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Background/Objectives: Campylobacter jejuni (CJ) is the etiological agent of the world's most common intestinal infectious food-borne disease, ranging from mild symptoms to fatal outcomes. The development of innovative synbiotics that inhibit the adhesion and reproduction of multidrug-resistant (MDR) CJ in animals and humans, thereby preserving intestinal homeostasis, is relevant. We have created a synbiotic based on the consortium of Lactobacillus crispatus 2029 (LC2029), Ligilactobacillus salivarius 7247 (LS7247), and a mannan-rich prebiotic (Actigen ® ). The purpose of this work was to study the in vitro anti-adhesive and antagonistic activities of the created synbiotic against MDR CJ strains, along with its role in preventing intestinal barrier dysfunction, which disrupts intestinal homeostasis. Methods: A complex of microbiological, immunological, and molecular biological methods was used. The ability of the LC2029 and LS7247 consortium to promote intestinal homeostasis in vitro was assessed by the effectiveness of controlling CJ-induced TLR4 activation, secretion of pro-inflammatory cytokines, development of intestinal barrier dysfunction, and production of intestinal alkaline phosphatase (IAP). Results: All MDR CJ strains showed marked adhesion to human Caco-2, pig IPEC-J2, chicken CPCE, and bovine BPCE enterocytes. For the first time, we found that the prebiotic and cell-free culture supernatant (CFS) from the consortium of LC2029 and LS7247 strains exhibit an additive effect in inhibiting the adhesion of MDR strains of CJ to human and animal enterocytes. CFS from the LC2029 and LS7247 consortium increased the permeability of the outer and inner membranes of CJ cells, which led to extracellular leakage of ATP and provided access to the peptidoglycan of the pathogen for the peptidoglycan-degrading bacteriocins nisin and enterolysin A produced by LS7247. The LC2029 and LS7247 consortium showed a bactericidal effect on CJ strains. Co-cultivation of the consortium with CJ strains resulted in a decrease in the viability of the pathogen by 6 log. CFS from the LC2029 and LS7247 consortium prevented the growth of CJ-induced TLR4 mRNA expression in enterocytes. The LC2029 and LS7247 consortium inhibited a CJ-induced increase in IL-8 and TNF-α production in enterocytes, prevented CJ-induced intestinal barrier dysfunction, maintained the transepithelial electrical resistance of the enterocyte monolayers, and prevented an increase in intestinal paracellular permeability and zonulin secretion. CFS from the consortium stimulated IAP mRNA expression in enterocytes. The LC2029 and LS7247 consortium and the prebiotic Actigen represent a new synergistic synbiotic with anti-CJ properties that prevents intestinal barrier dysfunction and preserves intestinal homeostasis. Conclusions: These data highlight the potential of using a synergistic synbiotic as a preventive strategy for creating feed additives and functional nutrition products based on it to combat the prevalence of campylobacteriosis caused by MDR strains in animals and humans.

The manuscript disputes the exclusive mono-infectious way of thinking, which presumes that for every infection only one pathogen is responsible and sufficient, when infectious vectors, close contact and reduced immunity meet. In situations involving heavily colonized anatomical sites such an approach often ends in insoluble contradictions. Upon critical reflection and evaluation of 20 years research on spatial organization of vaginal microbiota it is apparent, that in some situations, pathogens may act and operate in permanent, structurally organized consortia, whereas its individual components may be innocuous and innocent, failing to express any pathogenic effect. In these cases, consortia are the true pathogens responsible for many infectious conditions, which usually remain unrecognized as long as improperly diagnosed. The structure of such consortia can be unraveled using ribosomal fluorescence in situ hybridization (FISH). FISH methodology, that not only offers an ex vivo opportunity to recognize bacterial species, but provides unique physical insight into their specific role in the pathogenesis of polymicrobial infections. Ribosomal FISH technique applied to both, women with bacterial vaginosis (BV) and their male partners, has added significantly to our understanding of the pathogenesis of this condition and contributed to appreciating the mechanisms of polymicrobial, community-based infection, potentially leading to therapeutic advances., Competing Interests: Declaration of competing interest The authors have no conflict of interest (including financial commitments or ethical concerns). The data of this publication were not presented previously. The work has not been published previously and is not under consideration for publication elsewhere. No generative artificial intelligence was used. The publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out. If accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder., (Copyright © 2024. Published by Elsevier Masson SAS.)

