Streptococcus penaeicida sp. nov., isolated from a diseased farmed Pacific white shrimp (Penaeus vannamei).

Strain CAIM 1838T, isolated from the hepatopancreas of a cultured diseased Pacific white shrimp (Penaeus vannamei), was subjected to characterization by a polyphasic taxonomic approach. On the basis of 16S rRNA gene sequence analysis, strain CAIM 1838T was most closely related to Streptococcus bovimastitidis 99.3 % and to other species of the Pyogenes clade of Streptococcus with lower similarity values. Average nucleotide identity values and the genome-to-genome distance of strain CAIM 1838T, as compared with the type strains, confirmed the separate species status with closely related species of the genus Streptococcus and were all below the thresholds to delimit a species, 93.1 and 49.4 %, respectively. The DNA G+C content was 38.1 mol%. Differential phylogenetic distinctiveness together with phenotypic properties obtained in this study revealed that strain CAIM 1838T could be differentiated from the closely related species. Based on these results it is proposed that strain CAIM 1838T represents a novel species in the genus Streptococcus, for which the name Streptococcus penaeicida sp. nov is proposed (type strain, CAIM 1838T=CECT 8596T,=DSM26545T), is proposed.

Pelistega suis sp. nov., isolated from domestic and wild animals.

Biochemical and molecular genetic studies were performed on three novel Gram-stain-negative, catalase- and oxidase-positive, bacilli-shaped organisms isolated from the tonsils of two pigs and one wild boar. The micro-organism was identified as a species of the genus Pelistega based on its cellular morphological and biochemical tests. The closest phylogenetic relative of the novel bacilli was Pelistega indica HM-7T (98.2 % 16S rRNA gene sequence similarity to the type strain). groEL and gyrB sequence analysis showed interspecies divergence from the closest 16S rRNA gene phylogenetic relative, P. indica of 87.0.% and 69 %, respectively. The polyamine pattern contains predominantly putrescine and 2-hydroxyputrescine. The major quinone is ubiquinone Q-8 and in the polar lipid profile, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminolipid and an unidentified lipid are predominant. The novel bacterial isolate can be distinguished from P. indica by several biochemical characteristics, such as the production of l-pyrrolydonil arylamidase but not gamma-glutamyl-transferase, and the utilization of different carbon sources. Based on both phenotypic and phylogenetic findings, the novel bacterium is classified as representing a novel species of the genus Pelistega, for which the name Pelistega suis sp. nov. is proposed. The type strain is 3340-03T ( = CECT 8400T = CCUG 64465T).

Streptococcus pharyngis sp. nov., a novel streptococcal species isolated from the respiratory tract of wild rabbits.

Four isolates of an unknown Gram-stain-positive, catalase-negative coccus-shaped organism, isolated from the pharynx of four wild rabbits, were characterized by phenotypic and molecular genetic methods. The micro-organisms were tentatively assigned to the genus Streptococcus based on cellular morphological and biochemical criteria, although the organisms did not appear to correspond to any species with a validly published name. Comparative 16S rRNA gene sequencing confirmed their identification as members of the genus Streptococcus, being most closely related phylogenetically to Streptococcus porcorum 682-03(T) (96.9% 16S rRNA gene sequence similarity). Analysis of rpoB and sodA gene sequences showed divergence values between the novel species and S. porcorum 682-03(T) (the closest phylogenetic relative determined from 16S rRNA gene sequences) of 18.1 and 23.9%, respectively. The novel bacterial isolate could be distinguished from the type strain of S. porcorum by several biochemical characteristics, such as the production of glycyl-tryptophan arylamidase and α-chymotrypsin, and the non-acidification of different sugars. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be assigned to a novel species of the genus Streptococcus, and named Streptococcus pharyngis sp. nov. The type strain is DICM10-00796B(T) ( = CECT 8754(T) = CCUG 66496(T)).

Estimation of cultivable bacterial diversity in the cloacae and pharynx in Eurasian griffon vultures (Gyps fulvus).

