Garvicins AG1 and AG2: Two Novel Class IId Bacteriocins of Lactococcus garvieae Lg-Granada.

Lactococcus garvieae causes infectious diseases in animals and is considered an emerging zoonotic pathogen involved in human clinical conditions. In silico analysis of plasmid pLG50 of L. garvieae Lg-Granada, an isolate from a patient with endocarditis, revealed the presence of two gene clusters (orf46-47 and orf48-49), each one encoding a novel putative bacteriocin, i.e., garvicin AG1 (GarAG1; orf46) and garvicin AG2 (GarAG2; orf48), and their corresponding immunity proteins (orf47 and orf49). The chemically synthesised bacteriocins GarAG1 and GarAG2 presented inhibitory activity against pathogenic L. garvieae strains, with AG2 also being active against Listeria monocytogenes, Listeria ivanovii and Enterococcus faecalis. Genetic organisation, amino acid sequences and antimicrobial activities of GarAG1 and GarAG2 indicate that they belong to linear non-pediocin-like one-peptide class IId bacteriocins. Gram-positive bacteria that were sensitive to GarAG2 were also able to ferment mannose, suggesting that this bacteriocin could use the mannose phosphotransferase transport system (Man-PTS) involved in mannose uptake as a receptor in sensitive strains. Intriguingly, GarAG1 and GarAG2 were highly active against their own host, L. garvieae Lg-Granada, which could be envisaged as a new strategy to combat pathogens via their own weapons.

Spleen and head kidney differential gene expression patterns in trout infected with Lactococcus garvieae correlate with spleen granulomas.

Lactococcus garvieae is a significant pathogen in aquaculture with a potential zoonotic risk. To begin to characterize the late immune response of trout to lactococcosis, we selected infected individuals showing clinical signs of lactococcosis. At the time lactococcosis clinical signs appeared, infection by L. garvieae induced a robust inflammatory response in the spleen of rainbow trout, which correlated with abundant granulomatous lesions. The response in kidney goes in parallel with that of spleen, and most of the gene regulations are similar in both organs. A correlation existed between the early inflammatory granulomas in spleen (containing macrophages with internalized L. garvieae) and up-regulated gene sets, which defined the presence of macrophages and neutrophils. This is the first analysis of the immune transcriptome of rainbow trout following L. garvieae infection during the initiation of adaptive immune mechanisms and shows a transcriptome induction of antibody response by both IgM (+) and IgT (+) spleen B cells to respond to systemic infection. These results increase our understanding of lactococcosis and pave the way for future research to improve control measures of lactococcosis on fish farms.

Antimicrobial susceptibility of Pasteurella multocida isolated from sheep and pigs in Spain - Short communication.

Pasteurella multocida is responsible for economically important diseases in sheep and pigs. Antimicrobial susceptibility studies are essential for initiating rational and effective empirical therapy of P. multocida infections. In this study we investigated the antimicrobial susceptibility to 18 antimicrobial agents of 156 clinical isolates of P. multocida from sheep (n = 87) and pigs (n = 69) using the microdilution method. Both sheep and pig isolates exhibited low levels of resistance (≤ 15%) to ceftiofur, gentamicin, neomycin, spectinomycin, chlortetracycline, tulathromycin, florfenicol, danofloxacin, and enrofloxacin and trimethoprim/sulphamethoxazole, high resistance rates (> 15% up to 50%) to oxytetracycline, tilmicosin, and tiamulin, and very high resistance rates (> 50%) to tylosin tartrate, clindamycin, and sulphadimethoxine. However, sheep isolates exhibited significantly lower percentages of resistance and lower MIC90 values (P < 0.05) than pig isolates for most of the antimicrobials tested. In addition, sheep isolates exhibited also significantly lower phenotypic antimicrobial resistance diversity (8 resistotypes vs. 30 resistotypes). LAC-LIN-SUL-MAC was the resistotype most frequently detected in sheep (39.1%) and LIN-SUL-MAC in pig isolates (26.1%). The differences in susceptibility patterns could be influenced by the lower use of antimicrobials in the small ruminant industry compared with the pig farming industry.