In recent years, some new evidence on the role of Malassezia in late-onset sepsis in immunocompromised patients have been published, but there are still very few studies with special focus on newborns. The prevalence of Malassezia -associated conditions in 3519 newborn patients of general and surgical neonatal intensive care units (NICU) was assessed. All patients underwent pharyngeal and rectal swab screening for Malassezia spp. Identification of Malassezia spp. was carried out with the use of an adapted nutrient media, microscopic assessment of yeast cell morphology, and real-time PCR analysis. Malassezia furfur -induced invasive mycoses (IM) were developed 2.5 times more often in very low birth weight (VLBW) M. furfur -positive newborns, than in neonates with birth weight ≥1500 g, and affecting 15.8 % of VLBW infants. Funguria occurred 16 times more often in VLBW babies, but fungemia incidence was similar for both weight categories. Gastrointestinal (GI) colonization was found in 94.6 % of Malassezia -positive population, and in 8 % of all studied neonates. Among IM patients, death rate was 6.5 %. The specific pathogen was highly detectable by a combination of real-time PCR and an adapted nutrient media. Colonization with M. furfur in newborns was associated with low gestational age, VLBW, and long stay in NICU. The findings emphasize the need to monitor colonization and infection with M. furfur in neonates, staying in ICU for more than two weeks and to improve current diagnostic approaches by using real-time PCR and an adapted nutrient media for M. furfur isolation., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The data were presented at international conferences, including Priputnevich T.V. "Congress of the International Society of Human and Animal Mycology", Amsterdam, 2018 with the report "Morbidity of fungal infections caused by Malassezia furfur in neonatal intensive care units". I would like to comment on the data and its storage. Our center's servers store all patient information. With the help of special software developed by us, we can analyze almost all patients and provide this information upon request. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background/Objectives: The global emergence of antibiotic-resistant zooanthroponotic Escherichia coli strains, producing extended-spectrum beta-lactamases (ESBL-E) and persisting in the intestines of farm animals, has now led to the development of a pandemic of extra-intestinal infectious diseases in humans. The search for innovative probiotic microorganisms that eliminate ESBL-E from the intestines of humans and animals is relevant. Previously, we received three isolates of bifidobacteria: from milk of a calved cow (BLLT1), feces of a newborn calf (BLLT2) and feces of a three-year-old child who received fresh milk from this calved cow (BLLT3). Our goal was to evaluate the genetic identity of BLLT1, BLLT2, BLLT3 isolates using genomic DNA fingerprinting (GDF), to study the tolerance, adhesion, homeostatic and antibacterial activity of BLLT1 against ESBL-E. Methods: We used a complex of microbiological, molecular biological, and immunological methods, including next generation sequencing (NGS). Results: GDF showed that DNA fragments of BLLT2 and BLLT3 isolates were identical in number and size to DNA fragments of BLLT1. These data show for the first time the possibility of natural horizontal transmission of BLLT1 through with the milk of a calved cow into the intestines of a calf and the intestines of a child. BLLT1 was resistant to gastric and intestinal stresses and exhibited high adhesive activity to calf, pig, chicken, and human enterocytes. This indicates the unique ability of BLLT1 to inhabit the intestines of animals and humans. We are the first to show that BLLT1 has antibacterial activity against ESBL-E strains that persist in humans and animals. BLLT1 produced 145 ± 8 mM of acetic acid, which reduced the pH of the nutrient medium from 6.8 to 5.2. This had an antibacterial effect on ESBL-E. The genome of BLLT1 contains ABC-type carbohydrate transporter gene clusters responsible for the synthesis of acetic acid with its antibacterial activity against ESBL-E. BLLT1 inhibited TLR4 mRNA expression induced by ESBL-E in HT-29 enterocytes, and protected the enterocyte monolayers used in this study as a bio-model of the intestinal barrier. BLLT1 increased intestinal alkaline phosphatase (IAP) as one of the main molecular factors providing intestinal homeostasis. Conclusions: BLLT1 shows promise for the creation of innovative functional nutritional products for humans and feed additives for farm animals that will reduce the spread of ESBL-E strains in the food chain.

A cultural microbiological study of the vaginal microbiota of patients of reproductive age was carried out to isolate the species Lactobacillus iners with subsequent study of phenotypic features. The presence of two phenotypically different species variants was found in patients with bacterial vaginosis., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

We studied antimicrobial activity of epigallocatechin-3-gallate (EGCG), a green tea polyphenolic catechin, and its combined use with ceftazidime (CAZ) against bacterial strains of Klebsiella pneumoniae. EGCG exhibited no activity against strains of K. pneumoniae with a different sensitivity to CAZ. However, for a "sensitive" strain, a decrease in minimum inhibitory concentration (MIC) of CAZ (from 0.064 to 0.023 mg/liter) was revealed when CAZ was co-administered with EGCG. For a "resistant" stain, MIC of CAZ remained high, but activation of EGCG at its high concentrations was observed. Indirect evidence of antimicrobial effect of EGCG co-administered with CAZ on Klebsiella was obtained., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. Ligilactobacillus salivarius strain 7247 (LS7247) was isolated at the same time from the intestines and reproductive system of a healthy woman. The genomes of these strains contain genes responsible for the production of peptidoglycan-degrading enzymes and factors that increase the permeability of the outer membrane of Gram-negative pathogens. In this work, the anti- Salmonella and intestinal homeostatic features of the LF3872 and LS7247 consortium were studied. A multi-drug resistant (MDR) strain of Salmonella enteritidis (SE) was used in the experiments. The consortium effectively inhibited the adhesion of SE to intact and activated human, porcine, and chicken enterocytes and reduced invasion. The consortium had a bactericidal effect on SE in 6 h of co-culturing. A gene expression analysis of SE showed that the cell-free supernatant (CFS) of the consortium inhibited the expression of virulence genes critical for the colonization of human and animal enterocytes. The CFS stimulated the production of an intestinal homeostatic factor-intestinal alkaline phosphatase (IAP)-in Caco-2 and HT-29 enterocytes. The consortium decreased the production of pro-inflammatory cytokines IL-8, TNF-α, and IL-1β, and TLR4 mRNA expression in human and animal enterocytes. It stimulated the expression of TLR9 in human and porcine enterocytes and stimulated the expression of TLR21 in chicken enterocytes. The consortium also protected the intestinal barrier functions through the increase of transepithelial electrical resistance (TEER) and the inhibition of paracellular permeability in the monolayers of human and animal enterocytes. The results obtained suggest that a LF3872 and LS7247 consortium can be used as an innovative feed additive to reduce the spread of MDR SE among the population and farm animals.

