Differentiation Of Strains Research Articles

Evaluation of the discriminatory power of spoligotyping and 19-locus mycobacterial interspersed repetitive unit-variable number of tandem repeat analysis (MIRU-VNTR) of Mycobacterium bovis strains isolated from cattle in Algeria.

Bovine tuberculosis (bTB) caused by Mycobacterium (M.) bovis and M. caprae is a transmissible disease of livestock, notifiable to the World Organization for Animal Health (OIE). BTB particularly affects cattle and small ruminants and can be transmitted to humans thereby posing a significant threat to veterinary and public health worldwide. M. bovis is the principal cause of bTB in Algeria. In order to better understand the route of spreading and elaborate an eradication program, isolation and characterization of mycobacteria from Algerian cattle was performed. Sixty strains belonging to the M. tuberculosis complex were analyzed by spoligotyping, thereof 42 by 19-locus-MIRU-VNTR-typing. Spoligotyping revealed 16 distinguishable patterns (Hunter-Gaston discriminatory index [HGDI] of 0.8294), with types SB0120 (n = 20) and SB0121 (n = 13) being the most frequent patterns, representing 55% of the strains. Analyses based on 19-locus-MIRU-VNTR yielded 32 different profiles, five clusters and one orphan pattern, showing higher discriminatory power (HGDI = 0.9779) than spoligotyping. Seven VNTR-loci [VNTR 577 (alias ETR C), 2163b (QU11b), 2165 (ETR A), 2461 (ETR B), 3007 (MIRU 27), 2163a (QUB11a) and 3232 (QUB 3232)] were the most discriminative loci (HGDI ˃ 0.50). In conclusion, 19-locus-MIRU-VNTR yielded more information than spoligotyping concerning molecular differentiation of strains and better supports the elucidation of transmission routes of M. bovis between Algerian cattle herds.

Molecular detection of SARS-CoV-2 strains and differentiation of Delta variant strains.

The Delta variant of SARS-CoV-2 has now become the predominant strain in the global COVID-19 pandemic. Strain coverage of some detection assays developed during the early pandemic stages has declined due to periodic mutations in the viral genome. We have developed a real-time RT-PCR (RT-qPCR) for SARS-CoV-2 detection that provides nearly 100% strain coverage, and differentiation of highly transmissible Delta variant strains. All full or nearly full (≥28kb) SARS-CoV-2 genomes (n=403,812), including 6422 Delta and 280 Omicron variant strains, were collected from public databases at the time of analysis and used for assay design. The two amino acid deletions in the spike gene (S-gene, Δ156-157) that is characteristic of the Delta variant were targeted during the assay design. Although strain coverage for the Delta variant was very high (99.7%), detection coverage for non-Delta wild-type strains was 93.9%, mainly due to the confined region of design. To increase strain coverage of the assay, the design for CDC N1 target was added to the assay. In silico analysis of 403,812 genomes indicated a 95.4% strain coverage for the CDC N1 target, however, in combination with our new non-Delta S-gene target, total coverage for non-Delta wild-type strains increased to 99.8%. A human 18S rRNA gene was also analyzed and used as an internal control. The final four-plex RT-qPCR assay generated PCR amplification efficiencies between 95.4% and 102.0% with correlation coefficients (R2 ) of>0.99 for cloned positive controls; Delta and non-Delta human clinical samples generated PCR efficiencies of 93.4%-97.0% and R2 >0.99. The assay also detects 98.6% of 280 Omicron sequences. Assay primers and probes have no match to other closely related human coronaviruses, and did not produce a signal from samples positive to selected animal coronaviruses. Genotypes of selected clinical samples identified by the RT-qPCR were confirmed by Sanger sequencing.

Selection of Beneficial Bacterial Strains With Potential as Oral Probiotic Candidates.