Human Pasteurella multocida Infection with Likely Zoonotic Transmission from a Pet Dog, Spain.

We report a case of urinary tract infection caused by an unusual genotype (sequence type 211) of Pasteurella multocida associated with human infection. Molecular genetic analysis of P. multocida isolates obtained from the human patient and his pet strongly suggests a zoonotic transmission of this bacterium.

Characterization of Streptococcus pyogenes from Animal Clinical Specimens, Spain.

Streptococcus pyogenes appears to be almost exclusively restricted to humans, with few reports on isolation from animals. We provide a detailed characterization (emm typing, pulsed-field gel electrophoresis [PFGE], and multilocus sequence typing [MLST]) of 15 S. pyogenes isolates from animals associated with different clinical backgrounds. We also investigated erythromycin resistance mechanisms and phenotypes and virulence genes. We observed 2 emm types: emm12 (11 isolates) and emm77 (4 isolates). Similarly, we observed 2 genetic linages, sequence type (ST) 26 and ST63. Most isolates exhibited the M macrolide resistance phenotype and the mefA/ermB genotype. Isolates were grouped into 2 clones on the basis of emm-MLST-PFGE-virulence gene profile combinations: clone 1, characterized by the combined genotype emm12-ST36-pulsotype A-speG; and clone 2, characterized by the genotype emm77-ST63-pulsotype B-speC. Our results do not show conclusively that animals may represent a new reservoir of S. pyogenes but indicate the ability of human-derived S. pyogenes isolates to colonize and infect animals.

Jeotgalibaca porci sp. nov. and Jeotgalibaca arthritidis sp. nov., isolated from pigs, and emended description of the genus Jeotgalibaca.

Biochemical and molecular genetic studies were performed on two novel Gram-stain-positive, catalase-negative, coccus-shaped organisms isolated from liquid joint samples of two pigs. The micro-organisms were not identified as members of a recognized species based on results of cellular, morphological and biochemical tests. 16S rRNA gene sequence comparison studies allowed their identification as members of the genus Jeotgalibaca, but the organisms were different to Jeotgalibaca dankookensis, the single species of the genus. The two micro-organisms shared 96.3 and 96.9 % 16S rRNA gene sequence similarity values with their nearest phylogenetic relative, J. dankookensis. The novel bacterial isolates were distinguished from J. dankookensis using biochemical tests. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacteria be classified as representatives of two novel species of the genus Jeotgalibaca, Jeotgalibaca porci sp. nov. and Jeotgalibaca arthritidis sp. nov. The type strain of Jeotgalibaca porcisp. nov. is 1804-02T (=CECT 9156T=CCUG 69148T) and that of Jeotgalibaca arthritidissp. nov. is 1805-02T (=CECT 9157T=CCUG 69147T).

Streptococcus ovuberis sp. nov., isolated from a subcutaneous abscess in the udder of a sheep.

One unidentified, Gram-stain-positive, catalase-negative coccus-shaped organism was recovered from a subcutaneous abscess of the udder of a sheep and subjected to a polyphasic taxonomic analysis. Based on cellular morphology and biochemical criteria, the isolate was tentatively assigned to the genus Streptococcus, although the organism did not appear to match any recognized species. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus and showed that the nearest phylogenetic relatives of the unknown coccus corresponded to Streptococcus moroccensis and Streptococcus cameli (95.9 % 16S rRNA gene sequence similarity). The sodA sequence analysis showed less than 89.3 % sequence similarity with the currently recognized species of the genus Streptococcus. The novel bacterial isolate was distinguished from close relatives of the genus Streptococcusby using biochemical tests. A mass spectrometry profile was also obtained for the novel isolate using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a representative of a novel species of the genus Streptococcus, Streptococcus ovuberis sp. nov. The type strain of Streptococcus ovuberissp. nov. is VB15-00779T (=CECT 9179T=CCUG 69612T).

