Sequestration of membrane cholesterol by cholesterol-binding proteins inhibits SARS-CoV-2 entry into Vero E6 cells.

Membrane lipids and proteins form dynamic domains crucial for physiological and pathophysiological processes, including viral infection. Many plasma membrane proteins, residing within membrane domains enriched with cholesterol (CHOL) and sphingomyelin (SM), serve as receptors for attachment and entry of viruses into the host cell. Among these, human coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), use proteins associated with membrane domains for initial binding and internalization. We hypothesized that the interaction of lipid-binding proteins with CHOL in plasma membrane could sequestrate lipids and thus affect the efficiency of virus entry into host cells, preventing the initial steps of viral infection. We have prepared CHOL-binding proteins with high affinities for lipids in the plasma membrane of mammalian cells. Binding of the perfringolysin O domain four (D4) and its variant D4E458L to membrane CHOL impaired the internalization of the receptor-binding domain of the SARS-CoV-2 spike protein and the pseudovirus complemented with the SARS-CoV-2 spike protein. SARS-CoV-2 replication in Vero E6 cells was also decreased. Overall, our results demonstrate that the integrity of CHOL-rich membrane domains and the accessibility of CHOL in the membrane play an essential role in SARS-CoV-2 cell entry.

A scoping review on associations between paratuberculosis and productivity in cattle.

Paratuberculosis (PTB), or Johne's disease, is a disease with worldwide distribution caused by Mycobacterium avium subsp. paratuberculosis (MAP) that leads to chronic enteritis, primarily in ruminants. Even subclinical infection significantly reduces the animals' performance, and consequences of the disease lead to high economic losses for the cattle industry. To estimate the economic burden of bovine PTB and to evaluate the benefits of a potential control program, accurate estimates of the production effects associated with the disease are required. Therefore, the aim of this scoping review was to provide a comprehensive overview of associations between MAP infection and production parameters in cattle. The studies were collected from three electronic databases. Of the total 1,605 identified studies, 1,432 did not meet the set criteria in the title and abstract screening and a further 106 were excluded during full-text review. Finally, data on 34 different production parameters were extracted from 67 publications. Results show that the magnitude of reported performance losses varies depending on several factors, such as the type of diagnostic test applied, disease status or number of lactations. Studies reported a reduction in milk yield, changes in milk quality (e.g., higher somatic cell count, lower amount of produced milk fat and protein), reduced fertility (e.g., prolonged calving interval and service period, higher abortion rate and calving difficulties), reduced weaning weight, slaughter weight and slaughter value, or a higher risk for mastitis. Results from the studies included in our review show a median decrease of milk yield per infected cow of -452 kg/lactation for raw and -405 kg/lactation for modeled data. Similarly, the amount of produced milk protein fell by a median of -14.41 kg/lactation for modeled data and the amount of produced milk fat by a median of -13.13 kg/lactation. The reviewed studies revealed a prolonged calving interval by around 30 days and a 1.5 to 3 times higher likeliness of culling per lactation in PTB positive animals. Results from this scoping review provide evidence-based inputs for the development of economic models aiming at the estimation of the costs and benefits associated with different disease control scenarios for PTB.

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.

Assessment of Tuberculosis Biomarkers in Paratuberculosis-infected Cattle.

Introduction: Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis, respectively the causative agents of bovine tuberculosis (bTB) and bovine paratuberculosis (PTB), share a high number of antigenic proteins. This characteristics makes the differential diagnosis of the diseases difficult. The interferon gamma (IFN-γ), C-X-C motif chemokine ligand 10 (CXCL10), matrix metallopeptidase 9 (MMP9), interleukin 22 (IL-22) and thrombospondin 1 (THBS1) bovine genes have already been shown to be accurate transcriptional biomarkers of bTB. In order to improve the diagnosis of bTB and PTB, in the present study we evaluated the risk of false positivity of these bTB biomarkers in cattle with PTB. Material and Methods: The transcription of these genes was studied in 13 PTB-infected cattle, using Mycobacterium avium subsp. paratuberculosis (MAP)-stimulated peripheral blood mononuclear cells (PBMC). Results: Overall, the levels of IFN-γ, CXCL10, MMP9 and IL-22 transcripts in MAP-stimulated PBMC failed to differentiate animals with PTB from healthy animals. However, as bTB-afflicted cattle do, the MAP-infected group also displayed a lower level of THBS1 transcription than the non-infected animals. Conclusion: The results of this study add new specificity attributes to the levels of transcription of IFN-γ, CXCL10, MMP9 and IL-22 as biomarkers for bTB.

