Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

Antigenic and genetic variations in European and North American equine influenza virus strains (H3N8) isolated from 2006 to 2007

Equine influenza virus (EIV) surveillance is important in the management of equine influenza. It provides data on circulating and newly emerging strains for vaccine strain selection. To this end, antigenic characterisation by haemaggluttination inhibition (HI) assay and phylogenetic analysis was carried out on 28 EIV strains isolated in North America and Europe during 2006 and 2007. In the UK, 20 viruses were isolated from 28 nasopharyngeal swabs that tested positive by enzyme-linked immunosorbent assay. All except two of the UK viruses were characterised as members of the Florida sublineage with similarity to A/eq/Newmarket/5/03 (clade 2). One isolate, A/eq/Cheshire/1/06, was characterised as an American lineage strain similar to viruses isolated up to 10 years earlier. A second isolate, A/eq/Lincolnshire/1/07 was characterised as a member of the Florida sublineage (clade 1) with similarity to A/eq/Wisconsin/03. Furthermore, A/eq/Lincolnshire/1/06 was a member of the Florida sublineage (clade 2) by haemagglutinin (HA) gene sequence, but appeared to be a member of the Eurasian lineage by the non-structural gene (NS) sequence suggesting that reassortment had occurred. A/eq/Switzerland/P112/07 was characterised as a member of the Eurasian lineage, the first time since 2005 that isolation of a virus from this lineage has been reported. Seven viruses from North America were classified as members of the Florida sublineage (clade 1), similar to A/eq/Wisconsin/03. In conclusion, a variety of antigenically distinct EIVs continue to circulate worldwide. Florida sublineage clade 1 viruses appear to predominate in North America, clade 2 viruses in Europe.

Genetic relationships of Aeromonas strains inferred from 16S rRNA, gyrB and rpoB gene sequences

Genetic relationships among bacterial strains belonging to the genus Aeromonas were inferred from 16S rRNA, gyrB and rpoB gene sequences. Twenty-eight type or collection strains of the recognized species or subspecies and 33 Aeromonas strains isolated from human and animal specimens as well as from environmental samples were included in the study. As reported previously, the 16S rRNA gene sequence is highly conserved within the genus Aeromonas, having only limited resolution for this very tight group of species. Analysis of a 1.1 kb gyrB sequence confirmed that this gene has high resolving power, with maximal interspecies divergence of 15.2 %. Similar results were obtained by sequencing only 517 bp of the rpoB gene, which showed maximal interspecies divergence of 13 %. The topologies of the gyrB- and rpoB-derived trees were similar. The results confirm the close relationship of species within the genus Aeromonas and show that a phylogenetic approach including several genes is suitable for improving the complicated taxonomy of the genus.

Genetic diversity among Mycoplasma species bovine group 7: clonal isolates from an outbreak of polyarthritis, mastitis, and abortion in dairy cattle

A comprehensive genetic analysis of 60 Mycoplasma sp. bovine group 7 isolates from different geographic origins and epidemiological settings is presented. Twenty-four isolates were recovered from the joints of calves during sporadic episodes of polyarthritis in geographically distinct regions of Queensland and New South Wales, Australia, including two clones of the type strain PG5O. A further three Australian isolates were also recovered from the tympanic bulla, retropharyngeal lymph node and the lung and another three isolates had unconfirmed histories. Six isolates originated from Germany, Portugal, Nigeria, and France. Twenty-four epidemiologically related isolates of Mycoplasma sp. bovine group 7 were recovered from multiple tissue sites and body fluids of infected calves with polyarthritis, mastitic milk, and from the stomach contents, lung and liver from aborted foetuses in three large, centrally managed dairy herds in New South Wales, Australia. Restriction endonuclease analysis (REA) of genomic DNA differentiated 29 Cfol profiles among these 60 isolates and grouped all 24 epidemiologically related isolates in a defined pattern showing a clonal origin. Three isolates of this clonal cluster were recovered from mastitic milk and the synovial exudate of clinically-affected calves and appeared sporadically for periods up to 18 months after the initial outbreak of polyarthritis indicating a persistent, close association of the organism with cattle in these herds. The Cfol profile representative of the clonal cluster was distinguishable from profiles of isolates recovered from multiple, unrelated cases of polyarthritis in Queensland and New South Wales and from other countries. All 24 isolates from the clonal cluster possessed a plasmid (pBG7AU) with a molecular size of 1022 bp. DNA sequence analysis of pBG7AU identified two open reading frames sharing 81 and 99% DNA sequence similarity with hypothetical replication control proteins A and B respectively, previously described in plasmid pADB201 isolated from M. mycoides subspecies mycoides. Other isolates of bovine group 7, epidemiologically unrelated to the clonal cluster, including two clones of the type strain PG5O, possessed a similar-sized plasmid. These data confirm that Mycoplasma sp. bovine group 7 is capable of migrating to, and multiplying within, different tissue sites within a single animal and among different animals within a herd.