Carriage of Staphylococcus aureus by free-living wild animals in Spain.

The presence of methicillin-susceptible Staphylococcus aureus (MSSA) was analyzed in different free-living wild animals to assess the genetic diversity and predominant genotypes on each animal species. Samples were taken from the skin and/or nares, and isolates were characterized by spa typing, multilocus sequence typing (MLST) and antimicrobial susceptibility testing. The proportion of MSSA carriers were 5.00, 22.93, 19.78, and 17.67% in Eurasian griffon vulture, Iberian ibex, red deer, and wild boar, respectively (P = 0.057). A higher proportion of isolates (P = 0.000) were recovered from nasal samples (78.51%) than skin samples (21.49%), but the 9.26% of red deer and 18.25% of wild boar would have been undetected if only nasal samples had been tested. Sixty-three different spa types were identified, including 25 new spa types. The most common were t528 (43.59%) in Iberian ibex, t548 and t11212 (15.79% and 14.04%) in red deer, and t3750 (36.11%) in wild boar. By MLST, 27 STs were detected, of which 12 had not been described previously. The most frequent were ST581 for Iberian ibex (48.72%), ST425 for red deer (29.82%), and ST2328 for wild boar (42.36%). Isolates from Eurasian griffon vulture belong to ST133. Host specificity has been observed for the most frequent spa types and STs (P = 0.000). The highest resistance percentage was found against benzylpenicillin (average, 22.2%), although most of the S. aureus isolates were susceptible to all antimicrobial tested. Basically, MSSA isolates were different from those MRSA isolates previously detected in the same animal species.

Genotypic Comparison of Pasteurella multocida from Healthy Animals at Entry to the Feedlots with That and from Bovine Respiratory Disease-Affected Animals during the Fattening Period.

The aim of this study was to investigate the possible genotypic differences between commensal Pasteurella multocida isolates from apparently healthy animals (AHA) at the time of entry to feedlots and those from BRD-affected animals (BRD-AA). A total of 20 batches of beef calves in seven feedlots were followed-up during the fattening period. P. multocida was isolated from 28.1% of AHA and 22.9% of BRD-AA. All isolates belonged to the A: L3 genotype. Most isolates from clinical cases (81.0%) grouped into a PFGE cluster were significantly associated with BRD cases (OR, 24.9; 95% CI, 6.4-96.2). The whole genomes of 14 isolates representative of the pulsotypes most frequently detected in BRD-AA and AHA were sequenced and compared with 53 bovine genomes belonging to the identified ST13, ST79, and ST80 genotypes for a global comparison. No differences were found in the virulence-associated gene content between sequence types (STs) globally or between BRD-AA and AHA isolates in this study. Significantly, ST79 isolates harbored ARGs, conferring resistance to different antimicrobials, including macrolides and tetracyclines, which are commonly used for the treatment of BRD. Two Spanish ST79 isolates carried an ICE highly similar to ICE Tn7407, which was recently detected in Germany, suggesting that ST79 P. multocida isolates in Europe and North America may be associated with different ICEs.

16S-23S rRNA Internal Transcribed Spacer Region (ITS) Sequencing: A Potential Molecular Diagnostic Tool for Differentiating Lactococcus garvieae and Lactococcus petauri.

Lactococcus garvieae is the etiological agent of lactococcosis, a clinically and economically significant infectious disease affecting farmed rainbow trout. L. garvieae had been considered the only cause of lactococcosis for a long time; however, L. petauri, another species of the genus Lactococcus, has lately been linked to the same disease. The genomes and biochemical profiles of L. petauri and L. garvieae have a high degree of similarity. Traditional diagnostic tests currently available cannot distinguish between these two species. The aim of this study was to use the transcribed spacer (ITS) region between 16S rRNA and 23S rRNA as a potential useful molecular target to differentiate L. garvieae from L. petauri, saving time and money compared to genomics methods currently used as diagnostic tools for accurate discrimination between these two species. The ITS region of 82 strains was amplified and sequenced. The amplified fragments varied in size from 500 to 550 bp. Based on the sequence, seven SNPs were identified that separate L. garvieae from L. petauri. The 16S-23S rRNA ITS region has enough resolution to distinguish between closely related L. garvieae and L. petauri and it can be used as a diagnostic marker to quickly identify the pathogens in a lactococcosis outbreak.