This study aimed to select beneficial strains from the oral cavity of healthy volunteers and to evaluate these as potential oral probiotic candidates. The selection process was based on the isolation, differentiation, identification, and safety assessment of LAB strains, followed by a series of experiments for the selection of appropriate candidates with beneficial properties. In the screening procedure, 8 isolates from the oral cavity of a Caucasian volunteers were identified as Streptococcus (Str.) salivarius ST48HK, ST59HK, ST61HK, and ST62HK; Lactiplantibacillus plantarum (Lb.) (Lactobacillus plantarum) ST63HK and ST66HK; Latilactobacillus sakei (Lb.) (Lactobacillus sakei) ST69HK; and Lactobacillus (Lb.) gasseri ST16HK based on 16S rRNA sequencing. Physiological and phenotypic tests did not show hemolytic, proteinase, or gelatinase activities, as well as production of biogenic amines. In addition, screening for the presence of efaA, cyt, IS16, esp, asa1, and hyl virulence genes and vancomycin-resistant genes confirmed safety of the studied strains. Moreover, cell-to-cell antagonism indicated that the strains were able to inhibit the growth of tested representatives from the genera Bacillus, Enterococcus, Streptococcus, and Staphylococcus in a strain-specific manner. Various beneficial genes were detected including gad gene, which codes for GABA production. Furthermore, cell surface hydrophobicity levels ranging between 1.58% and 85% were determined. The studied strains have also demonstrated high survivability in a broad range of pH (4.0-8.0). The interaction of the 8 putative probiotic candidates with drugs from different groups and oral hygiene products were evaluated for their MICs. This is to determine if the application of these drugs and hygiene products can negatively affect the oral probiotic candidates. Overall, antagonistic properties, safety assessment, and high rates of survival in the presence of these commonly used drugs and oral hygiene products indicate Str. salivarius ST48HK, ST59HK, ST61HK, and ST62HK; Lb. plantarum ST63HK and ST66HK; Lb. sakei ST69HK; and Lb. gasseri ST16HK as promising oral cavity probiotic candidates.

Detection of multi-resistant clinical strains of E. coli with Raman spectroscopy

In recent years, we have seen a steady rise in the prevalence of antibiotic-resistant bacteria. This creates many challenges in treating patients who carry these infections, as well as stopping and preventing outbreaks. Identifying these resistant bacteria is critical for treatment decisions and epidemiological studies. However, current methods for identification of resistance either require long cultivation steps or expensive reagents. Raman spectroscopy has been shown in the past to enable the rapid identification of bacterial strains from single cells and cultures. In this study, Raman spectroscopy was applied for the differentiation of resistant and sensitive strains of Escherichia coli. Our focus was on clinical multi-resistant (extended-spectrum β-lactam and carbapenem-resistant) bacteria from hospital patients. The spectra were collected using both UV resonance Raman spectroscopy in bulk and single-cell Raman microspectroscopy, without exposure to antibiotics. We found resistant strains have a higher nucleic acid/protein ratio, and used the spectra to train a machine learning model that differentiates resistant and sensitive strains. In addition, we applied a majority of voting system to both improve the accuracy of our models and make them more applicable for a clinical setting. This method could allow rapid and accurate identification of antibiotic resistant bacteria, and thus improve public health.Graphical abstract

Phenotypic characterisation and strain differentiation of Vibrio alginolyticus isolates from Muttukadu brackishwater lagoon

Phenotypic characterisation and strain differentiation of Vibrio alginolyticus isolates from Muttukadu brackishwater lagoon

Modeling of rubber-cord layers under quasi-static loading

Modeling a pneumatic tire with a strong change in shape causes the problem of choosing an adequate model for rubber-cord plies. Generally, the classical method of asymptotic homogenization is not suitable due to physical and geometrical nonlinearity for the strain up to 15 %. The known models used for simulation the entire tire as well as rubber-cord plies are analyzed. A choice is made in favor of modeling the plies using the stress anisotropic potential, which is an anisotropic function of the strain tensor invariants. The relationship of such a constitutive law with a quasi-linear constitutive equation in terms of stress and strain differentials is indicated. A convenience of these two types of constitutive equations in terms of numerical implementation is also given. A modification of the effective properties definition for an inhomogeneous layer is explained. The difference from the standard effective moduli definition is clarified. The arrangement of the cords is supposed to be approximately periodic and all cords to be equivalent to the effective fiber. Two models of a rubber-cord plies are described under such assumption. These are a model of an orthotropic material and a model of a transversely isotropic material. Computational experiments, which make it possible to determine the material parameters of anisotropic potentials, are pointed out. Real tests with a sample of rubber ply under slow quasi-static loading were conducted. Significant hysteresis was detected. It is shown that an additive model combining a hyper elastic material with Maxwell viscoelastic model provides good accuracy in stress dependence on the strain rate. The numerical procedure developed to calculate solution to the quasi-static problem of tire deformation is described. It is implemented in home-made computer code. A numerical example on the tire simulation is given. That is so-called breaking test, in which strong deformation is achieved and the developed model is applied to.

