Novel multiplex-PCR test for Escherichia coli detection.

Escherichia coli is a diverse and ubiquitous strain of both commensal and pathogenic bacteria. In this study, we propose the use of multiplex polymerase chain reaction (PCR), using amplification of three genes (cydA, lacY, and ydiV), as a method for determining the affiliation of the tested strains to the E. coli species. The novelty of the method lies in the small number of steps needed to perform the diagnosis and, consequently, in the small amount of time needed to obtain it. This method, like any other, has some limitations, but its advantage is fast, cheap, and reliable identification of the presence of E. coli. Sequences of the indicated genes from 1,171 complete E. coli genomes in the NCBI database were used to prepare the primers. The developed multiplex PCR was tested on 47,370 different Enterobacteriaceae genomes using in silico PCR. The sensitivity and specificity of the developed test were 95.76% and 99.49%, respectively. Wet laboratory analyses confirmed the high specificity, repeatability, reproducibility, and reliability of the proposed test. Because of the detection of three genes, this method is very cost and labor-effective, yet still highly accurate, specific, and sensitive in comparison to similar methods. IMPORTANCE: Detection of E. coli from environmental or clinical samples is important due to the common occurrence of this species of bacteria in all human and animal environments. As commonly known, these bacteria strains can be commensal and pathogenic, causing numerous infections of clinical importance, including infections of the digestive system, urinary, respiratory, and even meninges, particularly dangerous for newborns. The developed multiplex polymerase chain reaction test, confirming the presence of E. coli in samples, can be used in many laboratories. The test provides new opportunities for quick and cheap analyses, detecting E. coli using only three pairs of primers (analysis of the presence of three genes) responsible for metabolism and distinguishing E. coli from other pathogens from the Enterobacteriaceae family. Compared to other tests previously described in the literature, our method is characterized by high specificity and sensitivity.

Are menstrual disorders in adolescent girls related to metabolic disorders?

INTRODUCTION: Menstrual disorders in adolescent girls are a common clinical problem. They are often accompanied by lipid and glucose metabolism disturbances. The aim of the study was to investigate to what extent the metabolic profile of adolescent girls relates to the severity of their menstrual disorders. MATERIAL AND METHODS: The study included 165 girls with menstrual disturbances and 49 regularly menstruating girls (REG) without clinical hyperandrogenism, matched for age and BMI. The subjects from the study group were divided into 2 subgroups: OLIGO - 111 girls with oligomenorrhea and SA - 54 girls with secondary amenorrhoea. In all girls, hormonal, lipid, and carbohydrate metabolism profiles were assessed. RESULTS: In the SA subgroup concentrations of total cholesterol (TC) and LDL were significantly higher than in the REG and OLIGO groups. Triglyceride (TG) concentration was also the highest in the SA group and significantly higher than in the REG group. The prevalence of lipid metabolism disorders was higher in the SA group (65%) vs. the REG (40%) and OLIGO (51%) groups. The subgroups did not differ significantly in terms of fasting and OGTT glucose and insulin as well as HOMA-IR. TyG index was significantly higher in the OLIGO and SA groups than in the REG group. BMI z-score correlated with TG, LDL, fasting and 120' OGTT glucose and insulin, HOMA-IR, and TyG and negatively with HDL. No relationship between hormonal concentration and metabolic disturbances was found. CONCLUSIONS: Adolescent girls with menstrual disorders are insulin resistant, regardless of PCOS diagnosis. The severity of menstrual disorders may be related to the incidence of lipid disorders in adolescent girls.

Staphylococcus aureus from Subclinical Cases of Mastitis in Dairy Cattle in Poland, What Are They Hiding? Antibiotic Resistance and Virulence Profile.

Bovine mastitis is a common disease worldwide, and staphylococci are one of the most important etiological factors of this disease. Staphylococcus aureus show adaptability to new conditions, by which monitoring their virulence and antibiotic resistance mechanisms is extremely important, as it can lead to the development of new therapies and prevention programs. In this study, we analyzed Staphylococcus aureus (n = 28) obtained from dairy cattle with subclinical mastitis in Poland. The sensitivity of the isolated strains to antibiotics were confirmed by the disc diffusion method. Additionally, minimum inhibitory concentration values were determined for vancomycin, cefoxitin and oxacillin. Genotyping was performed by two methods: PCR melting profile and MLVF-PCR (multiple-locus variable-number tandem-repeat fingerprinting). Furthermore, the presence of antibiotic resistance and virulence genes were checked using PCR reactions. The analyzed strains showed the greatest resistance to penicillin (57%), oxytetracycline (25%) and tetracycline (18%). Among the analyzed staphylococci, the presence of 9 of 15 selected virulence-related genes was confirmed, of which the icaD, clfB and sea genes were confirmed in all staphylococci. Biofilm was observed in the great majority of the analyzed bacteria (at least 70%). In the case of genotyping among the analyzed staphylococci (combined analysis of results from two methods), 14 patterns were distinguished, of which type 2 was the dominant one (n = 10). This study provides new data that highlights the importance of the dominance of biofilm over antibiotic resistance among the analyzed strains.

RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics.

Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.

Examining the Effects of an Anti-Salmonella Bacteriophage Preparation, BAFASAL®, on Ex-Vivo Human Gut Microbiome Composition and Function Using a Multi-Omics Approach.

Salmonella infections (salmonellosis) pose serious health risks to humans, usually via food-chain contamination. This foodborne pathogen causes major food losses and human illnesses, with significant economic impacts. Overuse of antibiotics in the food industry has led to multidrug-resistant strains of bacteria, and governments are now restricting their use, leading the food industry to search for alternatives to secure food chains. Bacteriophages, viruses that infect and kill bacteria, are currently being investigated and used as replacement treatments and prophylactics due to their specificity and efficacy. They are generally regarded as safe alternatives to antibiotics, as they are natural components of the ecosystem. However, when specifically used in the industry, they can also make their way into humans through our food chain or exposure, as is the case for antibiotics. In particular, agricultural workers could be repeatedly exposed to bacteriophages supplemented to animal feeds. To our knowledge, no studies have investigated the effects of such exposure to bacteriophages on the human gut microbiome. In this study, we used a novel in-vitro assay called RapidAIM to investigate the effect of a bacteriophage mixture, BAFASAL®, used in poultry farming on five individual human gut microbiomes. Multi-omics analyses, including 16S rRNA gene sequencing and metaproteomic, revealed that ex-vivo human gut microbiota composition and function were unaffected by BAFASAL® treatment, providing an additional measure for its safety. Due to the critical role of the gut microbiome in human health and the known role of bacteriophages in regulation of microbiome composition and function, we suggest assaying the impact of bacteriophage-cocktails on the human gut microbiome as a part of their safety assessment.

Comprehensive Evaluation of the Safety and Efficacy of BAFASAL® Bacteriophage Preparation for the Reduction of Salmonella in the Food Chain.

Bacteriophages are bacterial predators, which are garnering much interest nowadays vis-à-vis the global phenomenon of antimicrobial resistance. Bacteriophage preparations seem to be an alternative to antibiotics, which can be used at all levels of the food production chain. Their safety and efficacy, however, are of public concern. In this study, a detailed evaluation of BAFASAL® preparation was performed. BAFASAL® is a bacteriophage cocktail that reduces Salmonella in poultry farming. In vivo acute and sub-chronic toxicity studies on rats and tolerance study on targeted animals (chicken broiler) conducted according to GLP and OECD guidelines did not reveal any signs of toxicity, which could be associated with BAFASAL® administration. In addition, no evidences of genotoxicity were observed. The tolerance study with 100-times concentrated dose also did not show any statistically significant differences in the assessed parameters. The in vitro crop assay, mimicking normal feed storage and feed application conditions showed that BAFASAL® reduced the number of Salmonella bacteria in experimentally contaminated feed. Moreover, reductions were observed for all examined forms (liquid, powder, spray). Furthermore, the in vivo efficacy study showed that treatment with BAFASAL® significantly decreased Salmonella content in caeca of birds infected with Salmonella Enteritidis. Detailed examination of BAFASAL® in terms of safety and efficacy, adds to the body of evidence that bacteriophages are harmless to animals and effective in the struggle against bacteria.

Growing Trend of Fighting Infections in Aquaculture Environment-Opportunities and Challenges of Phage Therapy.

Phage therapy, a promising alternative to antimicrobial treatment of bacterial diseases, is getting more and more popular, especially due to the rising awareness of antibiotic resistance and restrictions in antibiotics' use. During recent years, we observed a growing trend of bacteriophages' application in aquaculture, which in each year reports high losses due to bacterial diseases. This review provides an update of the status of bacteriophage therapy for the treatment and prevention of infections in the aquatic environment. As it is still mostly in the scientific stage, there are a few constraints that may prevent effective therapy. Therefore, specific characteristics of bacteriophages, that can act in favor or against their successful use in treatment, were described. We underlined aspects that need to be considered: specificity of phages, bacterial resistance, safety, immune response of the host organism, formulation, administration and stability of phage preparations as well as bacteriophages' influence on the environment. The biggest challenge to overcome is finding the right balance between the desired and problematic characteristics of bacteriophages. Finally, regulatory approval challenges may be encountered by bacteriophage manufacturers. Even though there are still some technical constraints connected with the global use of bacteriophage therapy, it was concluded that it can be successfully applied in aquaculture.

Complete genome sequences of Aeromonas and Pseudomonas phages as a supportive tool for development of antibacterial treatment in aquaculture.