In this work, we describe the biodiversity of cloacal and pharynx culture-based bacteria (commensal and pathogenic), in 75 Eurasian griffon vultures (Gyps fulvus) from two geographic areas. We address the question of whether the cultivable microbiota of vultures is organised into assemblages occurring by chance. In addition, we assess bacterial diversity in both anatomic regions and geographic areas. Bacterial diversity was represented by 26 Gram-negative and 20 Gram-positive genera. The most common genera were Escherichia, Enterococcus, Staphylococcus, Clostridium and Lactococcus. Escherichia coli and Enterococcus faecalis were the most common species in cloacal and pharyngeal samples. Staphylococcus and Erysipelothrix were isolated from the pharynx and Salmonella and Corynebacterium from the cloacae, and no Campylobacter was isolated from the cloacal swabs. Ten cloacal swabs were positive for Salmonella, of which five isolates were Salmonella enterica serotype 4,(5),12:i:-, one isolate was S. enterica serotype Derby, three isolates were S. enterica serotype 61:k:1,5,7 and one isolate was S. enterica serotype Infantis. The null modelling approach revealed that the commensal bacteria of vultures are not structured in assemblages. On the other hand, differences in bacterial genus and species richness between cloacal and pharyngeal samples or between geographic areas were clear, with the pharynx in vultures from both geographic areas being richer. The results of this study indicate also that vultures can serve as a reservoir of certain pathogenic zoonotic bacteria. The dissemination of these zoonotic pathogens in wildlife could be prevented by periodic sanitary surveys.

Garvicin A, a novel class IId bacteriocin from Lactococcus garvieae that inhibits septum formation in L. garvieae strains.

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings.

Antimicrobial Susceptibility and Resistance Mechanisms in Mannheimia haemolytica Isolates from Sheep at Slaughter.

Mannheimia haemolytica is the main pathogen contributing to pneumonic pasteurellosis in sheep. The aim of this study was to investigate the antimicrobial resistance levels in M. haemolytica isolates from the lungs of slaughtered sheep and to examine the genetic resistance mechanisms involved. A total of 256 M. haemolytica isolates, 169 from lungs with pneumonic lesions and 87 from lungs without lesions, were analyzed by the disk diffusion method for 12 antimicrobials, and the whole genome of 14 isolates was sequenced to identify antimicrobial resistance determinants. Levels of phenotypic resistance ranged from <2% for 10 antimicrobials (amoxicillin, amoxicillin-clavulanic, ceftiofur, cefquinome, lincomycin/spectinomycin, gentamicin, erythromycin, florfenicol, enrofloxacin, and doxycycline) to 4.3% for tetracycline and 89.1% for tylosin. Six isolates carried tetH genes and four isolates carried, in addition, the strA and sul2 genes in putative plasmid sequences. No mutations associated with macrolide resistance were identified in 23 rDNA sequences, suggesting that the M. haemolytica phenotypic results for tylosin should be interpreted with care in the absence of well-established epidemiological and clinical breakpoints. The identification of strains phenotypically resistant to tetracycline and of several resistance genes, some of which were present in plasmids, highlights the need for continuous monitoring of susceptibility patterns in Pasteurellaceae isolates from livestock.

Corynebacterium conjunctivae: A New Corynebacterium Species Isolated from the Ocular Surface of Healthy Horses.

Twenty-two unidentified Gram-positive, rod-shaped organisms were recovered from the conjunctival surface of apparently healthy horses and subjected to a polyphasic taxonomic analysis. Based on cellular morphology and biochemical criteria, the isolates were tentatively assigned to the genus Corynebacterium, although they did not match any recognized species. Comparative 16S rRNA gene sequencing studies demonstrated that all of the isolates were phylogenetically members of the genus Corynebacterium. The isolates shared 99.4 to 100% 16S rRNA gene sequence similarity among the strains and 96.5% similarity with Corynebacterium tapiri 2385/12T, which was the closest phylogenetically related species. The DNA G+C content was 58.4 mol%. The major fatty acids were C15:0, C16:0, C17:1 ω8c and C18:1 ω9c, while the predominant mycolic acids consisted of C30:0, C32:0 and C34:0. The isolates were distinguished from related Corynebacterium species by a number of phenotypic properties. On the basis of phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from horses be classified in the genus Corynebacterium as Corynebacterium conjunctivae sp. nov. The type strain of C. conjunctivae is ICM19-01138T (DSM 109759T = CCUG 73728T).

Genome sequence of Lactococcus garvieae 8831, isolated from rainbow trout lactococcosis outbreaks in Spain.