Pathotyping of Newcastle Disease Virus: a Novel Single BsaHI Digestion Method of Detection and Differentiation of Avirulent Strains (Lentogenic and Mesogenic Vaccine Strains) from Virulent Virus.

ABSTRACTWe provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I “F gene” class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research.IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.

Development of multiplex reverse transcription-polymerase chain reaction for differentiation of strains of infectious bursal disease virus and primary screening of the virus in Thailand.

Background and Aim:A new set of primers (400 base pairs partial of VP2) was designed and used for the infectious bursal disease virus (IBDV) screening test. Using this new primer set, the enzymes MboI and BstNI were unable to differentiate the field and vaccine strains. As a result, a new simple, cheap, and appropriate tool for strain differentiation is required. The objective of this study was to develop the appropriate restriction fragment length polymorphism (RFLP) and multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the differentiation of classic IBDV (cIBDV) strains and very virulent IBDV (vvIBDV) strains in Thailand.Materials and Methods:Ninety seven bursa of Fabricius from 16 farms were collected from farms in the eastern and central regions of Thailand. RT-PCR screening showed that 82 samples were positive for IBDV and 15 samples were negative. Then, selected samples were sequenced from each farm with a positive test.Results:The sequencing results showed that samples from six of the farms were vvIBDV and samples from the other six farms were cIBDV. Although the whole genome sequencing was incomplete, both the sequencing results of segment A and segment B showed high similarity between cIBDV and vvIBDV. Restriction enzyme cutting site and primers for multiplex RT-PCR were hard to design. An RT-PCR-RFLP method was developed, but it failed to differentiate IBDV strains. However, the multiplex RT-PCR was able to differentiate cIBDV from vvIBDV. Four primers were used in the multiplex RT-PCR.Conclusion:These four primers were used together in one reaction at an annealing temperature of 45°C. Therefore, multiplex RT-PCR is a less complicated, cheaper, and less time-consuming method for the differentiation of cIBDV and vvIBDV strains.

Genetic Variability, Population Differentiation, and Correlations for Thermal Tolerance Indices in the Minute Wasp, Trichogramma cacoeciae.

Simple SummaryAugmentative biological control relies on the more or less frequent/abundant releases of biological control agents (BCAs) that have to be adapted to their short-term local environment including (micro-)climatic conditions. Thermal biology of BCAs is thus a key component for their success. The extent to which thermal tolerance indices may be relevant predictors of the field efficiency is however still poorly documented. Within this frame, we investigated the intraspecific variability for the ability to move at low temperatures in the minute wasp, Trichogramma cacoeciae. We collected, molecularly characterized, and compared for their thermal tolerance indices numerous strains originating from three contrasting geographic areas. Our findings evidenced both a geographic differentiation between strains for one of the thermal tolerance indices and a positive correlation between two of them, demonstrating the existence of an intraspecific variability. Temperature is a main driver of the ecology and evolution of ectotherms. In particular, the ability to move at sub-lethal low temperatures can be described through three thermal tolerance indices—critical thermal minimum (CTmin), chill coma temperature (CCT), and activity recovery (AR). Although these indices have proven relevant for inter-specific comparisons, little is known about their intraspecific variability as well as possible genetic correlations between them. We thus investigated these two topics (intraspecific variability and genetic correlations between thermal tolerance indices) using the minute wasp, Trichogramma cacoeciae. Strains from T. cacoeciae were sampled across three geographic regions in France—two bioclimatic zones along a sharp altitudinal cline in a Mediterranean context (meso-Mediterranean at low elevations and supra-Mediterranean at higher elevations) and a more northwestern area characterized by continental or mountainous climates. Our results evidenced a significant effect of both the longitude and the severity of the cold during winter months on CCT. Results were however counter-intuitive since the strains from the two bioclimatic zones characterized by more severe winters (northwestern area and supra-Mediterranean) exhibited opposite patterns. In addition, a strong positive correlation was observed between CCT and CTmin. Neither strain differentiation nor the covariations between traits seem to be linked with the molecular diversity observed on the part of the mitochondrial marker COI.