BACKGROUND: Aquaculture is the fastest growing sector of food production worldwide. However, one of the major reasons limiting its effectiveness are infectious diseases among aquatic organisms resulting in vast economic losses. Fighting such infections with chemotherapy is normally used as a rapid and effective treatment. The rise of antibiotic resistance, however, is limiting the efficacy of antibiotics and creates environmental and human safety concerns due to their massive application in the aquatic environment. Bacteriophages are an alternative solution that could be considered in order to protect fish against pathogens while minimizing the side-effects for the environment and humans. Bacteriophages kill bacteria via different mechanisms than antibiotics, and so fit nicely into the 'novel mode of action' concept desired for all new antibacterial agents. METHODS: The bacteriophages were isolated from sewage water and characterized by RFLP, spectrum of specificity, transmission electron microscopy (TEM) and sequencing (WGS). Bioinformatics analysis of genomic data enables an in-depth characterization of phages and the choice of phages. This allows an optimised choice of phage for therapy, excluding those with toxin genes, virulence factor genes, and genes responsible for lysogeny. RESULTS: In this study, we isolated eleven new bacteriophages: seven infecting Aeromonas and four infecting Pseudomonas, which significantly increases the genomic information of Aeromonas and Pseudomonas phages. Bioinformatics analysis of genomic data, assessing the likelihood of these phages to enter the lysogenic cycle with experimental data on their specificity towards large number of bacterial field isolates representing different locations. CONCLUSIONS: From 11 newly isolated bacteriophages only 6 (25AhydR2PP, 50AhydR13PP, 60AhydR15PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP) have a potential to be used in phage therapy due to confirmed lytic lifestyle and absence of virulence or resistance genes.

In silico Analysis of Virulence Associated Genes in Genomes of Escherichia Coli Strains Causing Colibacillosis in Poultry.

INTRODUCTION: Colibacillosis - the most common disease of poultry, is caused mainly by avian pathogenic Escherichia coli (APEC). However, thus far, no pattern to the molecular basis of the pathogenicity of these bacteria has been established beyond dispute. In this study, genomes of APEC were investigated to ascribe importance and explore the distribution of 16 genes recognised as their virulence factors. MATERIAL AND METHODS: A total of 14 pathogenic for poultry E. coli strains were isolated, and their DNA was sequenced, assembled de novo, and annotated. Amino acid sequences from these bacteria and an additional 16 freely available APEC amino acid sequences were analysed with the DIFFIND tool to define their virulence factors. RESULTS: The DIFFIND tool enabled quick, reliable, and convenient assessment of the differences between compared amino acid sequences from bacterial genomes. The presence of 16 protein sequences indicated as pathogenicity factors in poultry resulted in the generation of a heatmap which categorises genomes in terms of the existence and similarity of the analysed protein sequences. CONCLUSION: The proposed method of detection of virulence factors using the capabilities of the DIFFIND tool may be useful in the analysis of similarities of E. coli and other sequences deriving from bacteria. Phylogenetic analysis resulted in reliable segregation of 30 APEC strains into five main clusters containing various virulence associated genes (VAGs).

Differentiation of polyvalent bacteriophages specific to uropathogenic Proteus mirabilis strains based on the host range pattern and RFLP.

Urinary tract infections (UTIs) caused by P. mirabilis are difficult to cure because of the increasing antimicrobial resistance of these bacteria. Phage therapy is proposed as an alternative infection treatment. The aim of this study was to isolate and differentiate uropathogenic P. mirabilis strain specific polyvalent bacteriophages producing polysaccharide depolymerases (PDs). 51 specific phages were obtained. The plaques of 29 bacteriophages were surrounded by halos, which indicated that they produced PDs. The host range analysis showed that, except phages 58B and 58C, the phage host range profiles differed from each other. Phages 35 and 45 infected all P. mirabilis strains tested. Another 10 phages lysed more than 90% of isolates. Among these phages, 65A, 70, 66 and 66A caused a complete lysis of the bacterial lawn formed by 62% to 78% of strains. Additionally, phages 39A and 70 probably produced PDs. The phages' DNA restriction fragment length polymorphism (RFLP) analysis demonstrated that genomes of 51 isolated phages represented 34 different restriction profiles. DNA of phage 58A seemed to be resistant to selected EcoRV endonuclease. The 33 RFLP-EcoRV profiles showed a Dice similarity index of 38.8%. 22 RFLP patterns were obtained from single phage isolates. The remaining 12 restriction profiles consisted of 2 to 4 viruses. The results obtained from phage characterization based on the pattern of phage host range in combination with the RFLP method enabled effective differentiation of the studied phages and selection of PD producing polyvalent phages for further study.

PCR melting profile as a tool for outbreak studies of Salmonella enterica in chickens.