Phylogeography of Brucella suis biovar 2 with focus on Slovenian wildlife.

Brucella suis commonly infects swine but occasionally also other animal species and humans. Wild boars are the most important reservoir of B. suis biovar 2, continually infecting susceptible hosts through close contact. Nevertheless, the genetic diversity of B. suis in wildlife remains understudied. Here, we typed 17 Slovenian B. suis biovar 2 isolates obtained in 2017-2019 from wild boars (n = 16) and a hare (n = 1) using whole-genome sequencing (WGS). To assess the global phylogenetic diversity of B. suis, we compared them to 126 publicly available B. suis genomes. All Slovenian isolates fell within the biovar 2 lineage, confirming the previous multiplex PCR typing results. According to MLST-21, the wild boar isolates were of sequence types (STs) ST16 (n = 8) and ST153 (n = 8); the maximum genetic distance between isolates of the same ST was 28 wgMLST alleles. The ST153 isolates were restricted to the Slovenian-Croatian border and clustered together with the Croatian ST153 isolates from swine, indicating cross-border transmission of B. suis ST153 strain. The hare isolate was of ST40 and was genetically distant (≥ 489 alleles) from the wild boar isolates. The genome-wide phylogeny clearly separated different B. suis biovars. The present study is the first report on the population structure of B. suis in wildlife in Slovenia and shows that the Slovenian B. suis population is genetically heterogeneous. At the species level, B. suis biovars are clearly separated in the WGS-based phylogenetic tree and can therefore be reliably predicted using WGS.

Antimicrobial Resistance and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus from Two Pig Farms: Longitudinal Study of LA-MRSA.

Pigs were identified as the most important reservoir of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), mostly belonging to the emergent zoonotic clonal complex (CC) 398. Here, we investigated the presence of MRSA in sows and piglets over a period of several months in two pig farms (intensive farm A and family-run farm B). Isolates underwent antimicrobial susceptibility testing, PCR characterization and spa typing. We collected 280 samples, namely 206 nasal swabs from pigs and 74 environmental samples from pig housings at 12 consecutive time points. A total of 120/161 (74.5%) and 75/119 (63.0%) samples were MRSA-positive in farms A and B, respectively. All isolates harbored mecA but lacked mecC and PVL-encoding genes. The identified spa types (t571, t034, t1250 and t898 in farm A, t1451 and t011 in farm B) were indicative of CC398. Antimicrobial resistance patterns (all multidrug resistant in farm A, 57.2% in farm B) depended on the farm, suggesting the impact of farm size and management practices on the prevalence and characteristics of MRSA. Due to the intermittent colonization of pigs and the high contamination of their immediate environment, MRSA status should be determined at the farm level when considering preventive measures or animal trade between farms.

Clonal Spread of pESI-Positive Multidrug-Resistant ST32 Salmonella enterica Serovar Infantis Isolates among Broilers and Humans in Slovenia.

Salmonella enterica subsp. enterica serovar Infantis is the most prevalent serovar found in broilers and broiler meat and is among the top five serovars responsible for human infections in Europe. In 2008, a multidrug-resistant S. Infantis isolate emerged in Israel with a mosaic megaplasmid named pESI, associated with increased virulence, biofilm formation, and multidrug resistance. Since then, S. Infantis clones with pESI-like plasmids have been reported worldwide, replacing pESI-free clones. Here, we typed 161 S. Infantis isolates of poultry (n = 133) and human clinical (n = 28) origin using whole-genome sequencing. The isolates were collected between 2007 and 2021. In addition, we performed PacBio/Illumina sequencing for two representative pESI-like plasmids and compared them with publicly available sequences. All isolates belonged to sequence type 32 (ST32), except for one isolate that represented a novel single-locus variant of ST32. Core genome MLST (cgMLST) analysis revealed 14 clusters of genetically closely related isolates, of which four suggested broiler-to-human transmission of S. Infantis. pESI-like plasmids were present in 148/161 (91.9%) isolates; all were highly similar to the publicly available pESI-like sequences but lacked extended-spectrum beta-lactamase (ESBL) genes. PacBio/Illumina hybrid assembly allowed the reconstruction of two novel complete pESI variants. The present study revealed that the multidrug-resistant, pESI-positive S. Infantis clone became the predominant S. Infantis clone in Slovenian broilers and humans during the last decade. Continued surveillance of resistant S. Infantis clones along the food chain is needed to guide public health efforts. IMPORTANCE Salmonella Infantis clones with pESI-like plasmids harboring several virulence and resistance genes have been reported worldwide. In the present study, we compared the population structure of 161 Salmonella Infantis isolates obtained from humans and broilers in Slovenia from 2007 to 2021. Whole-genome sequencing showed that most human isolates clustered apart from broiler isolates, suggesting an alternative source of infection. Most isolates were multidrug resistant due to the presence of pESI-like plasmids, of which two variants (pS89 and pS19) were fully reconstructed using long-read sequencing. Both exhibited high similarity with the original Israeli pESI plasmid and German p2747 plasmid. The prototype plasmid pS89 harbored the typical pESI-associated resistance genes aadA1, qacEΔ1, sul1, and tet(A), which were absent in the truncated plasmid pS19.

