Impact of the biomass pretreatment and simulated gastrointestinal digestion on the digestibility and antioxidant activity of microalgae Chlorella vulgaris and Tetraselmis chuii.

Chlorella vulgaris and Tetraselmis chuii are two microalgae species already marketed because of their richness in high-value and health-beneficial compounds. Previous studies have demonstrated the biological properties of compounds isolated from both microalgae, although data are not yet available on the impact that pre-treatment and gastrointestinal digestion could exert on these properties. The aim of the present study was to analyze the impact of the biomass pre-treatment (freeze/thaw cycles plus ultrasounds) and simulated gastrointestinal digestion in the bioaccessibility and in vitro antioxidant activity (ABTS, ORAC, Q-FRAP, Q-DPPH) of the released digests. The cell wall from microalgae were susceptible to the pre-treatment and the action of saliva and gastric enzymes, releasing bioactive peptides and phenolic compounds that contributed to the potent antioxidant activity of digests through their radical scavenging and iron reduction capacities. Our findings suggest the potential of these microalgae against oxidative stress-associated diseases at both, intestinal and systemic level.

TRS-PCR profiles correlate with polymorphisms of the genomic o454-nlpD region, virulence factors repertoire, and phylogenetic groups among uropathogenic Escherichia coli strains isolated from patients from Lodz region, Poland.

Extraintestinal urinary tract infections are mainly caused by uropathogenic strains of E. coli. UPECs are a heterogeneous group of strains possessing various genes associated with virulence traits. It was demonstrated that changes in the composition of the o454-nlpD region and genetic variation in the mutS-rpoS chromosomal region in ExPEC strains are correlated with their virulence, particularly in those with the pattern III o454-nlpD region and belonging to phylogenetic group B2. In this study, we investigated the presence and distribution of the o454-nlpD genomic polymorphism in our collection of 124 uropathogenic E. coli strains, examining the correlation of o454-nlpD region types with the virulence factors studied. Our findings revealed a positive association between certain virulence factors in UPEC strains and the presence of pattern III in the o454-nlpD region. Additionally, all these strains were classified under phylogenetic group B2. We also showed that the highly pathogenic group of E. coli identified by examining the polymorphism of the o454-nlpD region coincides with the highly pathogenic group of uropathogens we identified in the averaged TRS-PCR analysis.

Mycobacterial Interspersed Repeat Unit-Variable Number Tandem Repeat Typing of Mycobacterium avium Strains Isolated from the Lymph Nodes of Free-Living Carnivorous Animals in Poland.

Non-tuberculous mycobacteria (NTM) are ubiquitous organisms, of which some, especially those of the Mycobacterium avium complex (MAC), may be opportunistic animal and human pathogens. Infection with NTM can interfere with tuberculosis (TB) diagnosis and induce zoonoses, especially in immunocompromised individuals. Diseases caused by NTM have become more readily recognized; however, they are likely still underestimated. In this study, we identified and genotyped Mycobacterium avium strains that were isolated during TB monitoring among free-living carnivorous animals from southeastern Poland. In 2011-2020, lymph node samples from 192 such animals were tested for mycobacteria. A total of 41 isolates of M. avium strains were detected with the use of IS901, IS900, IS1245, and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) identification. Thirty-three were identified as M. avium subsp. avium. These strains were derived from 1 beech marten (Martes foina), 1 common buzzard (Buteo buteo), 2 European badgers (Meles meles), 3 wolves (Canis lupus), and 26 red foxes (Vulpes vulpes). One strain isolated from a wolf was identified as M. avium subsp. hominissuis. The results show the widespread occurrence of MAC bacilli in the studied environment and additionally comprise new data on the molecular characteristics of M. avium subspecies carried by free-living southeastern Polish carnivores.

New Genetic Markers Differentiating IPEC and ExPEC Pathotypes-A New Approach to Genome-Wide Analysis Using a New Bioinformatics Tool.