Lactococcus garvieae is the etiological agent of lactococcosis, one of the most important disease threats to the sustainability of the rainbow trout farming industry. Here, we present the draft genome sequence of Lactococcus garvieae strain 8831, isolated from diseased rainbow trout, which is composed of 2,087,276 bp with a G+C content of 38%.

Genome sequence of Lactococcus garvieae 21881, isolated in a case of human septicemia.

Lactococcus garvieae is a Gram-positive bacterium considered an important opportunistic emerging human pathogen and also a well-recognized fish pathogen. Here, we present the draft genome sequence of Lactococcus garvieae strain 21881 (2,164,557 bp, with a G+C content of 37.9%), which represents the first report of a genome sequence on Lactococcus garvieae.

Pseudomonas composti sp. nov., isolated from compost samples.

Two unusual, Gram-negative, catalase- and oxidase-positive rods, designated C2(T) and C5, were isolated from compost samples. Comparative 16S rRNA gene sequencing studies demonstrated that both isolates were members of the genus Pseudomonas and belonged to the Pseudomonas aeruginosa group. Strain C2(T) was most closely related to Pseudomonas cuatrocienegasensis 1N(T) and Pseudomonas borbori R-20821(T) (97.9 and 97.8% 16S rRNA gene sequence similarity, respectively). However, phylogenetic analysis based on rpoD gene sequences revealed that both isolates could be discriminated from members of the P. aeruginosa group that exhibited >97% 16S rRNA gene sequence similarity. The DNA G+C content of strain C2(T) was 61.5 mol%. The major fatty acids of strain C2(T) were a summed feature (C(16:1)ω7c and/or iso-C(15:0) 2-OH), C(18:1)ω7c/12t/9t, C(16:0) and C(12:0), which supported the isolates' affiliation with the genus Pseudomonas. Moreover, strain C2(T) could be distinguished from its closest phylogenetic neighbours of the genus Pseudomonas by DNA-DNA hybridization studies and biochemical tests. On the basis of both phenotypic and phylogenetic findings, it is proposed that the isolates be classified as a novel species, with the name Pseudomonas composti sp. nov. The type strain is C2(T) (=CECT 7516(T)=CCUG 59231(T)).

Analysis of the genome content of Lactococcus garvieae by genomic interspecies microarray hybridization.

BACKGROUND: Lactococcus garvieae is a bacterial pathogen that affects different animal species in addition to humans. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study, the genomic content of L. garvieae CECT 4531 was analysed using bioinformatics tools and microarray-based comparative genomic hybridization (CGH) experiments. Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 were used as reference microorganisms. RESULTS: The combination and integration of in silico analyses and in vitro CGH experiments, performed in comparison with the reference microorganisms, allowed establishment of an inter-species hybridization framework with a detection threshold based on a sequence similarity of >or= 70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of the genes are related to ribosomal, sugar metabolism or energy conversion systems. Some of the identified genes, such as als and mycA, could be involved in the pathogenesis of L. garvieae infections. CONCLUSIONS: In this study, we identified 267 genes that were potentially present in L. garvieae CECT 4531. Some of the identified genes could be involved in the pathogenesis of L. garvieae infections. These results provide the first insight into the genome content of L. garvieae.

Utilization of lactose and presence of the phospho-ß-galactosidase (lacG) gene in Lactococcus garvieae isolates from different sources.

This study evaluates the utilization of lactose (Lac) and the presence of the phospho-ß-galactosidase (lacG) gene as markers for distinguishing between fish (Lac-/lacG-) and dairy isolates (Lac+/lacG+) of Lactococcus garvieae, using a panel of L. garvieae isolates from different sources. None of the fish isolates produced acid from lactose (Lac-), however Lac-/lacG- isolates were observed in pigs, cows, birds and humans. Most of the dairy isolates (77.8%) were Lac+/lacG+, but some dairy isolates did not produce acid from this sugar. Data in the present study show that the ability to metabolize lactose and the presence of the lacG gene are heterogeneously scattered among L. garvieae isolates of different sources. Therefore, the use of these criteria as markers to differentiate between L. garvieae isolates of dairy and fish origin should be considered with caution.

Genetic and virulence-phenotype characterization of serotypes 2 and 9 of Streptococcus suis swine isolates.