Previously, the protective role of the S-layer protein 2 (Slp2) of the vaginal Lactobacillus crispatus 2029 (LC2029) strain against foodborne pathogens Campylobacter jejuni , Salmonella enterica serovar Enteritidis, and Escherichia coli O157:H was demonstrated. We demonstrate the new roles of the Slp2-positive LC2029 strain and soluble Slp2 against C. albicans infections. We show that LC2029 bacteria can adhere to the surface of the cervical epithelial HeLa cells, prevent their contact with C. albicans , and block yeast transition to a pathogenic hyphal form. Surface-bound Slp2 provides the ability for LC2029 to co-aggregate with various C. albicans strains, including clinical isolates. C. albicans -induced necrotizing epithelial damage is reduced by colonization with the Slp2-positive LC2029 strain. Slp2 inhibits the adhesion of various strains of C. albicans to different human epithelial cells, blocks yeast transition to a pathogenic hyphal form, and prevents the colonization and pathogenic infiltration of mucosal barriers. Only Slp2 and LC2029 bacteria stimulate the production of protective human β-defensin 3 in various epithelial cells. These findings support the anti- Candida albicans potential of the probiotic LC2029 strain and Slp2 and form the basis for further research on their ability to prevent and manage invasive Candida infections.

The Ligilactobacillus salivarius 7247 (LS7247) strain, originally isolated from a healthy woman's intestines and reproductive system, has been studied for its probiotic potential, particularly against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) as well as its potential use in synbiotics. LS7247 showed high tolerance to gastric and intestinal stress and effectively adhered to human and animal enterocyte monolayers, essential for realizing its probiotic properties. LS7247 showed high anti- Salmonella activity. Additionally, the cell-free culture supernatant (CFS) of LS7247 exhibited anti- Salmonella activity, with a partial reduction upon neutralization with NaOH ( p < 0.05), suggesting the presence of anti- Salmonella factors such as lactic acid (LA) and bacteriocins. LS7247 produced a high concentration of LA, reaching 124.0 ± 2.5 mM after 48 h of cultivation. Unique gene clusters in the genome of LS7247 contribute to the production of Enterolysin A and metalloendopeptidase. Notably, LS7247 carries a plasmid with a gene cluster identical to human intestinal strain L. salivarius UCC118, responsible for class IIb bacteriocin synthesis, and a gene cluster identical to porcine strain L. salivarius P1ACE3, responsible for nisin S synthesis. Co-cultivation of LS7247 with SE and ST pathogens reduced their viability by 1.0-1.5 log, attributed to cell wall damage and ATP leakage caused by the CFS. For the first time, the CFS of LS7247 has been shown to inhibit adhesion of SE and ST to human and animal enterocytes ( p < 0.01). The combination of Actigen prebiotic and the CFS of LS7247 demonstrated a significant combined effect in inhibiting the adhesion of SE and ST to human and animal enterocytes ( p < 0.001). These findings highlight the potential of using the LS7247 as a preventive strategy and employing probiotics and synbiotics to combat the prevalence of salmonellosis in animals and humans caused by multidrug resistant (MDR) strains of SE and ST pathogens.

LF3872 was isolated from the milk of a healthy lactating and breastfeeding woman. Earlier, the genome of LF3872 was sequenced, and a gene encoding unique bacteriocin was discovered. We have shown here that the LF3872 strain produces a novel thermolabile class III bacteriolysin (BLF3872), exhibiting antimicrobial activity against antibiotic-resistant Staphylococcus aureus strains. Sequence analysis revealed the two-domain structural (lysozyme-like domain and peptidase M23 domain) organization of BLF3872. At least 25% residues of this protein are expected to be intrinsically disordered. Furthermore, BLF3872 is predicted to have a very high liquid-liquid phase separation. According to the electron microscopy data, the bacterial cells of LF3872 strain form co-aggregates with the S. aureus 8325-4 bacterial cells. LF3872 produced bacteriolysin BLF3872 that lyses the cells of the S. aureus 8325-4 mastitis-inducing strain. The sensitivity of the antibiotic-resistant S. aureus collection strains and freshly isolated antibiotic-resistant strains was tested using samples from women with lactation mastitis; the human nasopharynx and oral cavity; the oropharynx of pigs; and the cows with a diagnosis of clinical mastitis sensitive to the lytic action of the LF3872 strain producing BLF3872. The co-cultivation of LF3872 strain with various antibiotic-resistant S. aureus strains for 24 h reduced the level of living cells of these pathogens by six log. The LF3872 strain was found to be able to co-aggregate with all studied S. aureus strains. The cell-free culture supernatant of LF3872 (CSLF3872) induced S. aureus cell damage and ATP leakage. The effectiveness of the bacteriolytic action of LF3872 strain did not depend on the origin of the S. aureus strains. The results reported here are important for the creation of new effective drugs against antibiotic-resistant strains of S. aureus circulating in humans and animals.

