Assessment of MALDI-TOF MS as Alternative Tool for Streptococcus suis Identification

The accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying Streptococcus suis isolates obtained from pigs, wild animals, and humans was evaluated using a PCR-based identification assay as the gold standard. In addition, MALDI-TOF MS was compared with the commercial multi-tests Rapid ID 32 STREP system. From the 129 S. suis isolates included in the study and identified by the molecular method, only 31 isolates (24.03%) had score values ≥2.300 and 79 isolates (61.24%) gave score values between 2.299 and 2.000. After updating the currently available S. suis MALDI Biotyper database with the spectra of three additional clinical isolates of serotypes 2, 7, and 9, most isolates had statistically significant higher score values (mean score: 2.65) than those obtained using the original database (mean score: 2.182). Considering the results of the present study, we suggest using a less restrictive threshold score of ≥2.000 for reliable species identification of S. suis. According to this cut-off value, a total of 125 S. suis isolates (96.9%) were correctly identified using the updated database. These data indicate an excellent performance of MALDI-TOF MS for the identification of S. suis.

Fluoroquinolone efflux in Streptococcus suis is mediated by SatAB and not by SmrA

Streptococcus suis is an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia coli despite strong expression repression. Finally, site-directed insertion of smrA and satAB in the amy locus of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillus is a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.

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.

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.

Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific

Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

Discovery of stable and variable differences in the Mycobacterium avium subsp. paratuberculosis type I, II, and III genomes by pan-genome microarray analysis

Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.