Quantitative Risk Assessment of Exposure to Mycobacterium avium subsp. paratuberculosis (MAP) via Different Types of Milk for the Slovenian Consumer.

This study aimed to assess the risk of exposure to Mycobacterium avium subsp. paratuberculosis (MAP) via milk for the Slovenian consumer. MAP is suspected to be associated with several diseases in humans, therefore the risk of exposure should be better understood. The primary source of MAP for humans is thought to be cattle, in which MAP causes paratuberculosis or Johne's disease. We developed a stochastic quantitative risk assessment model using Monte Carlo simulations. Considering the assumptions and uncertainties, we estimated the overall risk of exposure to MAP via milk to be low. For people consuming raw milk from MAP positive farms, the risk was high. On-farm pasteurisation reduced the risk considerably, but not completely. The risk of exposure via pasteurised retail milk was most likely insignificant. However, with a higher paratuberculosis prevalence the risk would also increase. Given the popularity of raw milk vending machines and homemade dairy products, this risk should not be ignored. To reduce the risk, consumers should heat raw milk before consumption. To prevent a potential public health scare and safeguard farmers' livelihoods, a reduction in paratuberculosis prevalence should be sought. Our results show that culling clinically infected cows was insufficient to reduce milk contamination with MAP.

Modeling Paratuberculosis Transmission in a Small Dairy Herd Typical of Slovenia Suggests That Different Models Should Be Used to Study Disease Spread in Herds of Different Sizes.

This study aimed to investigate the possible dynamics of paratuberculosis or Johne's disease in a typical Slovenian dairy herd of about 17 cows. Paratuberculosis is a worldwide endemic disease of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is associated with significant economic losses. We developed a stochastic compartmental model with two pathways of disease progression, infections of adult cows and infections of young animals through horizontal and vertical transmission, and transmission through animal movements. The average proportions of subclinically and clinically infected cows were 4% and 0.47%, respectively. The prevalence within the herd, which included latently infected animals, averaged 7.13% and ranged from 0% to 70.59%. Under the given circumstances, the results showed a relatively high rate of spontaneous elimination (0.22 per herd per year) of the disease and a high rate of reinfection (0.18 per herd per year) facilitated by active animal trade. To our knowledge, this stochastic compartmental model is the first to be developed specifically to represent a small dairy herd and could apply to other countries with a similar structure of dairy farms. The results suggest that different models should be used to study MAP spread in herds of various sizes.

Corrigendum: Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe.

[This corrects the article DOI: 10.3389/fvets.2021.688078.].

Surveillance of the source of poultry infections with Enterococcus hirae and Enterococcus cecorum in Slovenia and E. hirae antibiotic resistance patterns.

Enterococcus cecorum and Enterococcus hirae can cause locomotor problems, septicaemia, and endocarditis in broiler chickens. Understanding transmission routes and resistance patterns are essential for effective treatment. The aim of this study was to follow the same animals from the breeder flock to the hatchery and up to 14-day-old broiler chickens on the farm to find the source of E. cecorum and E. hirae. During the production cycle, only faeces and organs of broilers were E. hirae positive in all three sampled farms in which recurrent enterococcal infections were previously confirmed. None of the isolates possessed virulence genes. Based on resistance profiles, a variety of different strains were present in faeces and organs of different broilers' ages. Samples from the breeder flock and hatchery were negative. Faecal shedding on the farm and tolerance of enterococci to the environmental conditions enable persistence of pathogenic enterococci in farm dust; therefore, adequate cleaning and disinfection after depopulation of the farms could prevent disease recurrence in the new cycle. Susceptibility testing of E. hirae isolates showed no resistance to the drugs of choice for the treatment of enterococcal infections in poultry.