The increasingly expanding genomic databases generate the need for new tools for their processing and further use. In the paper, a bioinformatics tool, which is a search engine of microsatellite elements-trinucleotide repeat sequences (TRS) in files of FASTA type-is presented. An innovative approach was applied in the tool, which consists of connecting-within one search engine-both mapping of TRS motifs and extracting sequences that are found between the mapped TRS motifs. Accordingly, we present hereby the tool called TRS-omix, which comprises a new engine for searching information on genomes and enables generation of sets of sequences and their number, providing the basis for making comparisons between genomes. In our paper, we showed one of the possibilities of using the software. Using TRS-omix and other IT tools, we showed that we were able to extract sets of DNA sequences that can be assigned only to the genomes of the extraintestinal pathogenic Escherichia coli strains or to the genomes of the intestinal pathogenic Escherichia coli strains, as well as providing the basis for differentiation of the genomes/strains belonging to each of these clinically essential pathotypes.

MIRU-VNTR Typing of Atypical Mycobacteria Isolated from the Lymph Nodes of Slaughtered Pigs from Poland.

No regulations currently require the excision of lymph nodes from pig carcasses or the thermal processing of pork before consumption. Therefore, the presence of anatomopathological lesions with signs of coagulation necrosis in lymph nodes from pigs during post-mortem inspection is concerning, as is the increasing incidence of mycobacteriosis in humans. Therefore, the aim of the present study is to verify whether mycobacteria can be isolated from tuberculous-like lesions in mandibular lymph nodes in slaughtered pigs, and whether further molecular analysis based on MIRU-VNRT, used to identify mycobacteria from the Mycobacterium avium complex, can indicate zoonotic potential. Forty of the fifty isolates from the lymph nodes with signs of coagulation necrosis were classified as Mycobacterium avium complex. MIRU-VNTR analysis allowed for the isolation of six strains, one of which was classified as M. avium subsp. paratuberculosis (MAP). Our findings confirm the presence of atypical mycobacteria in the lymph nodes of slaughtered pigs. While the isolated strains (other than MAP) do not pose a significant or direct health risk to consumers, further research and monitoring are necessary. Atypical mycobacteria can cause a wide range of diseases in children and compromised adults, and often show resistance to many classes of antibiotics, including those used to treat tuberculosis.

Molecular analysis of Pseudomonas aeruginosa strains isolated from cystic fibrosis patients.

Pseudomonas aeruginosa is a severe bacterial pathogen. Due to the genetic flexibility among strains, chronic airways infection can lead to mortality among cystic fibrosis (CF) patients. It is essential to develop patient-specific therapy which will rely on phenotypic and genomic diversity. The primary objective of this study was to assess the genomic variability of P. aeruginosa strains, using two different molecular techniques for tracking the epidemiological transmissions. This study applied a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) for an efficient genotyping of clinical P. aeruginosa strains isolated from CF patients and compared results with a TRS-PCR typing. The percentage similarity analysis was performed using the categorical multi-state coefficient and UPGMA method. Based on the MLVA and TRS-PCR group assessment, 43 P. aeruginosa strains/variants were detected among the 63 clinical isolates from eight CF patients. The study of P. aeruginosa isolates has revealed that during chronic bacterial infections, CF patients harbor different P. aeruginosa strains or variants within the same host over the years. P. aeruginosa genotypes diversity may result from infection with several strains and result from a microevolution process of an initially acquired strain. The TRS-PCR method proposed in this work can complement the MLVA scheme. It can also be used as a preliminary method for genetic typing of P. aeruginosa isolates in CF patients.

Case Study and Attempt of Treatment of Mycobacteriosis Caused by Mycobacterium avium in a Parental Flock of Meat-Breed Pigeons.