Conservation of ptfA gene encoded Type IV fimbrial protein among circulating Pasteurella multocida serogroup A strains causing pneumonia in sheep

Respiratory infections are most common in small ruminants. Differentiation of homogenous bacterial strains originated from similar clinical forms (acute or chronic) of infections in a particular or diverse host origin and regions posses a greater challenge for rapid epidemiological studies. Pasteurella multocida, being a multi-host pathogen with wide range of infections among small ruminants especially sheep, is of greater economical concern among small and marginal farmers. In our study, we report ptfA gene sequence based analysis of circulating P. multocida strains recovered from clinically ailing sheep either with pneumonia or septicaemia belonging to different geographical regions of Karnataka. All the 29 P. multocida strains were characterized by conventional methods as well as molecular methods which indicated homogeneity as they belonged to serogroup A and possessed highly conserved ptfA gene by phylogenetic analysis. The study highlighted the conservation of ptfA gene/fimbrial protein among P. multocida strains from identical/diverse clinical conditions and could be employed in rapid epidemiological studies in routine surveillance of circulating pathogenic bacterial strains as well as pasteurellosis outbreak investigations among animals and birds.

Reverse transcription-enzymatic recombinase amplification coupled with CRISPR-Cas12a for rapid detection and differentiation of PEDV wild-type strains and attenuated vaccine strains.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute watery diarrhea and vomiting in unweaned piglets, and is associated with high mortality, thus causing severe economic losses in the pig industry. Currently, although attenuated vaccines are commonly used in commercial pig farms in China, they do not completely protect against all mutated wild-type strains. Existing nucleic acid assays have high sensitivity and specificity, but the complexity of the assay process and expensive instrumentation hinder disease detection. Here, reverse transcription–enzymatic recombinase amplification (RT-ERA) was combined with the CRISPR-Cas12a system to develop a rapid diagnostic method to distinguish PEDV wild-type strains from attenuated vaccine strains. The protocol used crRNA and RT-ERA amplification primers against open reading frame 3 (ORF3), followed by Cas12a/crRNA complex detection of predefined target sequences at 37 °C for 30 min, thus producing results visible to the naked eye under LED blue light. The assay is highly sensitive and specific, detecting as few as two copies of the target gene per test and showing no cross-reactivity with other porcine pathogens. Overall, this integrated RT-ERA pre-amplification and Cas12a/crRNA cleavage assay is a practical tool for reliable and rapid detection of PEDV for diagnostic differentiation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03716-7.

Protein Biomarker Identification for the Discrimination of Brucella melitensis Field Isolates From the Brucella melitensis Rev.1 Vaccine Strain by MALDI-TOF MS.

Brucella melitensis Rev.1 is a live attenuated vaccine strain that is widely used to control brucellosis in small ruminants. For successful surveillance and control programs, rapid identification and characterization of Brucella isolates and reliable differentiation of vaccinated and naturally infected animals are essential prerequisites. Although MALDI-TOF MS is increasingly applied in clinical microbiology laboratories for the diagnosis of brucellosis, species or even strain differentiation by this method remains a challenge. To detect biomarkers, which enable to distinguish the B. melitensis Rev.1 vaccine strain from B. melitensis field isolates, we initially searched for unique marker proteins by in silico comparison of the B. melitensis Rev.1 and 16M proteomes. We found 113 protein sequences of B. melitensis 16M that revealed a homologous sequence in the B. melitensis Rev.1 annotation and 17 of these sequences yielded potential biomarker pairs. MALDI-TOF MS spectra of 18 B. melitensis Rev.1 vaccine and 183 Israeli B. melitensis field isolates were subsequently analyzed to validate the identified marker candidates. This approach detected two genus-wide unique biomarkers with properties most similar to the ribosomal proteins L24 and S12. These two proteins clearly discriminated B. melitensis Rev.1 from the closely related B. melitensis 16M and the Israeli B. melitensis field isolates. In addition, we verified their discriminatory power using a set of B. melitensis strains from various origins and of different MLVA types. Based on our results, we propose MALDI-TOF MS profiling as a rapid, cost-effective alternative to the traditional, time-consuming approach to differentiate certain B. melitensis isolates on strain level.

