fully consistent with the typical LOPAT profile for this bacterium. Conversely, most Slovenian Pss strains showed delayed oxidase activity, whilst Croatian Pss strains did not produce any fluorescent pigment when grown in vitro. For Pss molecular identification, both end-point and real-time PCR were used, as well as MALDI-TOF, which was additionally used for proteomic analysis and the subsequent species identification of a number of strains that showed deviations from expected LOPAT results. Pss was confirmed as a causal agent of olive knot disease in 46.6% of olive orchards screened. Overall, these data suggests a possible correlation of certain Pss features with the geographical origin and the ecological niche of Pss isolates.

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.

Challenging the "gold standard" of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses.

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the "gold standard" in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as "gold standard" and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.

Helicobacter labacensis sp. nov., Helicobacter mehlei sp. nov., and Helicobacter vulpis sp. nov., isolated from gastric mucosa of red foxes (Vulpes vulpes).

Six Helicobacter-like isolates were recovered from 15 gastric mucosa samples of red foxes (Vulpes vulpes) shot by hunters in the surroundings of Ljubljana, Slovenia. Gram-negative, tightly coiled, intensely motile, 7-15 µm long and ≤1 µm wide bacteria grew on the biphasic blood agar plates. By using a genus-specific polymerase chain reaction (PCR), all isolates were confirmed as Helicobacter sp. and subsequently subjected to whole-genome sequencing (WGS). Five isolates showed a genome-wide average nucleotide identity (ANI) value of <95 % to the previously described Helicobacter species and one isolate was classified as Helicobacter felis. In the five unidentified isolates, the 16S rRNA gene sequence similarity to the type strains of all Helicobacter species ranged from 98.6 to 98.9 %. Their taxonomic status was established using a polyphasic taxonomic approach comprising the core genome-based phylogeny, morphological and phenotypic characteristics, including an analysis of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra. Phylogeny revealed the existence of three novel and well-supported clusters, with Helicobacter bizzozeronii and Helicobacter baculiformis being the most closely related species. The isolates also differed from the previously described species in their MALDI-TOF profiles and some biochemical characteristics. In conclusion, the data presented herein indicate that the obtained isolates, excluding H. felis isolate, represent three novel Helicobacter species, for which the names Helicobacter labacensis sp. nov., Helicobacter mehlei sp. nov., and Helicobacter vulpis sp. nov. are proposed, with isolates L9T (=DSM 108823T=CRBIP 111719T), L15T (=DSM 108730T=CCUG 72910T) and L2T (=DSM 108727T=CCUG 72909T) as type strains, respectively.

Trichomonosis outbreak in a flock of canaries (Serinus canaria f. domestica) caused by a finch epidemic strain of Trichomonas gallinae.

In the present paper, an outbreak of trichomonosis in a flock of 15 breeding pairs of canaries is described. Trichomonosis was diagnosed on characteristic clinical signs, microscopic examination of crop/esophageal swabs, gross pathology and histopathology. Trichomonads were successfully grown in culture media and were characterized by multi-locus sequence typing. The three genomic loci ITS1-5.8S-ITS2, 18S rRNA and Fe-hydrogenase were analyzed. Molecular characterization confirmed the finch trichomonosis strain, identical to the strain that caused emerging disease in free-living passerine birds in Europe. Flock treatment with metronidazole (200mg/L) in drinking water for 5days was partially effective. After individual treatment with oral application of metronidazole (20mg/kg SID) for 5days no further clinical signs were observed in the flock over next 30 months.

New Approaches on Quantification of Campylobacter jejuni in Poultry Samples: The Use of Digital PCR and Real-time PCR against the ISO Standard Plate Count Method.

Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500-1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland-Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost-effective surveillance throughout all stages in the food production chain. As both rLOD and LOQ were beyond this limit, a modification of the procedure is suggested to include less sample dilution prior to DNA extraction to enable PCR-based quantification of C. jejuni at the proposed microbiological criteria.

High genetic similarity of ciprofloxacin-resistant Campylobacter jejuni in central Europe.

Campylobacteriosis is the leading zoonosis in the European Union with the majority of cases attributed to Campylobacter jejuni. Although the disease is usually self-limiting, some severe cases need to be treated with antibiotics, primarily macrolides and quinolones. However, the resistance to the latter is reaching alarming levels in most of the EU countries. To shed light on the expansion of antibiotic resistance in central Europe, we have investigated genetic similarity across 178 ciprofloxacin-resistant C. jejuni mostly isolated in Slovenia, Austria and Germany. We performed comparative genetic similarity analyses using allelic types of seven multilocus sequence typing housekeeping genes, and single nucleotide polymorphisms of a quinolone resistance determining region located within the DNA gyrase subunit A gene. This analysis revealed high genetic similarity of isolates from clonal complex ST-21 that carry gyrA allelic type 1 in all three of these central-European countries, suggesting these ciprofloxacin resistant isolates arose from a recent common ancestor and are spread clonally.