Molecular genetic analysis of Dichelobacter nodosus proteases AprV2/B2, AprV5/B5 and BprV/B in clinical material from European sheep flocks

Dichelobacter nodosus, the etiological agent of ovine footrot, exists both as virulent and as benign strains, which differ in virulence mainly due to subtle differences in the three subtilisin-like proteases AprV2, AprV5 and BprV found in virulent, and AprB2, AprB5 and BprB in benign strains of D. nodosus. Our objective was a molecular genetic epidemiological analysis of the genes of these proteases by direct sequence analysis from clinical material of sheep from herds with and without history of footrot from 4 different European countries. The data reveal the two proteases known as virulent AprV2 and benign AprB2 to correlate fully to the clinical status of the individuals or the footrot history of the herd. In samples taken from affected herds, the aprV2 gene was found as a single allele whereas in samples from unaffected herds several alleles with minor modifications of the aprB2 gene were detected. The different alleles of aprB2 were related to the herds. The aprV5 and aprB5 genes were found in the form of several alleles scattered without distinction between affected and non-affected herds. However, all different alleles of aprV5 and aprB5 encode the same amino acid sequences, indicating the existence of a single protease isoenzyme 5 in both benign and virulent strains. The genes of the basic proteases BprV and BprB also exist as various alleles. However, differences found in samples from affected versus non-affected herds do not reflect the currently known epitopes that are attributed to differences in biochemical activity. The data of the study confirm the prominent role of AprV2 in the virulence of D. nodosus and shed a new light on the presence of the other protease genes and their allelic variants in clinical samples.

Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk

Coagulase-negative staphylococci (CNS; n=417) were isolated from bovine milk and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nineteen different species were identified, and Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus sciuri were the most prevalent species. Resistance to oxacillin (47.0% of the isolates), fusidic acid (33.8%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), erythromycin (7.0%), sulfonamides (5%), trimethoprim (4.3%), clindamycin (3.4%), kanamycin (2.4%), and gentamicin (2.4%) was detected. Resistance to oxacillin was attributed to the mecA gene in 9.7% of the oxacillin-resistant isolates. The remaining oxacillin-resistant CNS did not contain the mecC gene or mecA1 promoter mutations. The mecA gene was detected in Staphylococcus fleurettii, Staphylococcus epidermidis, Staph. haemolyticus, and Staph. xylosus. Resistance to tetracycline was attributed to the presence of tet(K) and tet(L), penicillin resistance to blaZ, streptomycin resistance to str and ant(6)-Ia, and erythromycin resistance to erm(C), erm(B), and msr. Resistance to tiamulin and fusidic acid could not be attributed to an acquired resistance gene. In total, 15.1% of the CNS isolates were multidrug resistant (i.e., resistant to 2 or more antimicrobials). The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment. Methicillin-resistant CNS isolates were diverse, as determined by mecA gene sequence analysis, staphylococcal cassette chromosome mec typing, and pulsed-field gel electrophoresis. Arginine catabolic mobile element types 1 and 3 were detected in both methicillin-resistant and methicillin-susceptible Staph. epidermidis and were associated with sequence types ST59 and ST111. Because this study revealed the presence of multidrug-resistant CNS in a heterogeneous CNS population, we recommend antibiogram analysis of CNS in persistent infections before treatment with antimicrobials.

Multilocus sequence typing for characterization of Staphylococcus pseudintermedius

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs. Four housekeeping genes with allelic polymorphisms were identified and used to develop an expanded multilocus sequence typing (MLST) scheme. The new seven-locus technique shows S. pseudintermedius to have greater genetic diversity than previous methods and discriminates more isolates based upon host origin.

Magnetic resonance imaging and genetic investigation of a case of Rottweiler leukoencephalomyelopathy.