Natamycin is a macrolide polyene antibiotic, characterized by a potent broad spectrum antifungal activity and low toxicity. However, it is not used for the treatment of systemic mycoses due to its low bioavailability and low solubility in aqueous solutions. In order to create new semisynthetic antifungal agents for treatment of mycoses, a series of water-soluble amides of natamycin were synthesized. Antifungal activities of natamycin derivatives were investigated against Candida spp. , including a panel of Candida auris clinical isolates and filamentous fungi. Toxicity for mammalian cells was assayed by monitoring antiproliferative activity against human postnatal fibroblasts (HPF) and human embryonic kidney cells (HEK293). By comparing leakage of contents from ergosterol versus cholesterol containing vesicles, a ratio that characterizes the efficacy and safety of natamycin and its derivatives was determined (EI, efficiency index). Ability of all tested semisynthetic natamycines to prevent proliferation of the yeast Candida spp. cells was comparable or even slightly higher to those of parent antibiotic. Interestingly, amide 8 was more potent than natamycin ( 1 ) against all tested C. auris strains (MIC values 2 μg/mL vs 8 μg/mL, respectively). Among 7 derivatives, amide 10 with long lipophilic side chains showed the highest EI and strong antifungal activity in vitro but was more toxic against HPF. In vivo experiments with amide 8 showed in vivo efficacy on a mouse candidemia model with a larger LD 50 /ED 50 ratio in comparison to amphotericin B.

Objectives: Evaluation of clinical course of COVID-19 during pregnancy and maternal and perinatal outcomes of this pregnancy., Methods: 66 women with polymerase chain reaction (PCR) - confirmed SARS-CoV-2 and their 42 neonates were included in the prospective observational study. Demographic, epidemiological, clinical, laboratory and instrumental data of pregnancy, delivery, postpartum period, including pharmacotherapy and neonatal outcomes were analyzed., Results: 15 (22.7%) women were asymptomatic, 25 (38%) had mild disease, while moderate and severe forms were detected in 20 (30.2%) and 6 (9.1%) cases, respectively. Additional oxygenation was required in 6 (9%) cases: 4 (6%) received CPAP therapy and 2 (3%) - mechanical ventilation. Main clinical symptoms were cough (51.5%), anosmia (34.9%), and hyperthermia (33.3%). Laboratory changes included increased levels of lactate dehydrogenase (LDH), creatinine, d-dimer, and C-reactive protein (CRP), anemia, and leukopenia. All pregnant women received low molecular weight heparin and interferon alfa-2b according to the National clinical recommendations. Antimicrobial drugs included Amoxicillin/Clavulanic acid (46%) and macrolides (28%) or carbapenems in severe cases of disease. Spontaneous abortion was reported in 6.1% of cases. Eight preterm (19%) and 34 term deliveries (81%) occurred. The mean weight of neonates was (3283 ± 477) g, 1- and 5-min Apgar score was (7.8 ± 0.6) and (8.7 ± 0.5), respectively. No cases of neonatal COVID-19 infection were reported., Conclusions: Mostly, the manifestations of COVID-19 were mild. However, 9% of cases were severe, and could contribute to preterm delivery or maternal morbidity. Main predictors of severe COVID-19 course in pregnant women were a decrease in the levels of erythrocytes and lymphocytes and increase in the levels of alanine aminotransferase and CRP. Elimination of the virus in pregnant women required more time due to altered immunity. No evidence of vertical transmission during pregnancy and delivery was found. However, the possibility of this cannot be excluded.