Campylobacter jejuni contamination of broiler carcasses: Population dynamics and genetic profiles at slaughterhouse level.

Six slaughter batches deriving from six typical industrial broiler flocks were examined for the presence, quantity and genetic characteristics of contaminating Campylobacter jejuni (C. jejuni) during various stages of slaughtering and carcass processing. To assess the contamination dynamics of the carcasses, the analyses were always conducted on neck-skin samples from the same pre-selected and carefully marked carcasses in each batch. The skin samples were taken sequentially at three successive slaughter-line locations in the evisceration room, after three-day refrigeration and after three-day freezing procedure. Caecal samples from the same animals were also tested, as well as samples from the slaughterhouse environment before and after slaughtering. The samples were analysed by the ISO10272 isolation method; campylobacters from neck-skin samples were also quantified. Isolates were species-identified and genotyped by pulsed-field gel electrophoresis (PFGE). On average, the highest C. jejuni skin contamination was detected at the first sampling point (post-plucking), suggesting that the majority of Campylobacter contamination actually occurs before the entrance to the eviscerating room, probably during the preceding plucking stage. In two out of five positive batches, an additional increase in contamination was recorded after the evisceration step. An evident trend of increasing contamination level was detected when successive batches were compared at each of two initial sampling sites in the evisceration room, indicating an accumulation of contaminating C. jejuni at some point before the evisceration room. Three-day refrigeration and three-day freezing caused a 4.5- and 142-fold drop in mean C. jejuni CFU counts, respectively. All pre-slaughtering samples from the slaughterhouse environment were negative and all post-slaughtering samples, except water from the scalding tank, were positive. Pulsotypes were limited: altogether five different types were detected, typically one type per batch. The PFGE results from the slaughterhouse environment isolates indicate that cross-contamination is possible (multiple pulsotypes detected in e.g. eviscerating machine). Nevertheless, this was not confirmed in carcasses: analyses of neck-skin isolates suggest that carcasses are contaminated by their own caecal/farm/flock pulsotype.

Evidence of Anaplasma phagocytophilum in game animals from Slovenia.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen responsible for granulocytic anaplasmosis in mammalian hosts including humans. Wild animals may play an important role in the epidemiology of this disease. The aim of this study was to estimate the prevalence of infection with A. phagocytophilum among wildlife in Slovenia. Serum samples (n = 376) from the most important game species [red deer (Cervus elaphus), roe deer (Capreolus capreolus), wild boar (Sus scrofa), chamois (Rupicapra rupicapra) and brown bear (Ursus arctos)] were examined by A. phagocytophilum-specific indirect fluorescent-antibody assay (IFA) and wild boar spleen samples (n = 160) were tested by polymerase chain reaction (PCR). A. phagocytophilum-specific antibodies were found in 72% of sera and A. phagocytophilum DNA was present in 6.2% of spleens. The data indicate that A. phagocytophilum is present and widespread in Slovenian game animals and that game species are involved in the natural life cycle of A. phagocytophilum.

A pulsed-field gel electrophoresis study of the genetic diversity of Campylobacter jejuni and Campylobacter coli in poultry flocks in Slovenia.

Campylobacter jejuni and C. coli have recently become the most frequent cause of bacterial foodborne enteric infection in most industrialised countries. Consumption and handling of undercooked contaminated poultry meat was identified as an important risk factor for human campylobacteriosis. The aim of this study was to ascertain the genetic diversity of C. jejuni and C. coli strains isolated from poultry in Slovenia. A total of 68 isolates (42 C. jejuni , 26 C. coli ) from faeces (n = 48), meat (n = 15) and skin/carcasses (n = 5) of chicken (n = 60) and turkey samples (n = 5) were analysed by pulsed-field gel electrophoresis. Sma I macrorestriction discriminated between C. jejuni and C. coli isolates. C. jejuni isolates exhibited a higher degree of diversity compared to C. coli isolates. In the C. jejuni group, a number of small clusters were apparent, while C. coli strains formed less but larger clusters. Additional Kpn I digestion of selected isolates resulted in poor subtyping. Strains with identical or very similar profiles were found on different farms, either in the same or different regions and time periods. Some of the clones indicated possible cross-contamination at slaughterhouses.