BACKGROUND Leukoencephalomyelopathy is an inherited neurodegenerative disorder that affects the white matter of the spinal cord and brain and is known to occur in the Rottweiler breed. Due to the lack of a genetic test for this disorder, post mortem neuropathological examinations are required to confirm the diagnosis. Leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate levels is a rare, autosomal recessive disorder in humans that was recently described to have clinical features and magnetic resonance imaging (MRI) findings that are similar to the histopathologic lesions that define leukoencephalomyelopathy in Rottweilers. Leukoencephalopathy with brain stem and spinal cord involvement is caused by mutations in the DARS2 gene, which encodes a mitochondrial aspartyl-tRNA synthetase. The objective of this case report is to present the results of MRI and candidate gene analysis of a case of Rottweiler leukoencephalomyelopathy to investigate the hypothesis that leukoencephalomyelopathy in Rottweilers could serve as an animal model of human leukoencephalopathy with brain stem and spinal cord involvement. CASE PRESENTATION A two-and-a-half-year-old male purebred Rottweiler was evaluated for generalised progressive ataxia with hypermetria that was most evident in the thoracic limbs. MRI (T2-weighted) demonstrated well-circumscribed hyperintense signals within both lateral funiculi that extended from the level of the first to the sixth cervical vertebral body. A neurodegenerative disorder was suspected based on the progressive clinical course and MRI findings, and Rottweiler leukoencephalomyelopathy was subsequently confirmed via histopathology. The DARS2 gene was investigated as a causative candidate, but a sequence analysis failed to identify any disease-associated variants in the DNA sequence. CONCLUSION It was concluded that MRI may aid in the pre-mortem diagnosis of suspected cases of leukoencephalomyelopathy. Genes other than DARS2 may be involved in Rottweiler leukoencephalomyelopathy and may also be relevant in human leukoencephalopathy with brain stem and spinal cord involvement.

Occurrence and Genetic Characteristics of Third-Generation Cephalosporin-Resistant Escherichia coli in Swiss Retail Meat

Prevalence and genetic relatedness were determined for third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) detected in Swiss beef, veal, pork, and poultry retail meat. Samples from meat-packing plants (MPPs) processing 70% of the slaughtered animals in Switzerland were purchased at different intervals between April and June 2013 and analyzed. Sixty-nine 3GC-R-Ec isolates were obtained and characterized by microarray, PCR/DNA sequencing, Multi Locus Sequence Typing (MLST), and plasmid replicon typing. Plasmids of selected strains were transformed by electroporation into E. coli TOP10 cells and analyzed by plasmid MLST. The prevalence of 3GC-R-Ec was 73.3% in chicken and 2% in beef meat. No 3GC-R-Ec were found in pork and veal. Overall, the blaCTX-M-1 (79.4%), blaCMY-2 (17.6%), blaCMY-4 (1.5%), and blaSHV-12 (1.5%) β-lactamase genes were detected, as well as other genes conferring resistance to chloramphenicol (cmlA1-like), sulfonamides (sul), tetracycline (tet), and trimethoprim (dfrA). The 3GC-R-Ec from chicken meat often harbored virulence genes associated with avian pathogens. Plasmid incompatibility (Inc) groups IncI1, IncFIB, IncFII, and IncB/O were the most frequent. A high rate of clonality (e.g., ST1304, ST38, and ST93) among isolates from the same MPPs suggests that strains persist at the plant and spread to meat at the carcass-processing stage. Additionally, the presence of the blaCTX-M-1 gene on an IncI1 plasmid sequence type 3 (IncI1/pST3) in genetically diverse strains indicates interstrain spread of an epidemic plasmid. The blaCMY-2 and blaCMY-4 genes were located on IncB/O plasmids. This study represents the first comprehensive assessment of 3GC-R-Ec in meat in Switzerland. It demonstrates the need for monitoring contaminants and for the adaptation of the Hazard Analysis and Critical Control Point concept to avoid the spread of multidrug-resistant bacteria through the food chain.

