Streptococcus pyogenes appears to be almost exclusively restricted to humans, with few reports on... more 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...
The influence of potassium tellurite (PT) and brain heart infusion agar (Difco), two components o... more The influence of potassium tellurite (PT) and brain heart infusion agar (Difco), two components of modified Listeria selective agar medium (LSAMm), on the hemolytic phenotype of Listeria spp. was studied. L. monocytogenes and L. ivanovii displayed bigger zones of hemolysis on brain heart intusion agar compared with on Columbia agar base. The addition of PT increased the sizes of zones of hemolysis displayed by L. monocytogenes. This effect seemed to be produced by the enhancement of the cytolytic effect of listeriolysin O. PT decreased the hemolysis produced by L. ivanovii, and this effect seemed to be due to an inhibition of the sphingomyelinase C produced by this species.
International journal of systematic and evolutionary microbiology, 2018
Strain CAIM 1838, isolated from the hepatopancreas of a cultured diseased Pacific white shrimp (P... more Strain CAIM 1838, 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 1838 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 1838, 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 1838 could be differentiated from the closely related species. Based on these results it is proposed that strain CAIM 1838 r...
The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical sp... more The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical specimens of diseased pigs was evaluated. For this proposal, the MALDI BDAL Database (Bruker Daltoniks, Germany) was supplemented with the main spectrum profiles (MSP) of the reference strains of S. porci, S. porcorum and S. plurextorum associated with pneumonia and septicemia. Although these three species showed similar MALDI profiles, several peaks were recognized that can be useful for their differentiation: S. porci (4113, 6133, 7975 and 8228 m/z Da), S. plurextorum (3979, 4078, 4665, 6164, 6491, 6812, 7959 and 9330 m/z Da) and S. porcorum (3385, 3954, 4190, 6772, 7908, and 8381 m/z Da). After adding these MSPs, an evaluation was conducted to determine the accuracy of MALDI-TOF MS for the identification of streptococci from diseased pigs using 74 field isolates. Isolates were identified as S. suis, S. porcinus, S. dysgalactiae, S. hyovaginalis, S. porcorum, S. alactolyticus, S. hyointe...
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, Jan 19, 2015
Lactococcus garvieae is a gram-positive bacterium well-known as an important pathogen in aquacult... more 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 gene...
The aim of the present study was to compare the efficiency of two PCR techniques for the diagnosi... more The aim of the present study was to compare the efficiency of two PCR techniques for the diagnosis of small ruminant lentiviruses (SRLVs). Detection of the proviral genome by PCR, though sensitive, is difficult due to the heterogeneity of the SRLV genomes. One of the PCR techniques amplifies a fragment in the pol gene (pol-PCR) and the other PCR targets the LTR region of the proviral genome (LTR-PCR). Milk from 194 sheep and 163 goats from farms in the Central Spain was analyzed by both techniques and compared to results obtained by ELISA. When compared to the serologic assay, the agreement of both PCR techniques was very low (0.024 and 0.020 in sheep, and 0.124 and 0.114 in goats). In view of these results, it may be concluded that the efficacy of PCR for the diagnosis of SRLVs is low and a combination of PCR and ELISA should be used for diagnosis.
This study evaluated a multiplex real-time PCR method specific for the mecA, femA-SA and femA-SE ... more This study evaluated a multiplex real-time PCR method specific for the mecA, femA-SA and femA-SE genes for rapid identification of Staphylococcus aureus, Staphylococcus epidermidis and non-S. epidermidis coagulase-negative staphylococci (CoNS), and meticillin susceptibility testing directly in positive blood cultures that grew Gram-positive cocci in clusters. A total of 100 positive blood cultures produced: 39 S. aureus [12 meticillin-resistant S. aureus (MRSA), 31 % of all the S. aureus]; 30 S. epidermidis (56.6 % of the CoNS), 8 Staphylococcus capitis (15.1 %), 3 Staphylococcus saprophyticus (5.7 %), 4 Staphylococcus hominis (7.5 %), 3 Staphylococcus haemolyticus (5.7 %), 2 Staphylococcus warneri (3.8 %), 1 Staphylococcus cohnii (1.9 %) and 2 unidentified Staphylococcus spp. (3.8 %); and 1 Micrococcus luteus in pure culture. Two blood cultures had no growth on subculture and five blood cultures grew mixed CoNS. For the 95 blood cultures with pure growth or no growth on subculture,...