BACKGROUND: Salmonellosis is of great economic concern in all phases of the poultry industry, from production to marketing, leading to severe economic losses. Monitoring the source of the bacterial contamination has fundamental importance in the spreading of salmonellosis. RESULTS: We applied a ligation-mediated PCR method, PCR MP (PCR melting profile), to type S. enterica ssp. enterica ser. Enteritidis (56 strains) and 43 control strains classified to other serovars isolated from poultry. We demonstrated the PCR MP potential for salmonellosis spreading monitoring. Our rapid test presents higher discriminatory power (0.939 vs. 0.608) compared to current molecular subtyping tool such as pulsed-field gel electrophoresis (PFGE), which ineffectiveness underlies the high degree of clonality of S. Enteritidis. CONCLUSIONS: PCR MP was found to be a highly discriminating, sensitive and specific method that could be a valuable molecular tool, particularly for analyzing epidemiological links of limited number of S. enterica ser. Enteritidis strains.

ChoD and HsdD can be dispensable for cholesterol degradation in mycobacteria.

Cholesterol degradation is achieved through a complex metabolic pathway that starts with the oxidation of the 17-alkyl side chain and the steroid ring system. In bacteria, the oxidation of the 3ß-hydroxyl group and isomerization of the resulting cholest-5-en-3-one to cholest-4-en-3-one is catalyzed by hydroxysteroid dehydrogenase (HsdD) or cholesterol oxidase (ChoD). Genes encoding both enzymes were annotated in both fast and slow growing mycobacteria, however the enzymatic activity was confirmed for HsdD, exclusively. Here, we used homologous recombination to engineer multiple mutants, and directly show that both ChoD and HsdD are dispensable for cholesterol degradation in fast-growing Mycobacterium smegmatis mc(2)155 and slow-growing Mycobacterium tuberculosis H37Rv strains. The mutants deffective in the synthesis of ChoD, HsdD or both enzymes were able to grow in minimal media supplemented with cholesterol as a sole source of carbon and energy. Multiple mutants, defective in synthesis of ChoD, HsdD and ketosteroid dehydrogenase (KstD), showed attenuated growth in minimal medium supplemented with cholesterol and accumulated cholesterol degradation intermediates: androstendion (AD) and 9-hydroxy androstendion (9OHAD).

Direct and inverted repeats elicit genetic instability by both exploiting and eluding DNA double-strand break repair systems in mycobacteria.

Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the Mycobacterium smegmatis, Mycobacterium tuberculosis and Escherichia coli genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in M. smegmatis, direct repeats induced the perfect deletion of their intervening sequences ~1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the attB integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.

Postextrasystolic repolarization abnormalities in ST-U segment in patients with ventricular arrhythmias.

BACKGROUND: Changes in U-wave amplitude after premature ventricular contractions (PVC) are known as prognostic markers in the long QT syndrome dependent on bradycardia. The purpose of the study was to find correlation between postextrasystolic ST-U segment changes and a history of sustained ventricular tachycardia or ventricular fibrillation (VT/VF). METHODS: The ST-U segment configurations were taken from the 24-hour ambulatory ECG. The comparison of the morphology of these segments was performed between sinus beats preceding PVC's and first postextrasystolic beats. POPULATION: Two groups of patients were evaluated: 1) 32 patients with VT/VF history (VT/VF group), and 2) 36 patients with potentially malignant arrhythmia (structural heart disease with frequent PVCs and/or nonsustained VT- nsVT) (non-VT/VF group). RESULTS: We found T-wave changes in 8 patients (25%) from the VT/VF group and in 12 patients (33.3%) from the nonVT/VF group (P = NS) and U-wave changes in 13 patients (40.6%) and 3 patients (8.3%), respectively (P < 0.05). Other ECG indexes related to PVC's were also considered: RR interval, coupling interval (CI), prematurity index (PI), and postextrasystolic pause (PP). The analysis of these ECG indices revealed, when compared with patients without T-U-wave changes, that the occurrence of U-wave changes was significantly related to longer RR interval of the sinus rhythm preceding PVC: 1025 +/- 211 vs 918 +/- 200 ms (P < 0.05). The prematurity index was lowest in patients with U-wave changes: 0.54 +/- 0.12 vs 0.65 +/- 0.16 (P < 0.01) while postextrasystolic pauses leading to the postextrasystolic U-wave changes were significantly longer: 1383 +/- 223 vs 1130 +/- 247 ms (P < 0.001). CI did not differentiate patients: 556 +/- 108 vs 584 +/- 117 ms (P = NS). CONCLUSIONS: Postextrasystolic changes in ST-U segment configuration are dependent on bradycardia, low prematurity index of the PVC, and the lengthening of the postextrasystolic pause. U-wave changes more frequently appeared in patients with malignant arrhythmias. Follow-up study is needed to assess if they might be predictive for the occurrence or reoccurrence of arrhythmic episodes.