Genomic insights into Salmonella Choleraesuis var. Kunzendorf outbreak reveal possible interspecies transmission.

Salmonella enterica serovar Choleraesuis is a host-adapted serovar that causes serious infections in domestic pigs and wild boars. Here, we investigated an outbreak of salmonellosis in domestic pigs in Slovenia, 2018-2019. To assess the outbreak, 18 isolates from domestic pigs, wild boars, wild boar meat and a human patient underwent whole-genome sequencing (WGS). All isolates were of sequence type (ST) 145 and harbored no antimicrobial resistance genes or AMR-associated mutations. A single transmission cluster (≤ 6 alleles) of spatially (< 100 km) and temporally linked isolates was observed, comprising isolates of pig (n = 9), wild boar (n = 2) and human (n = 1) origin, and suggesting possible interspecies transmission. In all outbreak-related animal cases, septicemic salmonellosis was observed, accompanied in some cases by enteric symptoms. All pig isolates were linked to a single intensive breeding farm that distributed growers to small family farms. The same transport vehicles were used to distribute growers to family farms and also to transport livestock between neighboring countries. Both isolates that originated from the imported wild boar meat were genetically distant (≥ 122 alleles) from the outbreak cluster. The present results indicate the importance of screening domestic pigs and proper disinfection of transport vehicles to control the spread of S. Choleraesuis.

Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04-6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.

Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe.

The COST action "Standardising output-based surveillance to control non-regulated diseases of cattle in the European Union (SOUND control)," aims to harmonise the results of surveillance and control programmes (CPs) for non-EU regulated cattle diseases to facilitate safe trade and improve overall control of cattle infectious diseases. In this paper we aimed to provide an overview on the diversity of control for these diseases in Europe. A non-EU regulated cattle disease was defined as an infectious disease of cattle with no or limited control at EU level, which is not included in the European Union Animal health law Categories A or B under Commission Implementing Regulation (EU) 2020/2002. A CP was defined as surveillance and/or intervention strategies designed to lower the incidence, prevalence, mortality or prove freedom from a specific disease in a region or country. Passive surveillance, and active surveillance of breeding bulls under Council Directive 88/407/EEC were not considered as CPs. A questionnaire was designed to obtain country-specific information about CPs for each disease. Animal health experts from 33 European countries completed the questionnaire. Overall, there are 23 diseases for which a CP exists in one or more of the countries studied. The diseases for which CPs exist in the highest number of countries are enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis, bovine viral diarrhoea and anthrax (CPs reported by between 16 and 31 countries). Every participating country has on average, 6 CPs (min-max: 1-13) in place. Most programmes are implemented at a national level (86%) and are applied to both dairy and non-dairy cattle (75%). Approximately one-third of the CPs are voluntary, and the funding structure is divided between government and private resources. Countries that have eradicated diseases like enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis and bovine viral diarrhoea have implemented CPs for other diseases to further improve the health status of cattle in their country. The control of non-EU regulated cattle diseases is very heterogenous in Europe. Therefore, the standardising of the outputs of these programmes to enable comparison represents a challenge.

A Suggested Diagnostic Approach for Sporadic Anthrax in Cattle to Protect Public Health.

The repeated occurrence of anthrax in grazing animals should be a reminder of a widespread presence of Bacillus anthracis spores in the environment. Its rapid diagnosis is critical to protect public health. Here, we report a case of anthrax in cattle that was investigated using conventional and molecular methods. In 2015, six cows suddenly died within three days and the number of dead animals increased to a total of 12 within two weeks. At necropsy, anthrax was suspected. Therefore, spleen tissue samples were collected (from 6/12 animals) and laboratory tests (microscopy, cultivation, and real-time PCR) performed. The results of tissue staining for microscopy and cultivation were in congruence, while B. anthracis real-time PCR outperformed both. Spleen tissues from all six animals were real-time PCR-positive, while B. anthracis was successfully cultivated and detected by microscopy from the spleen of only three animals. Additionally, the ear tissue from another (1/12) cow tested positive by real-time PCR, supporting the suitability of ear clippings for molecular confirmation of B. anthracis. Genotyping of the isolates using multiple-locus variable-number tandem repeat analysis (MLVA) revealed a common source of infection as all three typed isolates had an indistinguishable MLVA genotype, which has not been observed previously in Europe. The results indicate that molecular testing should be selected as the first-line tool for confirming anthrax outbreaks in animals to ensure timely protection of public health.