Comparison of bacteria disintegration methods and their influence on data analysis in metabolomics

Metabolomic experiments usually contain many different steps, each of which can strongly influence the obtained results. In this work, metabolic analyses of six bacterial strains were performed in light of three different bacterial cell disintegration methods. Three strains were gram-negative (Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae), and three were gram-positive (Corynebacterium glutamicum, Bacillus cereus, and Enterococcus faecalis). For extraction, the methanol–water extraction method (1:1) was chosen. To compare the efficiency of different cell disintegration methods, sonication, sand mill, and tissue lyser were used. For bacterial extract metabolite analysis, 1H NMR together with univariate and multivariate analyses were applied. The obtained results showed that metabolite concentrations are strongly dependent on the cell lysing methodology used and are different for various bacterial strains. The results clearly show that one of the disruption methods gives the highest concentration for most identified compounds (e. g. sand mill for E. faecalis and tissue lyser for B. cereus). This study indicated that the comparison of samples prepared by different procedures can lead to false or imprecise results, leaving an imprint of the disintegration method. Furthermore, the presented results showed that NMR might be a useful bacterial strain identification and differentiation method. In addition to disintegration method comparison, the metabolic profiles of each elaborated strain were analyzed, and each exhibited its metabolic profile. Some metabolites were identified by the 1H NMR method in only one strain. The results of multivariate data analyses (PCA) show that regardless of the disintegration method used, the strain group can be identified. Presented results can be significant for all types of microbial studies containing the metabolomic targeted and non-targeted analysis.

Distinct conformers of amyloid beta accumulate in the neocortex of patients with rapidly progressive Alzheimer's disease

Amyloid beta (Aβ) deposition in the neocortex is a major hallmark of Alzheimer's disease (AD), but the extent of deposition does not readily explain phenotypic diversity and rate of disease progression. The prion strain–like model of disease heterogeneity suggests the existence of different conformers of Aβ. We explored this paradigm using conformation-dependent immunoassay (CDI) for Aβ and conformation-sensitive luminescent conjugated oligothiophenes (LCOs) in AD cases with variable progression rates. Mapping the Aβ conformations in the frontal, occipital, and temporal regions in 20 AD patients with CDI revealed extensive interindividual and anatomical diversity in the structural organization of Aβ with the most significant differences in the temporal cortex of rapidly progressive AD. The fluorescence emission spectra collected in situ from Aβ plaques in the same regions demonstrated considerable diversity of spectral characteristics of two LCOs—quatroformylthiophene acetic acid and heptaformylthiophene acetic acid. Heptaformylthiophene acetic acid detected a wider range of Aβ deposits, and both LCOs revealed distinct spectral attributes of diffuse and cored plaques in the temporal cortex of rapidly and slowly progressive AD and less frequent and discernible differences in the frontal and occipital cortex. These and CDI findings indicate a major conformational diversity of Aβ accumulating in the neocortex, with the most notable differences in temporal cortex of cases with shorter disease duration, and implicate distinct Aβ conformers (strains) in the rapid progression of AD.

Evoglow-Pp1 and mCherry proteins: a dual fluorescent labeling system for lactic acid bacteria.

Fluorescent proteins are widely used for cell and protein tracking. Most of these proteins show a high signal and need the presence of oxygen to emit fluorescence. Among them, the fluorescent protein mCherry stands up because of its bright signal and fast maturation. Furthermore, the anaerobic cyan-green fluorescent protein Evoglow-Pp1 allows fluorescent detection under anaerobic conditions. In this work, we modified the pNZ:TuR.aFP plasmid, which harbors the gene encoding Evoglow-Pp1 and the promoter of elongation factor Tu from Limosilactobacillus reuteri CECT925, to obtain a plasmid containing the mrfp gene encoding the monomeric mCherry (pNZ:TuR.mCherry). Moreover, both genes were cloned together (pNZ:TuR.aFP.mCherry) developing a chimeric protein; and with a stop codon between them (pNZ:TuR.aFP.STOP.mCherry) resulting in the expression of both Evoglow-Pp1 and mCherry proteins separately under the influence of the same promoter. Lactococcus lactis, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and L. reuteri strains were transformed with the previously mentioned plasmids, showing an excellent red (pNZ:TuR.mCherry), green (pNZ:TuR.aFP), and red combined with green (pNZ:TuR.aFP.mCherry and pNZ:TuR.aFP.STOP.mCherry) fluorescence signal. Both fluorescence emissions were stable in strains transformed with pNZ:TuR.aFP.STOP.mCherry, while differences in the red or green fluorescence emission were observed in some of the strains harboring pNZ:TuR.aFP.mCherry. Moreover, these plasmids allowed strains differentiation in a complex environment, such as fecal microbiota. Hence, we present the plasmid pNZ:TuR.aFP.STOP.mCherry as a useful tool for the labeling of lactobacilli strains, which would be functional under anoxic conditions, thanks to Evoglow-Pp1, while having the high brightness and good photostability of mCherry. KEY POINTS: • LAB transformed with pNZ:TuR.mCherry expressed the red fluorescent protein mCherry. • LAB transformed with pNZ:TuR.aFP.mCherry developed a fusion of both proteins Evoglow-Pp1 and mCherry. • LAB with pNZ:TuR.aFP.STOP.mCherry expressed both fluorescent proteins separately.