Zeitschrift f�r Lebensmittel-Untersuchung und -Forschung, 1990
An overlay technique is proposed for the identification and counting of haemolytic Listeria colon... more An overlay technique is proposed for the identification and counting of haemolytic Listeria colonies directly on selective plating media. The technique was applied to different Listeria-selective plating media. In pure culture studies with collection strains, the overlay technique was more efficient and reliable for detection haemolytic Listeria species compared with the incorporation of blood into the agar. The efficacy of the overlay technique for the direct detection of haemolytic colonies of Listeria from raw milk samples was related to agar selectivity. The best results were obtained with Listeria-selective agar medium modified (LSAMM). Catalase assay, together with reactions for aesculin and tellurite, were useful and reliable criteria for the identification of Listeria. All colonies on LSAMM which were positive for catalase, tellurite and aesculin while those displaying typical haemolysis corresponded in most cases to L. monocytogenes.
During the spring and summer of 2001, faeces from 166 wild reptiles (94 individuals) and amphibia... more During the spring and summer of 2001, faeces from 166 wild reptiles (94 individuals) and amphibians (72 individuals) from 21 different species found in central Spain were examined for the presence of Salmonella. Thirty-nine reptiles (41.5%) yielded 48 Salmonella isolates, whereas all the amphibians examined were negative. Subspecies Salmonella enterica enterica (I) accounted for up to 50% of isolates. Fourteen isolates (29.2%) belonged to subspecies diarizonae (IIIb), six isolates (12.5%) to subspecies salamae (II), and four isolates (8.3%) to subspecies arizonae (IIIa). Twenty-seven different serotypes were identified. Serotypes Anatum (12.5%), Herzliya (8.3%), Abony, 18:l,v:z, 9,12:z29:1,5 and 38:z10:z53 (6.2%/each) were the most frequently isolated. A high percentage (39.6%) of isolates belonged to serotypes previously associated with environmental sources. Also, 37.5% of isolates belonged to serotypes which had been related to human cases of salmonellosis. From these data, it is concluded that wild reptiles, but apparently not amphibians, may represent an important reservoir of Salmonella in nature and have potential implications for public health.
A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens in... more A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish ( Streptococcus iniae , Streptococcus difficilis , Streptococcus parauberis , and Lactococcus garvieae ). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae , 12.5 pg for S. difficilis , 50 pg for S. parauberis , and 30 pg for L. garvieae . The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.
Streptococcus pyogenes appears to be almost exclusively restricted to humans, with few reports on... more 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...
The influence of potassium tellurite (PT) and brain heart infusion agar (Difco), two components o... more The influence of potassium tellurite (PT) and brain heart infusion agar (Difco), two components of modified Listeria selective agar medium (LSAMm), on the hemolytic phenotype of Listeria spp. was studied. L. monocytogenes and L. ivanovii displayed bigger zones of hemolysis on brain heart intusion agar compared with on Columbia agar base. The addition of PT increased the sizes of zones of hemolysis displayed by L. monocytogenes. This effect seemed to be produced by the enhancement of the cytolytic effect of listeriolysin O. PT decreased the hemolysis produced by L. ivanovii, and this effect seemed to be due to an inhibition of the sphingomyelinase C produced by this species.
International journal of systematic and evolutionary microbiology, 2018
Strain CAIM 1838, isolated from the hepatopancreas of a cultured diseased Pacific white shrimp (P... more Strain CAIM 1838, 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 1838 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 1838, 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 1838 could be differentiated from the closely related species. Based on these results it is proposed that strain CAIM 1838 r...
The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical sp... more The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical specimens of diseased pigs was evaluated. For this proposal, the MALDI BDAL Database (Bruker Daltoniks, Germany) was supplemented with the main spectrum profiles (MSP) of the reference strains of S. porci, S. porcorum and S. plurextorum associated with pneumonia and septicemia. Although these three species showed similar MALDI profiles, several peaks were recognized that can be useful for their differentiation: S. porci (4113, 6133, 7975 and 8228 m/z Da), S. plurextorum (3979, 4078, 4665, 6164, 6491, 6812, 7959 and 9330 m/z Da) and S. porcorum (3385, 3954, 4190, 6772, 7908, and 8381 m/z Da). After adding these MSPs, an evaluation was conducted to determine the accuracy of MALDI-TOF MS for the identification of streptococci from diseased pigs using 74 field isolates. Isolates were identified as S. suis, S. porcinus, S. dysgalactiae, S. hyovaginalis, S. porcorum, S. alactolyticus, S. hyointe...
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, Jan 19, 2015
Lactococcus garvieae is a gram-positive bacterium well-known as an important pathogen in aquacult... more 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 gene...