Mycobacteriosis caused by Mycobacterium avium subsp. avium was observed in a parental loft of 70 meat-breed pigeons. It was decided to undertake treatment as the birds represented a substantial value to the owner. A multiagent therapy using azithromycin, marbofloxacin, and ethambutol was administered. After 4 mo of therapy, the desired results were not obtained. At the end of treatment, the birds were in poor general condition, with white blood cells above 20 g/L, and after clutching, 2-yr-old and older birds were euthanatized. Overall, postmortem lesions were found in 17 out of 49 necropsied individuals. Slide agglutination tests with a M. avium subsp. avium lysate were conducted in all examined pigeons. In 28 pigeons, blood count was conducted once a month during therapy, while in 24 pigeons, a tuberculin sensitivity test was conducted before the planned euthanatization. The tuberculin sensitivity test did not prove useful in the diagnosis of ill individuals. Slide agglutination yielded positive results in only four birds, all of which also had postmortem lesions. Blood count in a large number of cases allowed distinguishing between ill and healthy individuals, which was used for subsequent selection. The comparison of cultured strains with the (CCG)4-based PCR method showed the variation of M. avium isolates up to a maximum of 30%. The described case proves that the treatment of mycobacteriosis in pigeon flocks is not effective, mainly due to the high resistance to M. avium subsp. avium. In addition, therapy may contribute to an even greater increase in mycobacterial resistance to antibiotics, which may pose a potential risk to public health.

Numerical interpretation of TRS-PCR profiling results for Escherichia coli strains isolated from patients with bacteriuria in Lodz region, Poland.

With the multiplicity of existing methods to track E. coli infections, it still seems necessary to seek new, better and/or complementary ways for epidemiological investigations. Particularly, fast, cheap, effective and reproducible methods providing easily comparable results are needed. Our previous studies showed that the use of TRS-PCR is an effective molecular tool in E. coli epidemiology. In this paper, we have developed a unique classification scheme in which an individual TRS-PCR pattern is assigned a numerical value. This approach allows for rapid interpretation of the results obtained from several similarity dendrograms. Using this approach, based on CAC-PCR, GTG-PCR and CGG-PCR, we obtained 52, 86 and 99 different numerical types for the 124 analyzed uropathogenic E. coli strains, respectively. This allowed for the identification of 121 unique isolates differing in at least one TRS-PCR class. In this approach, we got numerical results, easy to sort and interpret, allowing easier analysis of these strains.

The genetic background of antibiotic resistance among clinical uropathogenic Escherichia coli strains.

The spreading mechanisms of antibiotic resistance are related to many bacterial and environment factors. The overuse of antibiotics is leading to an unceasing emergence of new multidrug resistant strains. This problem also concerns uropathogenic Escherichia coli strains, which is the most common pathogen causing urinary tract infections. The aim of this study was the genetic analysis of antibiotic resistance in comparison to the phenotypic background of E. coli strains. The characterized collection of E. coli strains isolated 10 years ago from the urine samples of patients with urinary tract infections was used for antimicrobial susceptibility testing (the disc diffusion method) and analysis of antibiotic resistance genes (PCR reaction, sequencing). Additionally, the presence of ESBL strains was analyzed. Fourteen genes were associated with resistance to beta-lactams, aminoglycosides, sulfonamides and quinolones. The genetic analysis revealed that blaTEM-1 and sul2 were present in almost all of the studied strains. Other drug-resistance genes were very rare or non-existent. Otherwise, the phenotypic resistance to fluoroquinolones was well correlated with the genotypic background of the studied bacteria. The presence of particular genes and specific mutations indicate a high bacterial potential to multidrug resistance. On the other hand, it needs to be emphasized that the standard disk diffusion test for the routine antimicrobial susceptibility analysis is still the best way to estimate the current situation of bacterial drug-resistance.

Predicting pathogenicity behavior in Escherichia coli population through a state dependent model and TRS profiling.