The aim of this study was to analyze the genetic characteristics and virulence phenotypes of Streptococcus suis, specifically, in clinical isolates of serotypes 2 and 9 (n = 195), obtained from diverse geographical areas across Spain. Pulsed-field gel electrophoresis (PFGE) typing identified 97 genetic profiles, 68% of which were represented by single isolates, indicative of a substantial genetic diversity among the S. suis isolates analyzed. Five PFGE profiles accounted for 33.3% of the isolates and were isolated from 38% of the herds in nine different provinces, indicative of the bacterium's widespread distribution in the Spanish swine population. Representative isolates of the most prevalent PFGE profiles of both serotypes were subjected to multilocus sequence typing (MLST) analysis. The results indicated that serotypes 2 and 9 have distinct genetic backgrounds. Serotype 2 isolates belong to the ST1 complex, a highly successful clone that has spread over most European countries. In accordance with isolates of this complex, most serotype 2 isolates also expressed the phenotype MRP(+)EF(+)SLY(+). Serotype 9 isolates belong to the ST61 complex, which is distantly related to the widespread European ST87 clone. Also, in contrast to most isolates of the European ST87 clone, which express the large variant MRP*, the majority of serotype 9 isolates (97.9%) did not express the protein.

Antimicrobial susceptibility and genetic characterization of Trueperella pyogenes isolates from pigs reared under intensive and extensive farming practices.

Trueperella pyogenes is an opportunistic pathogen associated with a variety of diseases and responsible for important economic losses for pig production. Minimal Inhibitory Concentration (MIC) and Pulsed Field Gel Electrophoresis (PFGE) typing analysis were used to determine the MIC distribution and to genetically characterize a total of 180 T. pyogenes isolates obtained from slaughtered pigs reared under intensive (TpIN, n = 89) and extensive (TpEX, n = 91) farming practices. Low MIC90 values for penicillin and amoxicillin (0.008 and 0.06 µg/ml, respectively), ceftiofur, gentamicin and enrofloxacin (1 µg/ml, respectively) were obtained, so they could be of choice for the empiric treatment of T. pyogenes infections. Except for the penicillin, amoxicillin and ceftiofur, a statistically significant difference was observed in the MIC distribution of all antimicrobials analysed between TpIN and TpEX isolates. Also, MIC90 values were higher in TpIN than in TpEX isolates for neomycin and streptomycin (32 µg/ml vs 8 µg/ml), sulfamethoxazole/trimethoprim (30.4/1.6 µg/ml vs 1.90/0.10 µg/ml) and tylosin (≥1024 µg/ml vs 1 µg/ml). A relatively lower genetic diversity was detected in TpIN in comparison with TpEX isolates (GD 0.42 and GD 0.47, respectively). All isolates were distributed in three clusters (A, B, C). TpIN isolates were statistically associated with cluster A (P = 0.0002; OR 3.21; CI95 1.74-5.93), whereas the TpEX were distributed throughout the dendrogram, showing more genetic diversity. These data suggest that the antimicrobial susceptibility and genetic variability of the T. pyogenes isolates could be influenced by the management systems.

Rapid differentiation of Staphylococcus aureus subspecies based on MALDI-TOF MS profiles.

Staphylococcus aureus encompasses 2 subspecies ( aureus and anaerobius) with significant differences in their epidemiology and pathogenicity. We evaluated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid identification of both subspecies using a panel of 52 S. aureus isolates (30 subsp. anaerobius and 22 subsp. aureus) recovered from different origins, countries, and years. The on-board library identification system correctly identified 42 of 52 (81%) S. aureus isolates at the species level with score values >2.0. Limited performance was observed for differentiation of S. aureus subspecies (particularly subsp. anaerobius). Visual inspection of MALDI-TOF MS profiles identified 5 subspecies-specific mass peaks ( m/ z 3430 and 6861 in S. aureus subsp. anaerobius, and m/ z 4046, 6890, and 8093 in S. aureus subsp. aureus) with 100% sensitivity and specificity values, which is potentially useful for differentiating these subspecies. The suitability of 3 models, Genetic Analysis (GA), Quick Classifier (QC), and Supervised Neural Network, for automatic identification of both subspecies was evaluated using the Recognition Capability (RC) and Cross Validation (CV) values provided by the on-board ClinProTools software. The GA and QC models reached RC and CV values of 100%. Both models were externally validated using a panel of 26 S. aureus isolates of both subspecies, with both models correctly classifying all isolates of both subspecies. MALDI-TOF MS coupled with ClinProTools software represents a rapid and simple approach for S. aureus subspecies discrimination.