Medical personnel are a group of people that often encounter infectious agents, leading to greater risk of contracting infectious diseases. Specific prevention of diseases in this group is a priority. The epidemiological effectiveness of COVID-19 prevention in the group of medical workers due to the emergence of new variants of concern of the SARS-CoV-2 virus has not been studied in sufficient depth. We conducted a study of the effectiveness of vaccine use to protect medical workers at a large medical center for obstetrics and gynecology in Moscow. Sputnik V and Sputnik Light were the main vaccines used for the prevention of COVID-19. The vaccines are based on a variant of the S-protein of the SARS-CoV-2 virus, with adenovirus serotypes 5 and 26 as the vector for delivery. Vaccination of employees occurred during the period in which the Delta variant was spreading. The overall epidemiological effectiveness was 81.7% (73.1-87.6%) during the period in which the Delta variant was dominant. During the period from the beginning of vaccination (26 November 2020) until 8 February 2022, the overall effectiveness was 89.1% (86.9-91.0%). As expected, the highest effectiveness during this period was obtained in the group that received the third and fourth doses-96.5% (75.0-99.5%). The severity of COVID-19 in the vaccinated group was significantly lower than in the unvaccinated group.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. The high-quality genome sequencing of LF3872 was performed, and a gene encoding a unique bacteriocin was discovered. It was established that the bacteriocin produced by LF3872 (BLF3872) belongs to the family of cell-wall-degrading proteins that cause cell lysis. The antibacterial properties of LF3872 were studied using test cultures of antibiotic-resistant Gram-positive and Gram-negative pathogens. Gram-positive pathogens ( Staphylococcus aureus strain 8325-4 and S. aureus strain IIE CI-SA 1246) were highly sensitive to the bacteriolytic action of LF3872. Gram-negative pathogens ( Escherichia coli , Salmonella strains, and Campylobacter jejuni strains) were more resistant to the bacteriolytic action of LF3872 compared to Gram-positive pathogens. LF3872 is a strong co-aggregator of Gram-negative pathogens. The cell-free culture supernatant of LF3872 (CSLF3872) induced cell damage in the Gram-positive and Gram-negative test cultures and ATP leakage. In the in vitro experiments, it was found that LF3872 and Actigen prebiotic (Alltech Inc., Nicholasville, KY, USA) exhibited synergistic anti-adhesive activity against Gram-negative pathogens. LF3872 has immunoregulatory properties: it inhibited the lipopolysaccharide-induced production of proinflammatory cytokines IL-8, IL-1β, and TNF-α in a monolayer of Caco-2 cells; inhibited the production of IL-12 and stimulated the production of IL-10 in immature human dendritic cells; and stimulated the production of TGF-β, IFN-γ, and IgA in the immunocompetent cells of intestinal Peyer's patches (PPs) in mice. These results indicate the possibility of creating a synbiotic based on LF3872 and a prebiotic derived from Saccharomyces cerevisiae cell wall components. Such innovative drugs and biologically active additives are necessary for the implementation of a strategy to reduce the spread of antibiotic-resistant strains of socially significant animal and human infections.

Background: Inefficiency of in vitro fertilization (IVF) programs can be caused by implantation failures. The uterine microbiota can influence the implantation process. However, it still remains unclear whether opportunistic microorganisms detected in the endometrium have a negative impact on the implantation success. The aim of our study was to evaluate the influence of the uterine microbiota on the embryo implantation success in patients undergoing assisted reproductive technologies., Methods: The study included 130 women diagnosed with infertility. The patients were divided into three groups: group I included women with the first IVF attempt (n = 39); group II included patients with recurrent implantation failure following embryo transfer with ovarian stimulation (n = 27); group III consisted of women with recurrent implantation failure following frozen-thawed embryo transfer (n = 64). We performed microbiological examination of the embryo transfer catheter which was removed from the uterine cavity after embryo transfer; cervical discharge of all the patients was studied as well. Thirty patients were selected for metagenomic sequencing., Results: The study showed that the uterine cavity is not free of microorganisms. A total of 44 species of microorganisms were detected: 26 species of opportunistic organisms and 18 species of commensals (14 species of lactobacilli and 4 species of bifidobacteria). Obligate anaerobic microorganisms and Gardnerella vaginalis were detected more frequently in group I compared to group III (strict anaerobes-15.4 and 1.6%; G. vaginalis-12.8 and 1.6%, respectively) (p < 0.05). However, this fact did not have a negative influence on the pregnancy rate: it was 51.3% in group I, it was 29.6% and 35.9% in women with recurrent implantation failures, respectively., Conclusion: Opportunistic microorganisms which were revealed in low or moderate titers (10 3 -10 5  CFU/ml) in the uterine cavity and cervical canal did not affect the pregnancy rate in the women in the study groups. The microflora of the uterine cavity and cervical canal differed in qualitative composition in 87.9% of patients, therefore, we can suggest that the uterine cavity may form its own microbiota. The microbiota of the uterine cavity is characterized by fewer species diversity compared to the microbiota of the cervical canal., (© 2022. The Author(s).)