Overview of Slovenian Control Programmes for Cattle Diseases Not Regulated by the European Union.

The European Union (EU) regulates the control of cattle diseases listed in categories A and B of the Animal Health Law (AHL). However, the control of other cattle diseases that have no, or limited EU regulation, is left to each member state. Slovenia has five control programmes (CPs) for non-EU regulated cattle diseases: bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), enzootic bovine leukosis (EBL), bluetongue and anthrax. Two (IBR and BVD) are voluntary and the others (EBL, anthrax and bluetongue) are compulsory. The three compulsory CPs are funded by the government. All the CPs are run by the government and laboratory tests are performed by the National Veterinary Institute. The rules for the CPs are laid down in Slovenian legislation. In addition, there is a national directive for the control of salmonellosis. Both BVD and IBR are endemic and have CPs based on increased biosecurity, testing and culling or vaccination, financed by the animal owners. Slovenia has been officially free of EBL since 2005 and carries out surveillance based on serological testing of a representative number of herds and inspection of carcasses at slaughter or necropsy. Vaccination is the main disease control measure for anthrax (sporadic) and bluetongue (currently perceived free-vaccination since 2017). Lack of motivation of farmers to participate in voluntary disease CPs and to implement and follow strict biosecurity measures are the most pressing issues in improving the health status of Slovenian cattle. An overview of the existing CPs and the circumstances leading to their implementation are presented.

Infectious Abortions in Small Ruminants: Challenges for Diagnosis and Public Health.

In this study, we report an abortion outbreak in a ruminant herd consisting of goats, sheep, and cows, with scenarios in two consecutive years. In early 2017, abortions occurred in ∼70% of goats and 66% tested positive for Coxiella burnetii (C. burnetii) and 40% of goats were positive for Chlamydophila abortus (C. abortus). In February 2018, the same herd reported an abortion rate of 75%, with 55% positive for C. burnetii, 36% for C. abortus, and 22% for Toxoplasma gondii. Six goat milk samples were positive for C. burnetii by molecular analysis. Three family members were positive for C. burnetii. C. burnetii could be considered as the main cause of abortions in the first and second year. Animals that undergo an infection and abortion are prone to secondary infections. Vaccination or other rapid interventions should be initiated to protect animals and humans.

Corrigendum: Overview of Slovenian Control Programmes for Selected Cattle Diseases, Listed Under Category C, D or E of the European Animal Health Law.

[This corrects the article DOI: 10.3389/fvets.2021.674515.].

Livestock-associated methicillin-resistant Staphylococcus aureus: Establishing links between animals and humans on livestock holdings.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) represents a concern in both human and veterinary medicine. The aim of this study was to investigate potential LA-MRSA transmission between animals and humans in rural settings. To this aim, a study was designed to include 14 farms in Slovenia, which were selected on the basis of a farmer (initial patient) with confirmed LA-MRSA infection and regular animal contacts. On all farms, the initial patients, their household members, animals and barn environment were analysed for the presence of LA-MRSA. In addition, the epidemiologically linked hospital-related LA-MRSA isolates were included to investigate possible nosocomial transmissions. On five farms, LA-MRSA was discovered both in animals and in humans. In total, 49 LA-MRSA isolates of different origins underwent whole-genome sequencing, antimicrobial susceptibility testing and spa typing. All 49 isolates belonged to the sequence type 398 (ST398), spa types t011 and t034, and harboured staphylococcal chromosomal cassette mec Vc. High levels of concordance between resistance phenotypes and genotypes were observed. No transmission pairs between animals and initial patients were discovered. However, several isolates originating from farm animals and other household members formed clusters with pairwise distances of ≤14 single nucleotide polymorphisms (SNPs), indicating recent transmission events. In addition, three closely related isolates (0 SNP) form hospitalized patients were observed, indicating a possible nosocomial transmission. Two hospital-related isolates harboured the immune evasion cluster genes, which are associated with adaptation to the human host; however, these two isolates differed in >30 SNPs from the remaining isolates. Characteristics of LA-MRSA from Slovenia reflect those observed previously in other European studies. Immune evasion cluster-positive LA-MRSA ST398 suggests its re-adaptation to the human host and calls for a closer monitoring of such emerging LA-MRSA lineages, in addition to monitoring and preventing the introduction of LA-MRSA from farms to hospitals where transmission is highly plausible.

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.