The zoonotic potential of Lactococcus garvieae: An overview on microbiology, epidemiology, virulence factors and relationship with its presence in foods.

Lactococcus garvieae is a relevant worldwide fish pathogen affecting various farmed and wild marine and freshwater species. It has also been isolated from other animals, such as ruminants with subclinical mastitis and pigs with pneumonia. From the early 90s, L. garvieae has been associated with different human infections, mainly endocarditis. During the last five years, human infections by this bacterium appear to be increasing, likely due to the improvement in microbiological methods for bacterial identification and the alertness of this bacterium by physicians. Human L. garvieae infections have been associated with the consumption or the handling of contaminated raw fish or seafood, and recently, a genetic study showed that meat, raw milk and dairy products may also be food sources of human L. garvieae infections. However, the status of L. garvieae as a potential zoonotic bacterium is still controversial to date. In this work, we describe four new human infections by L. garvieae in elderly and inmunocompromised patients, and we show an overview on L. garvieae microbiology, epidemiology, virulence factors and relationship with its presence in foods.

Screening of virulence-associated genes as a molecular typing method for characterization of Streptococcus suis isolates recovered from wild boars and pigs.

Streptococcus suis is an important zoonotic pathogen associated with a wide range of diseases in pigs, but has also been isolated from wild animals such as rabbits and wild boars. In the current study, 126 S. suis isolates recovered from pigs (n = 85) and wild boars (n = 41) were tested by polymerase chain reaction (PCR) for the presence of nine virulence-associated genes. S. suis isolates from wild boars were differentiated by the lower detection rates of the epf, sly, mrp, sao and dltA genes (0%, 2.4%, 2.4%, 4.8% and 21.9%, respectively) compared with the isolates from pigs (56.5%, 75.3%, 56.5%, 88.2.0% and 88.2%, respectively). The differences in the content of these virulence-associated genes were statistically significant (P < 0.05). There was a correlation between the variants saoM and saoL and serotypes 2 and 9, respectively (P < 0.05). Isolates were classified into 31 virulence-associated gene profiles (VPs). Ten VPs were detected among wild boar isolates and 22 VPs among pig isolates, with only two VPs common to wild boars and pigs. The predominant VPs among isolates from wild boars (VP1, VP7) were different from those observed in pig isolates (VP16 and VP26). VP16 was detected exclusively in clinical pig isolates of serotype 9 and VP26 was detected in 71.4% of the serotype 2 clinical pig isolates. Further multilocus sequence typing (MLST) analysis showed a significant correlation association between certain VPs and STs (VP16 and VP17 with ST123 and ST125 and VP26 with ST1). In conclusion, the current study showed that combination of virulence-associated gene profiling and MLST analysis may provide more information of the relatedness of the S. suis strains from different animal species that could be useful for epidemiological purposes.

Genetic analysis of human clinical isolates of Lactococcus garvieae: Relatedness with isolates from foods.

Lactococcus garvieae is a Gram-positive bacterium well-known as an important pathogen in aquaculture, and it is also a human pathogen of increasing clinical significance. Forty-three human L. garvieae isolates from clinical specimens were characterized by Multilocus Sequence Typing (MLST). Twenty-six different sequence types (STs) were identified among the human isolates, of which 20 were novel STs. Most human isolates clustered into four clonal complexes, with a predominance of CC3. Within CC3, ST10 was the most common genotype, indicating the existence of a circulating genetic lineage among the human isolates analyzed. The four CCs also grouped L. garvieae strains isolated from meat, dairy and fish, indicating a genetic overlap between isolates from human and these foods. Genetic relatedness among human and food L. garvieae isolates was confirmed by phylogenetic analysis based on the concatenated sequences of the seven MLST genes. These results represent the first evidence of genetic relatedness between isolates of L. garvieae of human and those isolated meat, milk and dairy products and suggest that, in addition to fish and seafood, these foods might represent important sources of human L. garvieae infections.