We have previously demonstrated the ability of the human vaginal strain Lactobacillus crispatus 2029 (LC2029) for strong adhesion to cervicovaginal epithelial cells, expression of the surface layer protein 2 (Slp2), and antagonistic activity against urogenital pathogens. Slp2 forms regular two-dimensional structure around the LC2029 cells,which is secreted into the medium and inhibits intestinal pathogen-induced activation of caspase-9 and caspase-3 in the human intestinal Caco-2 cells. Here, we elucidated the effects of soluble Slp2 on adhesion of proteobacteria pathogens inducing necrotizing enterocolitis (NEC), such as Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, and Pseudomonas aeruginosa ATCC 27853 to Caco-2 cells, as well as on growth promotion, differentiation, vascular endothelial growth factor (VEGF) production, and intestinal barrier function of Caco-2 cell monolayers. Slp2 acts as anti-adhesion agent for NEC-inducing proteobacteria, promotes growth of immature Caco-2 cells and their differentiation, and enhances expression and functional activity of sucrase, lactase, and alkaline phosphatase. Slp2 stimulates VEGF production, decreases paracellular permeability, and increases transepithelial electrical resistance, strengthening barrier function of Caco-2 cell monolayers. These data support the important role of Slp2 in the early postnatal development of the human small intestine enterocytes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We have previously demonstrated that human vaginal Lactobacillus crispatus 2029 (LC2029) strain is highly adhesive to cervicovaginal epithelial cells, exhibits antagonistic activity against genitourinary pathogens and expresses surface-layer protein (Slp). The aims of the present study were elucidation of Slp structural and immunomodulatory characteristics and its roles in protective properties of the whole vaginal LC2029 bacteria against foodborne pathogens. Enteric Caco-2 and colon HT-29 cell lines were used as the in vitro models of the human intestinal epithelial layer. LC2029 strain has two homologous surface-layer (S-layer) genes, slp1 and slp2. Whilst we found no evidence for the expression of slp1 under the growth conditions used, a very high level of expression of the slp2 gene was detected. C-terminal part of the amino sequence of Slp2 protein was found to be highly similar to that of the conserved C-terminal region of SlpA protein of L. crispatus Zj001 isolated from pig intestines and CbsA protein of L. crispatus JCM5810 isolated from chicken intestines, and was substantially variable at the N-terminal and middle regions. The amino acid sequence identity between SlpA and CbsA was as high as 84%, whilst the identity levels of these sequences with that of Slp2 were only 49% and 50% (respectively). LC2029 strain was found to be both acid and bile tolerant. Survival in simulated gastric and intestinal juices of LC2029 cells unable to produce Slp2 was reduced by 2-3 logs. Vaginal L. crispatus 1385 (LC1385) strain not expressing Slp was also very sensitive to gastric and intestinal stresses. Slp2 was found to be non-covalently bound to the surface of the bacterium, acting as an adhesin and facilitating interaction of LC2029 lactobacilli with the host immature or fully differentiated Caco-2 cells, as well as HT-29 cells. No toxicity to or damage of Caco-2 or HT-29 epithelial cells were detected after 24 h of colonization by LC2029 lactobacilli. Both Slp2 protein and LC2029 cells induced NF-kB activation in Caco-2 and HT-29 cells, but did not induce expression of innate immunity mediators Il-8, Il-1β, and TNF-α. Slp2 and LC2029 inhibited Il-8 production in Caco-2 and HT-29 cells induced by MALP-2 and increased production of anti-inflammatory cytokine Il-6. Slp2 inhibited production of CXCL1 and RANTES by Caco-2 cells during differentiation and maturation process within 15 days. Culturing Caco-2 and HT-29 cells in the presence of Slp2 increased adhesion of bifidobacteria BLI-2780 to these enterocytes. Upon binding to Caco-2 and HT-29 cells, Slp2 protein and LC2029 lactobacilli were recognized by toll-like receptors (TLR) 2/6. It was shown that LC2029 strain is a strong co-aggregator of foodborne pathogens Campylobacter jejuni, Salmonella enteritidis, and Escherichia coli O157:H used in this study. The Slp2 was responsible for the ability of LC2029 to co-aggregate these enteropathogens. Slp2 and intact LC2029 lactobacilli inhibited foodborne pathogen-induced activation of caspase-9 and caspase-3 as apoptotic biomarkers in Caco-2 and HT-29 cells. In addition, Slp2 and Slp2-positive LC2029 strain reduced adhesion of tested pathogenic bacteria to Caco-2 and HT-29 cells. Slp2-positive LC2029 strain but not Slp2 alone provided bactericidal effect on foodborne pathogens. These results suggest a range of mechanisms involved in inhibition of growth, viability, and cell-adhesion properties of pathogenic Proteobacteria by the Slp2 producing LC2029, which may be useful in treatment of necrotizing enterocolitis (NEC) in newborns and foodborne infectious diseases in children and adults, increasing the colonization resistance and maintaining the intestinal homeostasis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A new method for selection of bacterium antibiotic resistance genes is proposed and tested for solving the problems related to selection of primers for PCR assay. The method implies clustering of similar nucleotide sequences and selection of group primers for all genes of each cluster. Clustering of resistance genes for six groups of antibiotics (aminoglycosides, β-lactams, fluoroquinolones, glycopeptides, macrolides and lincosamides, and fusidic acid) was performed. The method was tested for 81 strains of bacteria of different genera isolated from patients (K. pneumoniae, Staphylococcus spp., S. agalactiae, E. faecalis, E. coli, and G. vaginalis). The results obtained by us are comparable to those in the selection of individual genes; this allows reducing the number of primers necessary for maximum coverage of the known antibiotic resistance genes during PCR analysis.

According to the results of analysis of whole genome sequencing, the presence of genes having resistance to β-lactam antibiotics in hospital-associated strains of Klebsiella pneumoniae was studied. The strains were isolated from neonatal intensive care units. The data obtained were compared with the results of antimicrobial susceptibility testing of isolated microorganisms. Among other strains resistant to cephalosporins, the dominance of genes of CTX-M-type extended-spectrum β-lactamases was shown. It was revealed that one of eight strains phenotypically resistant and moderately resistant to carbapenems have the blaOXA-48 carbapenemase gene.