First isolation and characterization of Chryseobacterium shigense from rainbow trout.

BACKGROUND: There have been an increasing number of infections in fish associated with different species of Chryseobacterium, being considered potentially emerging pathogens. Nevertheless the knowledge of the diversity of species associated with fish disease is partial due to the problems for a correct identification at the species level based exclusively on phenotypic laboratory methods. RESULTS: Chryseobacterium shigense was isolated from the liver, kidney and gills of diseased rainbow trout in different disease episodes that occurred in a fish farm between May 2008 and June 2009. Identity of the isolates was confirmed by 16 S rRNA gene sequencing and phenotypic characterization. Isolates represented a single strain as determined by random amplified polymorphic DNA analysis. CONCLUSIONS: This is the first description of the recovery of C. shigense from clinical specimens in trout, a very different habitat to fresh lactic acid beverage where it was initially isolated.

Comparative genomics and evolutionary analysis of Lactococcus garvieae isolated from human endocarditis.

Lactococcus garvieae is a well-known pathogen of fish, but is rarely involved in infections in humans and other mammals. In humans, the main clinical manifestation of L. garvieae infections is endocarditis usually related to the ingestion of contaminated food, such as undercooked fish and shellfish. This study presents the first complete genomic sequence of a clinical L. garvieae strain isolated from a patient with endocarditis and its comparative analysis with other genomes. This human isolate contains a circular chromosome of 2 099 060 bp and one plasmid of 50 557 bp. In comparison with other fully sequenced L. garvieae strains, the chromosomal DNA of L. garvieae Lg-Granada carries a low proportion of insertion sequence elements and a higher number of putative prophages. Our results show that, in general, L. garvieae is a highly recombinogenic species with an open pangenome in which almost 30 % of its genome has undergone horizontal transfers. Within the genus Lactococcus, L. lactis is the main donor of genetic components to L. garvieae but, taking Lg-Granada as a representative, this bacterium tends to import more genes from Bacilli taxa than from other Lactococcus species.

Detection and Antimicrobial Resistance of Enterobacteriaceae other than Escherichia Coli in Raccoons from the Madrid Region of Spain.

Introduction: Raccoons are an invasive alien species widely distributed in the Madrid region of Spain. These animals can carry a variety of enteric bacteria with associated antimicrobial resistance, which can infect humans and livestock. However, to our knowledge, the presence of non-E. coli Enterobacteriaceae in raccoons has not been previously studied. Material and Methods: We conducted a study to examine the species distribution of Enterobacteriaceae isolates other than E. coli, as well as their antimicrobial resistance, in the faeces of 83 raccoons in the Madrid region. Results: We detected 12 Enterobacteriaceae isolates other than E. coli belonging to seven different species: Citrobacter freundii (1 isolate), Citrobacter gillenii (3 isolates), Citrobacter murliniae (1 isolate), Citrobacter portucalensis (2 isolates), Enterobacter hormaechei subsp. hoffmannii (1 isolate), Hafnia paralvei (2 isolates) and Raoultella ornithinolytica (2 isolates). These isolates were found in 7 of the 83 (8.4%) animals studied. To our knowledge, this study is the first report of the presence of non-E. coli Enterobacteriaceae in raccoon faeces. All isolates but one were resistant to at least one of the 14 antimicrobials tested. Resistance to ampicillin (83.3%), amoxicillinclavulanic acid (50%) and cefoxitin (33.3%) was the most frequent. Conclusion: Our study indicates that raccoons are a potential source of infection with Enterobacteriaceae other than E. coli for humans and livestock in the Madrid region.