The Binary State Speciation and Extinction (BiSSE) model is a branching process based model that allows the diversification rates to be controlled by a binary trait. We develop a general approach, based on the BiSSE model, for predicting pathogenicity in bacterial populations from microsatellites profiling data. A comprehensive approach for predicting pathogenicity in E. coli populations is proposed using the state-dependent branching process model combined with microsatellites TRS-PCR profiling. Additionally, we have evaluated the possibility of using the BiSSE model for estimating parameters from genetic data. We analyzed a real dataset (from 251 E. coli strains) and confirmed previous biological observations demonstrating a prevalence of some virulence traits in specific bacterial sub-groups. The method may be used to predict pathogenicity of other bacterial taxa.

TRS-PCR profiling for discrimination of Escherichia coli strains isolated from children with diarrhea under 5 years of age in Lodz region, Poland.

Escherichia coli is one of the most frequently isolated gram-negative pathogens in cases of foodborne diseases and hospital infections. What is more, diarrheal diseases, including these associated with pathogenic E. coli strains, are leading causes of morbidity and mortality worldwide, especially among children. Improvements of the management of diarrheal diseases caused by these bacteria are in the spotlight of the World Health Organization. Therefore, there is still a need to develop new methods or improve ones that are commonly used to characterize and distinguish E. coli strains more precisely. In this work, TRS-based PCRs were effectively used for discrimination of 123 E. coli strains isolated from children with diarrhea in the Lodz region (Poland). The composite TRS-PCR approach, based on similarity comparisons of GTG-PCR and CGG-PCR fingerprints, enabled us to distinguish strains with very good efficacy. This was confirmed by the high diversity index (0.991) and high reproducibility of the band patterns obtained (95.0 %). These results showed the great variation in strains that may cause infections in children under 38 months. However, the stains were grouped in three separate clusters, which were different in terms of their phylogenetic affiliation and virulence factor repertoire. The obtained results support and are consistent with the need of public health surveillance for searching new and fast assays as far as children's health is concerned. TRS-PCR profiling is an effective tool for genotyping of E. coli strains isolated from children with diarrhea.

Usefulness of the (GTG)4-PCR for typing of monophasic Salmonella enterica isolates with antigenic shame l,4,[5],12:i:-.

INTRODUCTION: Monophasic Salmonella enterica strains presenting the antigenic shame 1,4,[5],12:i:- are becoming more prevalent. Accurate identification of such strains is hard with routine using biochemical and serological tests. Such strains can be identified with molecular tests. In this study we have tested the usefulness of(GTG)4-PCR for the diagnostic of such monophasic strains. This usefulness of this method was previously confirmed for genoserotyping of S. Enterica, Typhimurium, Infantis, Virchow, Hadar, Newport and Anatum. MATERIALS AND METHODS: 76 strains with antigenic shame l,4,[5],12:i:-, isolated in Poland in years 2007-12 were tested. Additionally (GTG)4-PCR patterns were obtained for reference strains of serotypes S. Lagos, S. Agama, S. Farsta, S. Tsevie, S. Glocester and S. Tumodi. (GTG)4-PCR was performed with DreamTaq DNA polymerase. Obtained patterns were analysed with BioNumerics software. RESULTS: No pattern specific for monophasic pattern was identified. Additionally it was also impossible to differentiate patterns obtained for S. Typhimurium, S. Farsta, S. Tsevie and S. Glocester. Only reference strains of serotypes S. Tumodi, Farsta and Agama has the distinguishable patterns of (GTG)4-PCR. CONCLUSIONS: Analysed (GTG)4-PCR method do not show the ability to distinguish S. enterica serotypes from group 04, H:i, including monophasic strains with the antigenic shame 1,4,[5],12:i:-.

TRS-based PCR as a potential tool for inter-serovar discrimination of Salmonella Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum.