Objective: Estimation of microbiocenosis of lower and upper genital tract in different morphological forms of chronic endometritis and endometrial polyps., Material and Methods: Histological examination of endometrial aspirates and microview of the lower and upper genital tract in 164 women of reproductive age with different character of menstrual and reproductive history., Results: The risk of endometrial colonization in, disturbance microecology of the vagina is 3.5 times higher than that, in patients with normosenosis (p<0.01, OP=3.5 [95%. CI 1.63-8.11])., Conclusion: The microbiological diagnosis can be considered as a component of comprehensive diagnostics necessary to choose the appropriate management of patients with CE and PE.

The urgency of the staphylococcus research is due to its ability to cause severe infections: softtissue infections, endocarditis, sepsis, toxic shock syndrome, and food poisoning. Coagulase-positive Staphylococcus aureus is the main infection agent of intrahospital infections. This agent has many factors of pathogenicity, which are well known. Among the coagulase-negative staphylococcus (CNS) strains, S. haemolyticus and S. epidermidis are clinically important, because they cause infections in patients with weak immune system. The mechanisms of the CNS pathogenicity are insufficiently understood. The goal of this work was to evaluate the potential pathogenicity of clinical strains of CNS from their capacity to create biofilms and the character of their interaction with human body cells by the example of the HT-29 cell culture. The research was carried out in laboratory strain S. aureus ATCC 29213 and clinical strains S. haemolyticus SH39, S. epidermidis SE36-1 isolated from the neonatal autopsy materials. The visual tests of biofilm formation by each strain and testing of the impact of the strains on the cell culture HT-29 was carried out in this work. The two species of CNS form biofilms at a higher rate than S. aureus. Upon incubation for 2 h of HT-29 cells with staphylococcus strains tested in this work, adhesion of bacteria on cell surface was observed. The adhesion was most pronounced in case of S. aureus ATCC 29213 and S. haemolyticus SH39. Upon 3 h of incubation with S. aureus ATCC 29213 and S. haemolyticus SH39, destruction of cell HT-29 monolayer was observed. The incubation for 24 h with the 3 strains tested in this work caused complete destruction of cell HT-29 monolayer. The maximal toxic effect on HT-29 cells was inherent in the strain S. haemolyticus SH39. The aggregate of the results obtained in this work indicates the presence of the pathogenicity factors in the strains S. haemolyticus SH39, which require additional research.

Aim: Comparative evaluation of species identification of microorganisms by MALDI-TOF mass-spectrometry and automatic biochemical analyzer VITEK2 Compact 30., Materials and Methods: Species identification of 18,400 isolates of microorganisms (staphylococci, streptococci, enterococci, enterobacteria, nonfermenting gram-negative bacteria, lactobacilli, anaerobes, yeast fungi, neisseriae), isolated from vagina of pregnant and non-pregnant women and from newborns, was carried out. Identification of the isolated microorganisms was carried out by automatic bacteriologic analyzer VITEK2 Compact30 (BioMerieuX, France) and MALDI-TOF-MS analysis method on AutofleXIII (Bruker Daltonics, Germany) mass-spectrometer., Results: Comparative identification of 2005 isolates of microorganisms was carried out. Sequencing of ribosomal RNA was used as a reference method. Authenticity of species identification my MALDI-TOF-MS analysis method was: for staphylococci (95.8%), enterococci (97.5%), enterobacteria (98.4%), nonfermenting gram-negative bacteria (93.6%), β-hemolytiC staphylococci (93.8%), lactobacilli (92.8%), yeast fungi (99.9%)., Conclusion: Introduction of MALDI-TOF-MS analysis technology into practical work of microbiological laboratories exceeds previously used methods of microbiological testing in terms of speed; cost and authenticity of identification of a wide spectrum of microorganisms.

We analyzed sensitivity of 123 vaginal lactobacillus strains to antibacterial substances. All lactobacillus strains were sensitive to ampicillin, cefazolin, cefotaxime, and vancomycin, and insensitive to metronidazole, trimethoprim/sulfamethoxazole, and levofloxacin. Lactobacillus strains demonstrated different sensitivity to gentamycin, clindamycin, erythromycin, ciprofloxacin, and tetracycline. The phenomenon of preferential selective influence of antibacterial drugs on the composition of lactobacilli of the vaginal microbiota, in which some lactobacilli survive as part of the vaginal microbiota and have a selective advantage over other types of lactobacilli, should be taken into account during treatment of vaginal infections and dysbiosis.