Molecular Epidemiology of Pasteurella multocida Associated with Bovine Respiratory Disease Outbreaks.

Studies that characterize bovine respiratory disease (BRD)-associated Pasteurella multocida isolates are scarce compared with research on isolates from other hosts and clinical backgrounds. In the present study, 170 P. multocida isolates from 125 BRD outbreaks were characterized by capsular and LPS typing as well as by virulotyping. Three capsular types (A, B, F) and three LPS genotypes (L2, L3, L6) were identified. Capsular and LPS typing revealed a very low genetic diversity (GD = 0.02) among P. multocida, with most isolates belonging to genotype A:L3 (97.6%). Virulotyping identified seven virulence-associated gene profiles, with two profiles including 95.9% of the isolates. A subset of isolates was further characterized by MLST and PFGE. The sequence types ST79 and ST13 were the most frequently identified and were grouped into the same clonal complex (CC13), a result that supports the clonal population structure of BRD-associated P. multocida isolates. PFGE typing also revealed a low genetic diversity (GD = 0.18), detecting a single pattern in 62.5% of the outbreaks in which multiple isolates were analyzed. Overall, 85.2% of the isolates belonged to pulsotypes with at least 80% genetic similarity, consistent with a clonal population structure observed by MLST analysis and corroborating the genetic relatedness of most P. multocida isolates associated with BRD in cattle.

Isolation and Genetic Characterization of Streptococcus iniae Virulence Factors in Adriatic Sturgeon (Acipenser naccarii).

The first case of infection of Streptococcus iniae in Adriatic sturgeon (Acipenser naccarii) was recently reported in a raceway system located in Northern Italy. A second episode of infection in sturgeons with absence of mortality and evident clinical signs, was registered in November 2020 in the same farm and is reported in this study. Histopathological changes observed in infected organs are described. The strains isolated in the two episodes were compared using molecular analysis based on PCR, phylogeny and virulence factors analysis. Not all the major virulence factors were detected for the two strains; in particular the strains 78697, isolated in November, lacks cpsD, compared to the strains 64844, isolated in September. Moreover, genetic variations were reported for lctO and pmg genes. These findings let us hypothesize a different virulence of the strains in accordance with clinical findings related to the sturgeons.

Characterization of some bacterial strains isolated from animal clinical materials and identified as Corynebacterium xerosis by molecular biological techniques.

Eighteen Corynebacterium xerosis strains isolated from different animal clinical specimens were subjected to phenotypic and molecular genetic studies. On the basis of the results of the biochemical characterization, the strains were tentatively identified as C. xerosis. Phylogenetic analysis based on comparative analysis of the sequences of 16S rRNA and rpoB genes revealed that the 18 strains were highly related to C. xerosis, C. amycolatum, C. freneyi, and C. hansenii. There was a good concordance between 16S rRNA and partial rpoB gene sequencing results, although partial rpoB gene sequencing allowed better differentiation of C. xerosis. Alternatively, C. xerosis was also differentiated from C. freneyi and C. amycolatum by restriction fragment length polymorphism analysis of the 16S-23S rRNA gene intergenic spacer region. Phenotypic characterization indicated that besides acid production from D-turanose and 5-ketogluconate, 90% of the strains were able to reduce nitrate. The absence of the fatty acids C(14:0), C(15:0), C(16:1)omega 7c, and C(17:1)omega 8c can also facilitate the differentiation of C. xerosis from closely related species. The results of the present investigation demonstrated that for reliable identification of C. xerosis strains from clinical samples, a combination of phenotypic and molecular-biology-based identification techniques is necessary.

A Summer Mortality Outbreak of Lactococcosis by Lactococcus garvieae in a Raceway System Affecting Farmed Rainbow Trout (Oncorhynchus mykiss) and Brook Trout (Salvelinus fontinalis).