Simple sequence repeat markers reflect the biological phenotype differentiation and genetic diversity of Colletotrichum gloeosporioides strains from Capsicum annuum L. in China

AbstractPepper anthracnose, caused by fungi of the Colletotrichum complex, is one of the most important diseases in pepper production. Recent studies have revealed the rich genetic diversity of different Colletotrichum species originating from Capsicum annuum L. However, for Colletotrichum gloeosporioides, the dominant species from Capsicum annuum L., the relationships between the genetic diversity and biological phenotype differentiation remain unclear. Here, we developed a highly specific simple sequence repeat (SSR)‐PCR system, which showed rich intraspecific genetic diversity in the tested Colletotrichum gloeosporioides strains. Moreover, we characterized the biological phenotypes of significantly differentiated strains, including their morphological and sporulation traits, and their pathogenicity. These results help us to elucidate the relationship between genetic diversity and biological phenotype differentiation in Colletotrichum gloeosporioides strains from Capsicum annuum L. Our findings will be useful for the prevention and control of pepper anthracnose and for pepper resistance breeding.

Discrimination of hospital isolates of Acinetobacter baumannii using repeated sequences and whole genome alignment differential analysis

An optimized method for bacterial strain differentiation, based on combination of Repeated Sequences and Whole Genome Alignment Differential Analysis (RS&WGADA), is presented in this report. In this analysis, 51 Acinetobacter baumannii multidrug-resistance strains from one hospital environment and patients from 14 hospital wards were classified on the basis of polymorphisms of repeated sequences located in CRISPR region, variation in the gene encoding the EmrA-homologue of E. coli, and antibiotic resistance patterns, in combination with three newly identified polymorphic regions in the genomes of A. baumannii clinical isolates. Differential analysis of two similarity matrices between different genotypes and resistance patterns allowed to distinguish three significant correlations (p < 0.05) between 172 bp DNA insertion combined with resistance to chloramphenicol and gentamycin. Interestingly, 45 and 55 bp DNA insertions within the CRISPR region were identified, and combined during analyses with resistance/susceptibility to trimethoprim/sulfamethoxazole. Moreover, 184 or 1374 bp DNA length polymorphisms in the genomic region located upstream of the GTP cyclohydrolase I gene, associated mainly with imipenem susceptibility, was identified. In addition, considerable nucleotide polymorphism of the gene encoding the gamma/tau subunit of DNA polymerase III, an enzyme crucial for bacterial DNA replication, was discovered. The differentiation analysis performed using the above described approach allowed us to monitor the distribution of A. baumannii isolates in different wards of the hospital in the time frame of several years, indicating that the optimized method may be useful in hospital epidemiological studies, particularly in identification of the source of primary infections.

Genetic Diversity of Botrytis cinerea Revealed by Multilocus Sequencing, and Identification of B. cinerea Populations Showing Genetic Isolation and Distinct Host Adaptation.