Genetic Relatedness, Antimicrobial and Biocide Susceptibility Comparative Analysis of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius from Portugal

Forty methicillin-resistant and -susceptible Staphylococcus pseudintermedius (MRSP and MSSP, respectively) from colonization and infection in dogs and cats were characterized for clonality, antimicrobial, and biocide susceptibility. MSSP were genetically more diverse than MRSP by multi-locus sequence typing and pulsed-field gel electrophoresis. Three different spa types (t06, t02, t05) and two SCCmec types (II-III and V) were detected in the MRSP isolates. All MRSP and two MSSP strains were multidrug-resistant. Several antibiotic resistance genes (mecA, blaZ, tet(M), tet(K), aac(6')-Ie-aph(2')-Ia, aph(3')-III, ant(6)-Ia, sat4, erm(B), lnu(A), dfr(G), and catpC221) were identified by microarray and double mutations in the gyrA and grlA genes and a single mutation in the rpoB gene were detected by sequence analysis. No differences were detected between MSSP and MRSP in the chlorhexidine acetate (CHA) minimum inhibitory concentrations (MICs). However, two MSSP had elevated MIC to triclosan (TCL) and one to benzalkonium chloride and ethidium bromide. One MSSP isolate harboured a qacA gene, while in another a qacB gene was detected. None of the isolates harboured the sh-fabI gene. Three of the biocide products studied had high bactericidal activity (Otodine(®), Clorexyderm Spot Gel(®), Dermocanis Piocure-M(®)), while Skingel(®) failed to achieve a five log reduction in the bacterial counting. S. pseudintermedius have become a serious therapeutic challenge in particular if methicillin- resistance and/or multidrug-resistance are involved. Biocides, like CHA and TCL, seem to be clinically effective and safe topical therapeutic options.

Temporal sequence of hard and soft tissue healing around titanium dental implants.

The objective of the present review was to summarize the evidence available on the temporal sequence of hard and soft tissue healing around titanium dental implants in animal models and in humans. A search was undertaken to find animal and human studies reporting on the temporal dynamics of hard and soft tissue integration of titanium dental implants. Moreover, the influence of implant surface roughness and chemistry on the molecular mechanisms associated with osseointegration was also investigated. The findings indicated that the integration of titanium dental implants into hard and soft tissue represents the result of a complex cascade of biological events initiated by the surgical intervention. Implant placement into alveolar bone induces a cascade of healing events starting with clot formation and continuing with the maturation of bone in contact with the implant surface. From a genetic point of view, osseointegration is associated with a decrease in inflammation and an increase in osteogenesis-, angiogenesis- and neurogenesis-associated gene expression during the early stages of wound healing. The attachment and maturation of the soft tissue complex (i.e. epithelium and connective tissue) to implants becomes established 6-8 weeks following surgery. Based on the findings of the present review it can be concluded that improved understanding of the mechanisms associated with osseointegration will provide leads and targets for strategies aimed at enhancing the clinical performance of titanium dental implants.

Mining microsatellite markers from public expressed sequence tags databases for the study of threatened plants

Background Simple Sequence Repeats (SSRs) are widely used in population genetic studies but their classical development is costly and time-consuming. The ever-increasing available DNA datasets generated by high-throughput techniques offer an inexpensive alternative for SSRs discovery. Expressed Sequence Tags (ESTs) have been widely used as SSR source for plants of economic relevance but their application to non-model species is still modest. Methods Here, we explored the use of publicly available ESTs (GenBank at the National Center for Biotechnology Information-NCBI) for SSRs development in non-model plants, focusing on genera listed by the International Union for the Conservation of Nature (IUCN). We also search two model genera with fully annotated genomes for EST-SSRs, Arabidopsis and Oryza, and used them as controls for genome distribution analyses. Overall, we downloaded 16 031 555 sequences for 258 plant genera which were mined for SSRsand their primers with the help of QDD1. Genome distribution analyses in Oryza and Arabidopsis were done by blasting the sequences with SSR against the Oryza sativa and Arabidopsis thaliana reference genomes implemented in the Basal Local Alignment Tool (BLAST) of the NCBI website. Finally, we performed an empirical test to determine the performance of our EST-SSRs in a few individuals from four species of two eudicot genera, Trifolium and Centaurea. Results We explored a total of 14 498 726 EST sequences from the dbEST database (NCBI) in 257 plant genera from the IUCN Red List. We identify a very large number (17 102) of ready-to-test EST-SSRs in most plant genera (193) at no cost. Overall, dinucleotide and trinucleotide repeats were the prevalent types but the abundance of the various types of repeat differed between taxonomic groups. Control genomes revealed that trinucleotide repeats were mostly located in coding regions while dinucleotide repeats were largely associated with untranslated regions. Our results from the empirical test revealed considerable amplification success and transferability between congenerics. Conclusions The present work represents the first large-scale study developing SSRs by utilizing publicly accessible EST databases in threatened plants. Here we provide a very large number of ready-to-test EST-SSR (17 102) for 193 genera. The cross-species transferability suggests that the number of possible target species would be large. Since trinucleotide repeats are abundant and mainly linked to exons they might be useful in evolutionary and conservation studies. Altogether, our study highly supports the use of EST databases as an extremely affordable and fast alternative for SSR developing in threatened plants.