Lactococcosis is a fish disease of major concern in Mediterranean countries caused by Lactococcus garvieae. The most susceptible species is the rainbow trout (Oncorhynchus mykiss), suffering acute disease associated with elevated mortalities compared to other fish species. References reported that other salmonids are also susceptible to the disease, but no mortality outbreak has been described to date. The aim of this study was to present a mortality outbreak that occurred in brook trout (Salvelinus fontinalis) farmed in northwestern Italy during the summer of 2018. Fish exhibited clinical signs, such as exophthalmos, diffused hemorrhages localized in the ocular zone, hemorrhagic enteritis, and enlarged spleen. L. garvieae was isolated in all fish. Molecular and epidemiological characterization of the isolates, through Pulsed Field Gel Electrophoresis (PFGE), confirmed the initial hypothesis of water as vehicle of infection favoring transmission between rainbow trout farmed in upstream compartments and brook trout located in downstream tanks. Moreover, several environmental conditions affected and promoted the outbreak, among them the high-water temperature, which probably induced a physiological stress in brook trout, being way above the optimal temperature for this species, increasing the susceptibility to infection.

Potentially human-virulent Vibrio vulnificus isolates from diseased great pompano (Trachinotus goodei).

Vibrio vulnificus is an opportunistic human pathogen responsible for the majority of seafood-associated deaths worldwide and is also a relevant fish pathogen for the aquaculture industry. In addition to infections in aquatic livestock, V. vulnificus also represents a risk to aquarium animals. For the first time, this work describes an important mortality outbreak in Trachinotus goodei in a zoo aquarium, with the isolation of Vibrio vulnificus (Vv) from the internal organs of the diseased fish. The isolates were identified by MALDI-TOF MS, serotyped and characterized by pulsed-field gel electrophoresis (PFGE). Although the isolates from great pompanos did not belong to pathovar piscis (formerly biotype 2) or to any of the fish-related serovars, they all had identical phenotypes, antimicrobial susceptibility profiles and PFGE patterns, which together with their isolation in pure culture from internal organs is strongly indicative of their clinical significance. Moreover, Vv isolates harboured important genetic markers of human virulence potential: they had the clinical variant of the vcg gene, gave the 338 bp DNA amplification product of the pilF gene and resisted the bactericidal activity of human serum. All these results strongly suggest that these Vv isolates should be considered potentially virulent for humans. These results extend the range of fish species affected by V. vulnificus, confirm the threat that this pathogen represents to aquatic animals and highlight the risk that this bacterial pathogen poses to human health.

Ovine Mannheimia haemolytica isolates from lungs with and without pneumonic lesions belong to similar genotypes.

This study investigated the genetic characteristics of 121 ovine Mannheimia haemolytica isolates from lungs with (n = 75) and without pneumonic lesions (n = 46) using multilocus sequence typing (MLST), virulence-associated gene typing and pulsed-field gel electrophoresis (PFGE). Twelve STs were identified with most isolates (81%) belonged to ST16, ST28 and ST8. Analysis of the M. haemolytica MLST Database indicate a wide distribution of these genotypes in small ruminants, never reported in bovine isolates. This could suggest the adaptation of certain genetic lineages of M. haemolytica to small ruminants. e-BURST analysis grouped most STs into three clonal complexes (CC2, CC8 and CC28), consistent with a clonal population structure of M. haemolytica. Virulence-associated gene typing identified five virulence profiles in 64% and 65.1% of the M. haemolytica isolates from lungs with and without pneumonic lesions, respectively. These data suggest that M. haemolytica isolates from the lungs with and without pneumonic lesions are genetically homogeneous. By PGFE analysis a high level of genetic diversity was observed not only within isolates from lungs without pneumonic lesions but also among isolates from pneumonic lesions (GD 0.69 and GD 0.66, respectively; P > 0.05). These results indicate that multiple strains of M. haemolytica may be associated with individual cases of pneumonia in sheep.