Botrytis cinerea is a world-wide occurring plant pathogen, causing pre- and post-harvest gray mold rot on a large number of fruit, vegetable, and flower crops. B. cinerea is closely related to Botrytis pseudocinerea, another broad host range species which often occurs in sympatry with B. cinerea, and to several host-specific species including Botrytis fabae and Botrytis calthae. B. cinerea populations have been shown to be genetically heterogeneous, and attempts have been made to correlate genetic markers to virulence and host adaptation. Here, we present the development of a multilocus sequence typing (MLST) scheme, with 10 genes selected for high variability and phylogenetic congruence, to evaluate the genetic diversity of B. cinerea, B. fabae, and B. pseudocinerea. Using PacBio-assisted simultaneous mass sequencing of PCR products, MLST analysis of about 100 strains from diverse geographical origins and years of isolation was performed, which resulted in high-resolution strain differentiation and robust species separation. Several B. cinerea strains formed an as yet unknown population, referred to as group B, which was well separated from all other B. cinerea strains. Furthermore, the gene cluster for biosynthesis of the phytotoxin botcinic acid was missing in B. cinerea B strains. B. cinerea strains from the monocot Iris pseudacorus were found to form a genetically distinct population, and contained an intact gene cluster for production of the red pigment bikaverin, which is usually degenerated in B. cinerea. Remarkably, these strains were much more aggressive on Iris than other B. cinerea strains, which is the first unequivocal example for host specialization in B. cinerea. Our data reveal new insights into the genetic diversity of B. cinerea and provide evidence for intraspecific differentiation and different degrees of host adaptation of this polyphagous necrotrophic pathogen.

Multiplexed detection and differentiation of bacterial enzymes and bacteria by color-encoded sensor hydrogels

We report on the fabrication and characterization of color-encoded chitosan hydrogels for the rapid, sensitive and specific detection of bacterial enzymes as well as the selective detection of a set of tested bacteria through characteristic enzyme reactions. These patterned sensor hydrogels are functionalized with three different colorimetric enzyme substrates affording the multiplexed detection and differentiation of α-glucosidase, β-galactosidase and β-glucuronidase. The limits of detection of the hydrogels for an observation time of 60 min using a conventional microplate reader correspond to concentrations of 0.2, 3.4 and 4.5 nM of these enzymes, respectively. Based on their different enzyme expression patterns, Staphylococcus aureus strain RN4220, methicillin-resistant S. aureus (MRSA) strain N315, both producing α-glucosidase, but not β-glucuronidase and β-galactosidase, Escherichia coli strain DH5α, producing β-glucuronidase and α-glucosidase, but not β-galactosidase, and the enterohemorrhagic E. coli (EHEC) strain E32511, producing β-galactosidase, but none of the other two enzymes, can be reliably and rapidly distinguished from each other. These results confirm the applicability of enzyme sensing hydrogels for the detection and discrimination of specific enzymes to facilitate differentiation of bacterial strains. Patterned hydrogels thus possess the potential to be further refined as detection units of a multiplexed format to identify certain bacteria for future application in point-of-care microbiological diagnostics in food safety and medical settings.

Yersinia pestis strains isolated in natural plague foci of Caucasus and Transcaucasia in the context of the global evolution of species

BackgroundPlague is a highly dangerous vector-borne infectious disease that has left a significant mark on history of humankind. There are 13 natural plague foci in the Caucasus, located on the territory of the Russian Federation, Azerbaijan, Armenia and Georgia. We performed whole-genome sequencing of Y. pestis strains, isolated in the natural foci of the Caucasus and Transcaucasia. Using the data of whole-genome SNP analysis and Bayesian phylogeny methods, we carried out an evolutionary-phylogeographic analysis of modern population of the plague pathogen in order to determine the phylogenetic relationships of Y. pestis strains from the Caucasus with the strains from other countries. ResultsWe used 345 Y. pestis genomes to construct a global evolutionary phylogenetic reconstruction of species based on whole-genome SNP analysis. The genomes of 16 isolates were sequenced in this study, the remaining 329 genomes were obtained from the GenBank database. Analysis of the core genome revealed 3315 SNPs that allow differentiation of strains. The evolutionary phylogeographic analysis showed that the studied Y. pestis strains belong to the genetic lineages 0.PE2, 2.MED0, and 2.MED1. It was shown that the Y. pestis strains isolated on the territory of the East Caucasian high-mountain, the Transcaucasian high-mountain and the Priaraksinsky low-mountain plague foci belong to the most ancient of all existing genetic lineages - 0.PE2. ConclusionsOn the basis of the whole-genome SNP analysis of 345 Y. pestis strains, we describe the modern population structure of the plague pathogen and specify the place of the strains isolated in the natural foci of the Caucasus and Transcaucasia in the structure of the global population of Y. pestis. As a result of the retrospective evolutionary-phylogeographic analysis of the current population of the pathogen, we determined the probable time frame of the divergence of the genetic lineages of Y. pestis, as well as suggested the possible paths of the historical spread of the plague pathogen.