Genetic stability of Schmallenberg virus in vivo during an epidemic, and in vitro, when passaged in the highly susceptible porcine SK-6 cell line

Schmallenberg virus (SBV), an arthropod-borne orthobunyavirus was first detected in 2011 in cattle suffering from diarrhea and fever. The most severe impact of an SBV infection is the induction of malformations in newborns and abortions. Between 2011 and 2013 SBV spread throughout Europe in an unprecedented epidemic wave. SBV contains a tripartite genome consisting of the three negative-sense RNA segments L, M, and S. The virus is usually isolated from clinical samples by inoculation of KC (insect) or BHK-21 (mammalian) cells. Several virus passages are required to allow adaptation of SBV to cells in vitro. In the present study, the porcine SK-6 cell line was used for isolation and passaging of SBV. SK-6 cells proved to be more sensitive to SBV infection and allowed to produce higher titers more rapidly as in BHK-21 cells after just one passage. No adaptation was required. In order to determine the in vivo genetic stability of SBV during an epidemic spread of the virus the nucleotide sequence of the genome from seven SBV field isolates collected in summer 2012 in Switzerland was determined and compared to other SBV sequences available in GenBank. A total of 101 mutations, mostly transitions randomly dispersed along the L and M segment were found when the Swiss isolates were compared to the first SBV isolated late 2011 in Germany. However, when these mutations were studied in detail, a previously described hypervariable region in the M segment was identified. The S segment was completely conserved among all sequenced SBV isolates. To assess the in vitro genetic stability of SBV, three isolates were passage 10 times in SK-6 cells and sequenced before and after passaging. Between two and five nt exchanges per genome were found. This low in vitro mutation rate further demonstrates the suitability of SK-6 cells for SBV propagation.

Genetic variability and limited clonality of Mycoplasma hyorhinis in pig herds

Mycoplasma hyorhinis is a common inhabitant of the upper respiratory tract and tonsils of pigs. Its role as a possible pathogen remains controversial. In order to gain more insight into the epidemiology and population structure of M. hyorhinis we genetically characterized 60 isolates by multi locus sequence typing (MLST). The M. hyorhinis strains originated from Swiss and German pig herds with knowledge on the clinical background. The MLST scheme of Tocqueville et al. (J. Clin. Microbiol. 2014) was optimized, primers for the six MLST gene fragments were newly designed to allow amplification and sequencing with a single protocol. A total of 27 ST were observed with the 60 strains, 26 of those were previously unknown types. Generally identical genotypes were observed within a farm but they differed between farms. The identical genotype was also observed in three different Swiss farms. On the other Hand different genotypes within a farm were found with three German farms. The Swiss isolates formed a distinct cluster but otherwise there was no geographical nor a clinical association with specific Clusters observed. Data shows a high variability of M. hyorhinis comparable to what is observed for Mycoplasma hyopneumoniae. Similar to this pathogen the population structure of M. hyorhinis also shows some limited clonality with predominant genotypes within an animal and a single farm but different ones between farms. The comparable population structure of M. hyopneumoniae and M. hyorhinis could indicate a similar evolution of the two species in the common pig host.

Genetic Codes with No Dedicated Stop Codon: Context-Dependent Translation Termination

The prevailing view of the nuclear genetic code is that it is largely frozen and unambiguous. Flexibility in the nuclear genetic code has been demonstrated in ciliates that reassign standard stop codons to amino acids, resulting in seven variant genetic codes, including three previously undescribed ones reported here. Surprisingly, in two of these species, we find efficient translation of all 64 codons as standard amino acids and recognition of either one or all three stop codons. How, therefore, does the translation machinery interpret a “stop” codon? We provide evidence, based on ribosomal profiling and “stop” codon depletion shortly before coding sequence ends, that mRNA 3′ ends may contribute to distinguishing stop from sense in a context-dependent manner. We further propose that such context-dependent termination/readthrough suppression near transcript ends enables genetic code evolution.