The aim of the present study was to compare the efficiency of two PCR techniques for the diagnosi... more The aim of the present study was to compare the efficiency of two PCR techniques for the diagnosis of small ruminant lentiviruses (SRLVs). Detection of the proviral genome by PCR, though sensitive, is difficult due to the heterogeneity of the SRLV genomes. One of the PCR techniques amplifies a fragment in the pol gene (pol-PCR) and the other PCR targets the LTR region of the proviral genome (LTR-PCR). Milk from 194 sheep and 163 goats from farms in the Central Spain was analyzed by both techniques and compared to results obtained by ELISA. When compared to the serologic assay, the agreement of both PCR techniques was very low (0.024 and 0.020 in sheep, and 0.124 and 0.114 in goats). In view of these results, it may be concluded that the efficacy of PCR for the diagnosis of SRLVs is low and a combination of PCR and ELISA should be used for diagnosis.
This study evaluated a multiplex real-time PCR method specific for the mecA, femA-SA and femA-SE ... more This study evaluated a multiplex real-time PCR method specific for the mecA, femA-SA and femA-SE genes for rapid identification of Staphylococcus aureus, Staphylococcus epidermidis and non-S. epidermidis coagulase-negative staphylococci (CoNS), and meticillin susceptibility testing directly in positive blood cultures that grew Gram-positive cocci in clusters. A total of 100 positive blood cultures produced: 39 S. aureus [12 meticillin-resistant S. aureus (MRSA), 31 % of all the S. aureus]; 30 S. epidermidis (56.6 % of the CoNS), 8 Staphylococcus capitis (15.1 %), 3 Staphylococcus saprophyticus (5.7 %), 4 Staphylococcus hominis (7.5 %), 3 Staphylococcus haemolyticus (5.7 %), 2 Staphylococcus warneri (3.8 %), 1 Staphylococcus cohnii (1.9 %) and 2 unidentified Staphylococcus spp. (3.8 %); and 1 Micrococcus luteus in pure culture. Two blood cultures had no growth on subculture and five blood cultures grew mixed CoNS. For the 95 blood cultures with pure growth or no growth on subculture,...
Zeitschrift f�r Lebensmittel-Untersuchung und -Forschung, 1990
An overlay technique is proposed for the identification and counting of haemolytic Listeria colon... more An overlay technique is proposed for the identification and counting of haemolytic Listeria colonies directly on selective plating media. The technique was applied to different Listeria-selective plating media. In pure culture studies with collection strains, the overlay technique was more efficient and reliable for detection haemolytic Listeria species compared with the incorporation of blood into the agar. The efficacy of the overlay technique for the direct detection of haemolytic colonies of Listeria from raw milk samples was related to agar selectivity. The best results were obtained with Listeria-selective agar medium modified (LSAMM). Catalase assay, together with reactions for aesculin and tellurite, were useful and reliable criteria for the identification of Listeria. All colonies on LSAMM which were positive for catalase, tellurite and aesculin while those displaying typical haemolysis corresponded in most cases to L. monocytogenes.
During the spring and summer of 2001, faeces from 166 wild reptiles (94 individuals) and amphibia... more During the spring and summer of 2001, faeces from 166 wild reptiles (94 individuals) and amphibians (72 individuals) from 21 different species found in central Spain were examined for the presence of Salmonella. Thirty-nine reptiles (41.5%) yielded 48 Salmonella isolates, whereas all the amphibians examined were negative. Subspecies Salmonella enterica enterica (I) accounted for up to 50% of isolates. Fourteen isolates (29.2%) belonged to subspecies diarizonae (IIIb), six isolates (12.5%) to subspecies salamae (II), and four isolates (8.3%) to subspecies arizonae (IIIa). Twenty-seven different serotypes were identified. Serotypes Anatum (12.5%), Herzliya (8.3%), Abony, 18:l,v:z, 9,12:z29:1,5 and 38:z10:z53 (6.2%/each) were the most frequently isolated. A high percentage (39.6%) of isolates belonged to serotypes previously associated with environmental sources. Also, 37.5% of isolates belonged to serotypes which had been related to human cases of salmonellosis. From these data, it is concluded that wild reptiles, but apparently not amphibians, may represent an important reservoir of Salmonella in nature and have potential implications for public health.
A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens in... more A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish ( Streptococcus iniae , Streptococcus difficilis , Streptococcus parauberis , and Lactococcus garvieae ). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae , 12.5 pg for S. difficilis , 50 pg for S. parauberis , and 30 pg for L. garvieae . The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.
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Papers by Jose Fernandez-garayzabal