In the present study, a multiple-locus variable number tandem repeat analysis (MLVA) was used to assess the molecular epidemiology of Neisseria gonorrhoeae clinical isolates originating from different regions of Russia. MLVA, based on seven loci, was performed on 218 isolates that were previously tested for susceptibility to penicillin, tetracycline and ciprofloxacin and for the presence of certain genetic determinants of drug resistance. In total, 83 MLVA types were identified, indicating that MLVA is a highly discriminatory technique with a Hunter-Gaston discriminatory index of 0.963 (95% CI, 0.950-0.977). MLVA type 16 was shown to be the most prevalent type and is undoubtedly associated with a multidrug resistant phenotype. The spread of MLVA type 16 from Moscow to Irkutsk suggests that this type has a highly successful transmission rate. Hierarchical cluster analysis of the MLVA profiles classified 208 isolates (95%) into six large groups (containing more than 10 isolates). Clusters differed in geographical characteristics and susceptibility profiles. MLVA cluster A comprised in total 34 isolates and was unambiguously associated with multidrug resistance. Most isolates in cluster A carried mutations in penA, ponA, rpsJ, mtrR, gyrA, and parC genes. MLVA cluster D was associated with resistance to penicillin and with mutations in ponA and rpsJ genes and the presence of plasmid-borne bla(TEM-1) gene. MLVA clusters B, C and E were associated with susceptibility to ciprofloxacin and had a lack of mutations in ponA, rpsJ, gyrA, and parC genes. We conclude that MLVA will be a useful tool for N. gonorrhoeae epidemiological studies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The problem of hospital-acquired infections due to coagulase-negative staphylococci (CoNS) in neonatal intensive care units is crucial over the last 20 years in the world. Neonates with very low or extremely low body weight belong to a special group of risks by the CoNS infection. However, in Russia CoNS up to now are frequently considered as contaminants and not as the main etiologic factors of pneumonia and sepsis in extremely premature infants. It was shown that hospital strains of CoNS causing fatal infections in extremely premature infants are always present in intensive care units.

We applied complex genetic analysis for evaluation of tetracycline-resistance markers in 129 clinical strains of Neisseria gonorrhoeae from Central, Privolzhskii, and Siberian regions. For detection of mutations in rpsJ gene and MtrRCDE locus we first used minisequence reaction followed by identification of products by MALDI-TOF mass spectrometry. The incidence of detection of resistance markers among the analyzed strains were: tetM--3.1%, mutations in genes rpsJ--82.2%, penB--62.8%, and mtrR--54.3%. The analyzed genetic markers were not detected in 17.5% strains. tetM gene was detected in only 12.5% strains from the Central Region. No differences were revealed in regional distribution of other genotypes. Genotypes tetM(pres), rpsJ(mut), mtrR(mut), and rpsJ(mut), penB(mut), mtrR(mut) reliably predict tetracycline resistance. Microbiological and genetic testing of tetracycline resistance yielded similar results.

We carried out complex genetic analysis of clinical samples containing N. gonorrhoeae DNA, the genotype and profile of drug resistance of this agent were evaluated. Changes in genes responsible for the formation of N. gonorrhoeae resistance to penicillins, fluoroquinolones, and spectinomycin were detected during minisequencing with subsequent MALDI-TOF mass spectrometry. The sensitivity of gonococcus was evaluated directly in the clinical sample without culturing.

A complex method for detection of genetic markers of N. gonorrhoeae resistance to penicillin was developed. Mutations in penA and ponA genes were detected by minisequencing reaction with subsequent detection of reaction products by MALDI-TOF mass spectrometry. This approach was tested on 31 clinical strains of N. gonorrhoeae with minimum inhibitory concentration of penicillin from 0.03 to 8 microg/ml and higher. Mutations in penA and ponA genes in moderately resistant strains were shown (minimum inhibitory concentration up to 0.5 microg/ml) and mutations in penA, ponA, and penB genes in resistant strains (minimum inhibitory concentration more than 1.0 microg/ml). beta-Lactamase genes were detected in 4 strains with high resistance (minimum inhibitory concentration 4-8 and more microg/ml). Correlation between microbiological resistance and presence of respective mutations in the studied locuses was detected.

For many known mechanisms of the drug resistance in microorganisms are described genetic markers (specific changes in the genome of microorganism, in the majority of the cases representing single nucleotide polymorphism). The search for the new methods, which make possible to identify single nucleotide changes with the greater effectiveness and at smaller prime is actual for the solution of the problem of the identification of the resistant strains. In this work a new approach of the determination of single nucleotide polymorphisms is proposed. It is based on the reactions of mini-sequencing and/or sequencing with the subsequent Matrix-Assisted Laser Desorption/Ionisation Time Of Flight Mass-Spectrometry (MALDI-TOF MS) of the reaction products. The approach was tested on a clinical group of Neisseria gonorrhoeae strains to investigate specific single nucleotide polymorphisms in genes gyrA and parC (the genetic markers of the bacterium fluoroquinolone resistance). The results of the nucleotide polymorphism deter- mination was completely agreed with the data, obtained earlier with the use of a "gold standard" (sequencing with the classical gel-electrophoresis separation of the reaction products). There is specific interest in the method of sequencing of the short DNA sequences using MALDI-TOF MS. The new high-throughput approach of the single nucleotide polymorphisms determination in bacterial genes considerably increases the effectiveness of the methods of microorganism's identification, genotyping and determining the genetic markers of the drug resistance.