Salmonella enterica subsp. enterica comprises a number of serovars, many of which pose an epidemiological threat to humans and are a worldwide cause of morbidity and mortality. Most reported food infection outbreaks involve the serovars Salmonella Enteritidis and Salmonella Typhimurium. Rapid identification to determine the primary sources of the bacterial contamination is important to the improvement of public health. In recent years, many DNA-based techniques have been applied to genotype Salmonella. Herein, we report the use of a manual TRS-PCR approach for the differentiation of the Salmonella enterica subspecies enterica serovars in a single-tube assay. One hundred seventy Salmonella strains were examined in this work. These consisted of serovars S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum. Five of the TRS-primers, N6(GTG)4, N6(CAC)4, N6(CGG)4, N6(CCG)4 and N6(CTG)4, perfectly distinguished the S. Enteritidis and S. Typhimurium serovars, and the N6(GTG)4 primer additionally grouped the other five frequently isolated serovars. In our opinion, the TRS-PCR methodology could be recommended for a quick and simple DNA-based test for inter-serovar discrimination of Salmonella strains.

Comparison of the (CCG)4-based PCR and MIRU-VNTR for molecular typing of Mycobacterium avium strains.

Mycobacterium avium, a member of the group of non-tuberculous mycobacteria, is most often responsible for the serious diseases in humans and is frequently isolated from NTM-caused pulmonary events. In this connection the epidemiological aspect is also of great importance. Here we present a useful genetic assay that uses (CCG)(4)-based PCR for genotyping M. avium. After applying this test to 33 strains of M. avium, we found a discriminatory index of 0.979. The accuracy of this analysis was supported by a reasonable reproducibility of 95.1%. These results were compared with the Mycobacterial Intergenic Repeat Unit-Variable Number Tandem Repeats (MIRU-VNTR) typing scheme which had slightly lower discriminatory index of 0.945 however, the method was able to cluster different strains compared to CCG-PCR. Taking into account high discriminatory index and reproducibility, this test scheme has the potential as a screening tool in the investigation of M. avium infections, especially if combined with MIRU-VNTR.

Trinucleotide repeat sequence-based PCR as a potential approach for genotyping Mycobacterium gordonae strains.

Diseases that are caused by non-tuberculous mycobacteria (NTM) continue to pose difficult clinical problems, and the epidemiological aspect of NTM-caused diseases is of great importance. In the case of Mycobacterium gordonae there is no adequate genotyping scheme. Here we present a potential rapid and reproducible genetic assay that uses trinucleotide repeat sequence-based PCR (TRS-PCR) for genotyping M. gordonae. The proposed method constitutes a useful single-primer PCR screen for genotyping this species. Among 10 TRS-containing primers, after applying (CAC)4-based PCR to 36 strains of M. gordonae, we found a discriminatory index of 0.975. The accuracy of this analysis was supported by a reasonable reproducibility of 92%. These results were compared with the Enterobacterial Repetitive Intergenic Consensus Sequences (ERIC)-PCR typing scheme which had lower discriminatory index of 0.93 and its reproducibility was only 86.3%.

(CGG)4-based PCR as a novel tool for discrimination of uropathogenic Escherichia coli strains: comparison with enterobacterial repetitive intergenic consensus-PCR.

Urinary tract infections are one of the most frequent bacterial diseases in humans, and Escherichia coli is most often the relevant pathogen. A specific pathotype of E. coli, known as uropathogenic E. coli (UPEC), often causes serious and difficult-to-treat infections of the urinary tract. We propose a new single-tube screening tool that uses an (N)(6)(CGG)(4) primer to generate fingerprint profiles that allow rapid discrimination and epidemiology of this group of bacteria. We found 71 different CGG-PCR profiles among 127 E. coli strains, while enterobacterial repetitive intergenic consensus (ERIC)-PCR of the same group yielded only 28 profiles. Additionally, the (CGG)(4)-based PCR test turned out to be very effective for clustering UPEC strains exhibiting multiple virulence genes and usually belonging to the B2 phylogenetic group, and it separated these strains from E. coli strains lacking most of the UPEC-specific virulence factors. Since the reproducibility of the CGG-PCR screen is higher than that of ERIC-PCR, our test should be a valuable means of increasing the discriminatory power of current UPEC typing schemes.

SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli.

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS- strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences.