Genetic Determinants Of Antibiotic Resistance Research Articles

Characteristics of <i>Streptococcus pneumoniae</i> carriage in the pediatric population

Objective: to investigate the regional peculiarities of Streptococcus pneumoniae carriage in the pediatric population and characterize the dominant serotypes of the pathogen.
 Materials and methods. The clinical study group consisted of 509 healthy children attending preschool institutions. Examination of nasopharyngeal samples for the detection of S. pneumonae was carried out by classical bacteriological and molecular biological methods. The serotype was determined by real-time PCR. Genome-wide sequencing of the serogroups 15 and 11 isolates and bioinformatic analysis were performed.
 Results. The S. pneumoniae bacterial carriers in the group of healthy children was detected in 207 children (40.7%), while the frequency of detection of S. pneumoniae in urban children living in Kazan was significantly higher than in children living in rural area and amounted to 53.4 and 31.1%, respectively (p 0.05). Among children vaccinated with the 13-valent pneumococcal conjugate vaccine (PCV-13), S. pneumoniae carriers were not detected in 57.5% of cases. There were no significant differences in the degree of nasopharyngeal contamination depending on the vaccination status. Analysis of the serotype composition indicates the predominance of vaccine serotypes (57.7%), while the share of serotypes included in the PСV-13 vaccine accounts for only 24.7%, the share of non-vaccine serotypes was 32.1%, untyped — 10.2%. In unvaccinated children, vaccine serotypes that are part of the PCV-13 and 23-valent polysaccharide pneumococcal vaccine prevailed (PPSV-23): 6ABCD (21%), 11 AD (15%), 14 (13%). In vaccinated children, serotypes not included in the active vaccines dominated: 15AF (17.4%), 23A (19.2%), as well as 11AD (19.6%) (11А is included in PPSV-23). The 27 Kz isolate (serotype 15C) belonged to one of the most common sequence types ST1025. The 105_Kz isolate (serotype 11D) belonged to another common sequence type ST 62.
 Conclusion. In order to improve epidemiological surveillance of pneumococcal infection, it is necessary to introduce the monitoring of circulating clonal complexes of dominant S. pneumoniae serogroups and analyze the genetic determinants of antibiotic resistance and virulence depending on the sequence type.

Antibiotic resistance determination using Enterococcus faecium whole-genome sequences: a diagnostic accuracy study using genotypic and phenotypic data

DNA sequencing could become an alternative to invitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. In this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan14,2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and invitro culture-based AST phenotypes. Isolates were obtained from various sources in 11 countries worldwide between 2000 and 2018. We included isolates tested with broth microdilution, Vitek 2, and disc diffusion, and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations and measured inhibition diameters, and harmonised primarily using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing. A bioinformatics pipeline was developed to process raw sequencing reads, identify antibiotic resistance genetic determinants, and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus, and LRE-Finder, to assess the accuracy of genotypic predictions against phenotypic resistance. We curated a catalogue of 228 genetic markers involved in resistance to 12 antibiotics in E faecium. Very accurate genotypic predictions were obtained for ampicillin (sensitivity 99·7% [95% CI 99·5-99·9] and specificity 97·9% [95·8-99·0]), ciprofloxacin (98·0% [96·4-98·9] and 98·8% [95·9-99·7]), vancomycin (98·8% [98·3-99·2] and 98·8% [98·0-99·3]), andlinezolid resistance (after re-testing false negatives: 100·0% [90·8-100·0] and 98·3% [97·8-98·7]). High sensitivity was obtained for tetracycline (99·5% [99·1-99·7]), teicoplanin (98·9% [98·4-99·3]), and high-level resistance to aminoglycosides (97·7% [96·6-98·4] for streptomycin and 96·8% [95·8-97·5] for gentamicin), although at lower specificity (60-90%). Sensitivity was expectedly low for daptomycin (73·6% [65·1-80·6]) and tigecycline (38·3% [27·1-51·0]), for which the genetic basis of resistance is not fully characterised. Compared with other antibiotic resistance databases and bioinformatic tools, our curated database was similarly accurate at detecting resistance to ciprofloxacin and linezolid and high-level resistance to streptomycin and gentamicin, but had better sensitivity for detecting resistance to ampicillin, tigecycline, daptomycin, and quinupristin-dalfopristin, and better specificity for ampicillin, vancomycin, teicoplanin, and tetracycline resistance. In a validation dataset of 382 isolates, similar or improved diagnostic accuracies were also achieved. To our knowledge, this work represents the largest published evaluation to date of the accuracy of antibiotic susceptibility predictions from E faecium genomes. The results and resources will facilitate the adoption of whole-genome sequencing as a tool for the diagnosis and surveillance of antimicrobial resistance in E faecium. A complete characterisation of the genetic basis of resistance to last-line antibiotics, and the mechanisms mediating antibiotic resistance silencing, are needed to close the remaining sensitivity and specificity gaps in genotypic predictions. Wellcome Trust, UK Department of Health, British Society for Antimicrobial Chemotherapy, Academy of Medical Sciences and the Health Foundation, Medical Research Council Newton Fund, Vietnamese Ministry of Science and Technology, and European Society of Clinical Microbiology and Infectious Disease.

A Novel IncX Plasmid Mediates High-Level Oxytetracycline and Streptomycin Resistance in Erwinia amylovora from Commercial Pear Orchards in California.

Isolates of the fire blight pathogen Erwinia amylovora with high-level resistance to oxytetracycline (minimal inhibitory concentration [MIC] > 100 μg/ml) and to streptomycin (MIC > 100 μg/ml) were recovered from four commercial pear orchards in California between 2018 and 2020. The two representative oxytetracycline- and streptomycin-resistant (OxyTcR-SmR) strains 32-10 and 33-1 were as virulent as the antibiotic susceptible strain 13-1 in causing blossom blight of pear and were recovered more than 50% of the time 7 days after co-inoculation to pear flowers with strain 13-1. In the field, inoculation of strain 32-10 to pear flowers that were pretreated with oxytetracycline at 200 μg/ml did not reduce disease compared with an untreated control. Four OxyTcR-SmR strains were subjected to draft genome sequencing to identify the genetic determinants of antibiotic resistance and their location. A 43.6-kb IncX plasmid, designated pX11-7, was detected in each of the four strains, and this plasmid encoded the tetracycline-resistance gene tetB and the streptomycin-resistance gene pair strAB within a large putatively mobile genetic element consisting of the transposon Tn10 that had inserted within the streptomycin-resistance transposon Tn6082. We also determined that pX11-7 was conjugative and was transferred at a rate that was 104 to 105 higher into an E. amylovora strain isolated in California compared with an E. amylovora strain that was isolated in Michigan. The occurrence of high levels of resistance to both oxytetracycline and streptomycin in E. amylovora strains from commercial pear orchards in California significantly limits the options for blossom blight management in these locations.

Genetic determinants of antibiotic resistance in enterobacteria isolated during microbiological monitoring in the perinatal center

Relevance. Currently, studies of the prevalence of antibiotic resistance and its genetic characteristics are focused primarily on the adult population, although infection with multiple drug infection has been registered as etiological agents of a general infection in obstetric and gynecological and pediatric institutions. The study of the prevalence of genetic determinants of antibiotic resistance is an important area of scientific research.Aim. To analyze the results of the studies carried out to identify the genetic determinants of antibiotic resistance of enterobacteria isolated during microbiological monitoring in the perinatal center.Materials and methods. The genetic profile of antibiotic resistance was studied in ESBL-producing strains isolated from 45 women and 35 children examined at the departments of the Federal State Budgetary Institution «NII OMM» of the Ministry of Health of Russia. To determine the determinants of antibiotic resistance, 80 non-duplicate strains of 7 species of the Enterobacteriaceae family were studied. DNA of bacterial cells was isolated from a daily culture of microorganisms using the PROBA-NK kit, detection of the tem, ctx-M-1, shv genes; oxa-40-like, oxa-48-like, oxa-23-like, oxa-51-like, imp, kpc, ges, ndm, vim were carried out using the diagnostic kit «BacResista GLA» on the detecting amplifier DT-48 (DNA -technology, Russia). To assess the statistical significance of differences in the frequency of occurrence of genes, Pearson's c2 test with Yates' correction was used.Results and discussion. When analyzing the results of studies on the molecular genetic detection of antibiotic resistance determinants, which we conducted in 2022, it was found that 8 genovariants were found in bacterial strains isolated from patients of the departments of the Research Institute of OMM in Yekaterinburg, providing resistance to beta-lactam antibiotics. The dominant genome, as in 2021, remains blaCTX-M-1, found in 29 cases. The blaTEM gene was identified both in association with other genes and as a single variant in Escherichiae coli and Klebsiella pneumoniae strains. Of the eight strains of K. pneumoniae, 4 were found to have three antibiotic resistance genes blaCTX-M, blaTEM, blaSHV, strains with a genetic profile of blaCTX-M, blaTEM, blaSHV, blaNDM were isolated once; and blaTEM, blaSHV, blaKPC. In one strain of K. pneumoniae, phenotypically showing resistance to AB, no genetic determinants of AB resistance were found. In addition to resistance to beta-lactam antibiotics, the strains demonstrate resistance to such groups of antibacterial drugs as fluoroquinolones, phosphonic acid derivatives (fosfomycin), and aminoglycosides. The data obtained indicate that the intestines of newborns during their stay at the stationary stage of nursing in some cases are colonized by strains of enterobacteria with multidrug resistance. Consequently, children are a reservoir of resistant microorganisms and can be sources of pathogens of infectious diseases in families and children's organized groups.

The Prevalence of Genetic Determinants of Antibiotic Resistance, which are of Particular Epidemiological Consequences, in the Microbiota of the Oropharyngeal Swabs in Patients with Cystic Fibrosis

Relevance. Antibiotic resistance of microorganisms can contribute to the chronicity of the inflammatory process, lead to an increase in the cost of treating patients and make it difficult to eradicate the pathogen. Patients with cystic fibrosis constantly require medical supervision, regular visits to medical institutions, and therefore there is a high risk of infection with nosocomial antibiotic- resistant strains. In addition, the necessary intake of antibacterial drugs provides an advantage for the reproduction of resistant microorganisms.Aim. Comparison of the frequency of detection of antibiotic resistance determinants in oropharyngeal swabs in children with cystic fibrosis and conditionally healthy children using molecular biological methods.Materials and methods. A PCR study of oropharyngeal discharge from 100 children with cystic fibrosis and 100 children from the control (healthy comparison subject) group was performed. Genetic antibiotic resistance locus: metallo-b-lactamases of the VIM, IMP and NDM groups; carbapenemase genes of the KPC and OXA-48 groups; extended-spectrum beta-lactamase genes of the CTX-M group and the mecA gene were detected by polymerase chain reaction (PCR) with hybridization-fluorescence detection.Results and discussion. As a result of the analysis, a statistically significant increase in the frequency of detection of genetic determinants of antibiotic resistance in the microbiota of the oropharyngeal discharge in children with cystic fibrosis was found compared with healthy children (p<0.001). The chances of detecting antibiotic resistance loci in the discharge of the oropharynx among children with cystic fibrosis are 38.5 times higher than among healthy children (95% CI: 5.1-289.5). In 28% of children with cystic fibrosis, DNA of the genetic determinants of antibiotic resistance was detected in the microbiome of the discharge of the oropharynx. A high percentage of the presence of genetic determinants of antibiotic resistance may be the reason for the ineffectiveness of antibiotic therapy.Conclusion. Due to the high occurrence in the microbiome of the oropharyngeal discharge of patients with cystic fibrosis of genetic antibiotic resistance locus that are of particular clinical and/or epidemiological significance, and the high risk of the spread of antibiotic-resistant strains outside medical institutions, it is necessary to include this group of patients in regular epidemiological monitoring.

Phenotypic and genotypic resistance to antibiotics in Staphylococcus aureus strains isolated from cattle milk in Northern Kazakhstan.

Staphylococcus aureus is the most frequent and ubiquitous cause of mastitis in cows. In recent decades, antibiotic resistance has rapidly spread among infectious disease pathogens in Kazakhstan and globally. This study examined the phenotypic and genotypic resistance of S. aureus strains obtained from cattle milk to antibiotics. In 2021 and 2022, 675 cow milk samples were collected from 16 dairy farms in Northern Kazakhstan. Staphylococcus aureus was identified using culture and biochemical methods. The nature of antibiotic resistance was determined by the disk diffusion (DD) method. The distribution of antibiotic resistance genes was determined by polymerase chain reaction. Among the obtained S. aureus isolates, high levels of resistance to β-lactam antibiotics (100%), tetracyclines (95.4%), fluoroquinolones (95.4%), and macrolides (60.92%) were observed. Meanwhile, the lowest levels of resistance were identified for sulfonamides (21.84%) and aminoglycosides (27.59%). All the obtained isolates were positive for the nuc gene encoding thermonuclease. The blaZ, ermC, and tetK genes were detected in 45.9%, 77%, and 83.9% of the studied S. aureus isolates, respectively. The results indicate a high prevalence of antibiotic resistance in S. aureus isolated from cows with clinical and subclinical forms of mastitis in Northern Kazakhstan. In addition, the prevalence of resistance was higher when evaluated by the DD method than when detecting the specific antibiotic resistance genes blaZ, tetK, and ermC, indicating the need for deeper analysis of the phenotypic and genetic determinants of antibiotic resistance.

Comparative genomics of two Vietnamese Helicobacter pylori strains, CHC155 from a non-cardia gastric cancer patient and VN1291 from a duodenal ulcer patient

Helicobacter pylori is involved in the etiology and severity of several gastroduodenal diseases; however, plasticity of the H. pylori genome makes complete genome assembly difficult. We report here the full genomes of H. pylori strains CHC155 and VN1291 isolated from a non-cardia gastric cancer patient and a duodenal ulcer patient, respectively, and their virulence demonstrated by in vitro infection. Whole-genome sequences were obtained by combining long- and short-reads with a hybrid-assembly approach. Both CHC155 and VN1291 genome possessed four kinds of genomic island: a cag pathogenicity island (cagPAI), two type 4 secretion system islands within an integrative and conjugative element (tfs ICE), and prophage. CHC155 and VN1291 carried East Asian-type cagA and vacA s1m1, and outer membrane protein genes, including two copies of oipA. Corresponded to genetic determinants of antibiotic resistance, chromosomal mutations were identified in CHC155 (rdxA, gyrA, and 23S rRNA) and VN1291 (rdxA, 23S rRNA, and pbp1A). In vitro infection of AGS cells by both strains induced the cell scattering phenotype, tyrosine phosphorylation of CagA, and promoted high levels of IL8 secretion, indicating fully intact phenotypes of the cagPAI. Virulence genes in CHC155 and VN1291 genomes are crucial for H. pylori pathogenesis and are risk factors in the development of gastric cancer and duodenal ulcer. Our in vitro studies indicate that the strains CHC155 and VN1291 carry the pathogenic potential.

Uncovering Genetic Determinants of Antibiotic Resistance in Helicobacter pylori through Next-Generation Sequencing and Genome-Wide Association Study

Uncovering Genetic Determinants of Antibiotic Resistance in Helicobacter pylori through Next-Generation Sequencing and Genome-Wide Association Study

Pangenomic analysis of Coxiella burnetii unveils new traits in genome architecture.

Coxiella burnetii is the etiological agent of Q fever, a worldwide zoonosis able to cause large outbreaks. The disease is polymorphic. Symptomatic primary infection is named acute Q fever and is associated with hepatitis, pneumonia, fever, and auto-immune complications while persistent focalized infections, mainly endocarditis, and vascular infections, occur in a minority of patients but are potentially lethal. In order to evaluate the genomic features, genetic diversity, evolution, as well as genetic determinants of antibiotic resistance, pathogenicity, and ability to cause outbreaks of Q fever, we performed a pangenomic analysis and genomic comparison of 75 C. burnetii strains including 63 newly sequenced genomes. Our analysis demonstrated that C. burnetii has an open pangenome, unique genes being found in many strains. In addition, pathogenicity islands were detected in all genomes. In consequence C. burnetii has a high genomic plasticity, higher than that of other intracellular bacteria. The core- and pan-genomes are made of 1,211 and 4,501 genes, respectively (ratio 0.27). The core gene-based phylogenetic analysis matched that obtained from multi-spacer typing and the distribution of plasmid types. Genomic characteristics were associated to clinical and epidemiological features. Some genotypes were associated to specific clinical forms and countries. MST1 genotype strains were associated to acute Q fever. A significant association was also found between clinical forms and plasmids. Strains harboring the QpRS plasmid were never found in acute Q fever and were only associated to persistent focalized infections. The QpDV and QpH1 plasmids were associated to acute Q fever. In addition, the Guyanese strain CB175, the most virulent strain to date, exhibited a unique MST genotype, a distinct COG profile and an important variation in gene number that may explain its unique pathogenesis. Therefore, strain-specific factors play an important role in determining the epidemiological and clinical manifestations of Q fever alongside with host-specific factors (valvular and vascular defects notably).

Whole-genome sequencing-based characteristics of Escherichia coli Rize-53 isolate from Turkey.

Urinary tract infections (UTIs) are one of the most common infectious diseases. Inappropriate and excessive administration of antibiotics has led to the increased antibiotic resistance in the pathogens that cause UTIs. This work focused on identifying genetic determinants of antibiotic resistance in a clinical isolate of UTI-causing Escherichia coli. A clinical isolate of E. coli resistant to β-lactam, tetracycline and aminoglycoside antibiotics was analyzed using whole-genome sequencing (WGS) to identify genes that contribute to its resistance. The clinical isolate was obtained from a urine sample of a UTI patient in Turkey and identified via 16S rDNA sequencing. Antimicrobial susceptibility test was performed for 17 antibiotics using VITEK® 2 and the results were confirmed using minimum inhibitory concentration assay. Whole-genome sequencing of the isolate was performed using Illumina sequencing and analyzed with bioinformatic tools for multilocus sequence typing, replicon types, virulence factors, and antimicrobial resistance genes. Whole-genome datum was submitted to the National Center for Biotechnology Information (NCBI; accession No. JAKSGM000000000). The isolate was only found to be resistant to piperacillin in the β-lactam class of antibiotics. While the isolate was also resistant to aminoglycoside and tetracycline antibiotics, it was sensitive to other antibiotics tested. Ten antibiotic resistance genes were identified in the genome of the isolate: blaOXA-1, blaOXA-2, aac(6')-II, aac(6')-Ib-cr, tetB, catB3, qacE, sitABCD, mdfA, and sul-2. Clonal subtype (ST) and serotype of the isolate were identified as ST2141 and O107/H39, respectively. Plasmid replicon typing was used to identify 5 plasmid types in the genome of E. coli Rize-53 (Col(BS512), IncC, IncIA, IncFIB(AP1918), and IncFII(pRSB107)); however, none of the resistance genes were encoded on the plasmid. Genetic determinants of resistance to tetracycline, β-lactam and aminoglycoside antibiotics were identified using WGS in a uropathogenic E. coli from ST2141 lineage and O107:H39 serotype, isolated in Turkey.

Prevalence of antibiotic resistance genes bla-CTX-M, bla-SHV, bla-TEM in enterobacteria strains isolated from perinatal center patients

Relevance A common mechanism of resistance is the synthesis of enzymes that inactivate the antibiotic (AB). Since its discovery at the end of the 20th century, ESBLs have spread widely throughout the world. Representatives of this group of complex protein compounds have common properties of hydrolysis of ß-lactam antibiotics and differ from each other in amino acid sequence. The genetic determinants that determine their synthesis are the bla-CTX-M, bla-SHV, and bla-TEM genes. Aims. To assess the prevalence of bla-CTX-M, bla-SHV, bla-TEM genes in antibiotic-resistant strains of enterobacteria isolated from samples of patients of the perinatal center. Materials & Methods. We studied 135 strains of ESBL-producing enterobacteria isolated during microbiological monitoring from January 01, 2020 to December 31, 2021. Biological material (feces (92), blood (1), urine (5), cervical canal discharge (33), last (4)) came from patients of the perinatal center (43 women and 92 newborns), examined both for clinical indications and during local microbiological monitoring. Species identification of isolated microorganisms was carried out by bacteriological method, detection of antibiotic resistance genes was carried out using real-time PCR. Results. In 87 cases (64.4%), the studied genetic determinants of antibiotic resistance were successfully identified. Of the 26 studied strains of Klebsiella pneumonia, 24 (92.3%) were able to establish the determinants of resistance to AB, 50% of which are represented by bla-CTX-M. Out of 66 Escherichia coli, 59 (89.33%) had a genetic profile of antibiotic resistance, which was also dominated by bla-CTX-M (67.80%). However, in the Enterobacter cloacae group, it was possible to determine the presence of the studied genes only in 8.10% of cases. K. pneumoniae carrying three antibiotic resistance genes bla-CTX-M, bla-SHV, bla-TEM was isolated once from a sample of the cervical canal of a woman aged 27 years. E. coli strains carrying the CTX-M gene were isolated from 3 mother-child pairs out of 135 cases: in mothers – from the discharge of the cervical canal in the third trimester of pregnancy, in newborns – from feces in the first week of life, which can indicate possible transmission of an AB-resistant microorganism either in utero or through the birth canal. Conclusions. Thus, in the course of the study, determinants of antibiotic resistance of enterobacteria isolated from patients of the perinatal center were identified in 64.4% of cases. The dominant gene leading to the occurrence of AD among ESBL is the bla-CTX-M gene. The number of strains with an established genetic profile of resistance to AB is less among Enterobacter cloacae (p<0.001). In order to study the genetic profile in more detail and to identify the mechanisms for the formation of antibiotic resistance, it is necessary to expand the detection of observed genes

Characteristics of Stenotrophomonas maltophilia isolates from cystic fibrosis patients in Russia.

Stenotrophomonas maltophilia is a common opportunistic microorganism and an important respiratory pathogen in cystic fibrosis (CF). The aim of this study was to determine antimicrobial resistance phenotypes, sequence-types (ST) and genetic determinants of antibiotic resistance in S. maltophilia strains recovered from CF patients in Russia. S. maltophilia isolates recovered from 170 CF patients were analyzed. Minimum inhibitory concentrations of antibacterial agents were determined using Sensititre Gram Negative GNX2F plates and the results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) criteria. Whole-genome sequencing (WGS) was performed on MGISEQ-2000 platform. SPAdes software, Galaxy, ResFinder, Integrall and PubMLST were used for analysis of WGS data. S. maltophilia strains were identified from 24/170 (14%) CF patients. In total, 25 isolates were detected, two strains were isolated from the same patient. The isolates belonged to 17 different STs, including 5 new STs; ST4 was the most prevalent ST. Resistance to ceftazidime was observed in 60% of strains, to ticarcillin-clavulanate - in 32%, to levofloxacin - in 24%, to trimethoprim/sulfamethoxazole - in 12% of strains. All isolates were susceptible to minocycline. All ST4 isolates were resistant or intermediate to ceftazidime and ticarcillin-clavulanate. In two isolates, the sul1 gene was detected. In one isolate, sul1 was part of a class 1 integron. The detected integron also contained the blaGES-7 and aac(6')-Ib-cr genes. The ST4 sequence-type was the most prevalent ST among S. maltophilia strains recovered from CF patients in Russia. Antibiotic resistance genes, including sul1, blaGES-7, aac(6')-Ib-cr, were detected in single strains.

GENOTYPIC AND PHENOTYPIC CHARACTERIZATION OF BACTERIOCINOGENIC LACTIC ACID BACTERIAL STRAINS FOR POSSIBLE BENEFICIAL, VIRULENCE, AND ANTIBIOTIC RESISTANCE TRAITS

Lactic acid bacteria (LAB) are a diverse group of microorganisms that can be found in natural habitats and are used in a wide range of industries, especially in the food sector. They are regarded "Generally Recognized as Safe" and are commonly used as a starter culture, probiotics, and biopreservative. However, some LAB genera are excluded from the "Qualified Presumption of Safety" (QPS) list due to the possibility that they have virulence and antibiotic resistance genetic determinants. The objective of this study was to investigate the beneficial, virulence, and antibiotic resistance-related genes in 10 well-characterized bacteriocinogenic LAB strains. The auto-aggregation, co-aggregation, cell surface hydrophobicity, and bile salt tolerance were also evaluated to examine their potential as bacteriocinogenic probiotics and/or starter culture. Results showed that all tested strains showed abilities for auto-aggregation at 4oC and 37oC, co-aggregation with S. aureus JCM8704, S. typhimurium BIOTECH1826, and E. coli DH5α, and significant cell-surface hydrophobicity. However, only a few strains were able to withstand the media treated with 0.3% bile salt. Among the tested bacteriocinogenic LAB strains, L. lactis IO-1 and C. divergens V41 had the maximum values for auto-aggregation at 4oC and 37oC, respectively. C. divergens V41 also exhibited the highest percentage for cell surface hydrophobicity. E. faecium NKR-5-3 showed the highest co-aggregation percentages with all indicator strains. Our findings also showed that the tested isolates presented distinct combinations of virulence-related genes. Only two of ten bacteriocinogenic LAB strains exhibited presence of multiple virulence genes. Lactococcus sp. QU12 was found to have a high frequency of beneficial and virulence genes, with 2 out of 7 genes present encoding beneficial factor and 11 out of 13 genes encoding virulence factor. Lactococcus sp. QU12 and L. lactis IO-1 were also positive for tetracycline resistance gene tetM and aminoglycoside resistance gene aphA-2, respectively, and transposon genes. Moreover, only a few LAB isolates tested positive for 2 out of 8 antibiotic resistance classes. Although few, the substantial danger of these genes being transferred and acquired cannot be overlooked as this could potentially cause serious health concerns in the future. These results suggest that despite the promising properties of bacteriocinogenic LAB, careful safety evaluation of these strains should be a prerequisite before using these in food systems.

Diagnosis and Management of Bloodstream Infections With Rapid, Multiplexed Molecular Assays.

Bloodstream infection is a major health concern, responsible for considerable morbidity and mortality across the globe. Prompt identification of the responsible pathogen in the early stages of the disease allows clinicians to implement appropriate antibiotic therapy in a timelier manner. Rapid treatment with the correct antibiotic not only improves the chances of patient survival, but also significantly reduces the length of hospital stay and associated healthcare costs. Although culture has been the gold standard and most common method for diagnosis of bloodstream pathogens, it is being enhanced or supplanted with more advanced methods, including molecular tests that can reduce the turnaround time from several days to a few hours. In this article, we describe two rapid, molecular bloodstream infection panels that identify the most common pathogens and associated genetic determinants of antibiotic resistance – the Luminex® VERIGENE® Gram-Positive Blood Culture Test and the VERIGENE® Gram-Negative Blood Culture Test. We conducted a search on PubMed to retrieve articles describing the performance and impact of these tests in the clinical setting. From a total of 48 articles retrieved, we selected 15 for inclusion in this review based on the type and size of the study and so there would be minimum of three articles describing performance and three articles describing the impact post-implementation for each assay. Here we provide a comprehensive review of these publications illustrating the performance and clinical utility of these assays, demonstrating how genotypic tests can benefit diagnostic and antimicrobial stewardship efforts.

Prevalence and Diversity of Antibiotic Resistant Escherichia coli From Anthropogenic-Impacted Larut River.

Aquatic environments, under frequent anthropogenic pressure, could serve as reservoirs that provide an ideal condition for the acquisition and dissemination of antibiotic resistance genetic determinants. We investigated the prevalence and diversity of antibiotic-resistant Escherichia coli by focusing on their genetic diversity, virulence, and resistance genes in anthropogenic-impacted Larut River. The abundance of E. coli ranged from (estimated count) Est 1 to 4.7 × 105 (colony-forming units per 100 ml) CFU 100 ml−1 to Est 1 to 4.1 × 105 CFU 100 ml−1 with phylogenetic group B1 (46.72%), and A (34.39%) being the most predominant. The prevalence of multiple antibiotic resistance phenotypes of E. coli, with the presence of tet and sul resistance genes, was higher in wastewater effluents than in the river waters. These findings suggested that E. coli could be an important carrier of the resistance genes in freshwater river environments. The phylogenetic composition of E. coli and resistance genes was associated with physicochemical properties and antibiotic residues. These findings indicated that the anthropogenic inputs exerted an effect on the E. coli phylogroup composition, diversification of multiple antibiotic resistance phenotypes, and the distribution of resistance genes in the Larut River.

WGS CHARACTERIZATION OF ENTEROCOCCUS FAECALIS H1041 ISOLATED FROM THE TRADITIONAL BULGARIAN GREEN CHEESE

In this study, Enterococcus faecalis H1041 which was isolated from the traditional Bulgarian green cheese was characterized in depth by a whole-genome sequencing performed on the Illumina HiSeq platform using 2 x 150 bases paired-end reads mode. The bioinformatic analysis performed only with free online tools revealed that the strain resembles other cheese-born E. faecalis strains by its antibiotics’ resistances and virulence genotype, thus its presence within the green cheese should not be considered as a health risk. Only two antibiotic resistance genetic determinants were found, but they both carry mutations, and probably are not functional. Furthermore, 18 virulence factor genetic determinants were found, but only 6 of them possessed 100% identities with the canonical sequences, and being more or less intrinsic for the Enterococcus genus. None of them were found on contigs that contain sequences with known plasmid origin of replication. Enterolysin A gene was also detected which most probably plays role in ripening process of the Bulgarian green cheese.

GENOTYPIC AND PHENOTYPIC CHARACTERIZATION OF BACTERIOCINOGENIC LACTIC ACID BACTERIAL STRAINS FOR POSSIBLE BENEFICIAL, VIRULENCE, AND ANTIBIOTIC RESISTANCE TRAITS

Lactic acid bacteria (LAB) are a diverse group of microorganisms that can be found in natural habitats and are used in a wide range of industries, especially in the food sector. They are regarded Generally Recognized as Safe and are commonly used as a starter culture, probiotics, and biopreservative. However, some LAB genera are excluded from the Qualified Presumption of Safety (QPS) list due to the possibility that they have virulence and antibiotic resistance genetic determinants. The objective of this study was to investigate the beneficial, virulence, and antibiotic resistance-related genes in 10 well-characterized bacteriocinogenic LAB strains. The auto-aggregation, co-aggregation, cell surface hydrophobicity, and bile salt tolerance were also evaluated to examine their potential as bacteriocinogenic probiotics and/or starter culture. Results showed that all tested strains showed abilities for auto-aggregation at 4oC and 37oC, co-aggregation with S. aureus JCM8704, S. typhimurium BIOTECH1826, and E. coli DH5α, and significant cell-surface hydrophobicity. However, only a few strains were able to withstand the media treated with 0.3% bile salt. Among the tested bacteriocinogenic LAB strains, L. lactis IO-1 and C. divergens V41 had the maximum values for auto-aggregation at 4oC and 37oC, respectively. C. divergens V41 also exhibited the highest percentage for cell surface hydrophobicity. E. faecium NKR-5-3 showed the highest co-aggregation percentages with all indicator strains. Our findings also showed that the tested isolates presented distinct combinations of virulence-related genes. Only two of ten bacteriocinogenic LAB strains exhibited presence of multiple virulence genes. Lactococcus sp. QU12 was found to have a high frequency of beneficial and virulence genes, with 2 out of 7 genes present encoding beneficial factor and 11 out of 13 genes encoding virulence factor. Lactococcus sp. QU12 and L. lactis IO-1 were also positive for tetracycline resistance gene tetM and aminoglycoside resistance gene aphA-2, respectively, and transposon genes. Moreover, only a few LAB isolates tested positive for 2 out of 8 antibiotic resistance classes. Although few, the substantial danger of these genes being transferred and acquired cannot be overlooked as this could potentially cause serious health concerns in the future. These results suggest that despite the promising properties of bacteriocinogenic LAB, careful safety evaluation of these strains should be a prerequisite before using these in food systems.

Prevalence dynamics of uropatogens and antimicrobial resistance determinants in children's significant bacteriuria in 2017 and 2019: a monocenter study

Urinary tract infections are the second most common infections in children with spreading of antimicrobial resistance among uropathogens currently poses a high epidemiological threat.Purpose. Analysis of species prevalence and the presence of genetic determinants of antibiotic resistance.Materials and methods. In the study 215 midstream urine samples were retrospectively analyzed. Samples were obtained during 2017 and 2019 from patients aged 4 weeks to 17 years at the National Medical Research Center for Children's Health Federal State Autonomous Institution of the Ministry of Health of the Russian Federation.Results. Species of pathogen were identified in 93 samples, while the bacterial composition of other samples was classified as «intestinal flora» (n = 17), «coccus flora» (n = 16) or «mixed flora» (n = 89). The most common types of uropathogens in monopathogenic infections in 2017 and 2019 were Escherichia coli (37.5% and 29.2%, respectively). Among infections caused by multiple pathogens, the most common etiological agents were Pseudomonas aeruginosa and Staphylococcus spp. Among all studied samples, 31.9% contained CTX-M-like genes, 5% VIM genes, 1.8% NDM genes, and 3.0% — OXA-48-like genes, and 5.6% of samples contained two and more genetic determinants associated with resistance, with the most prevalent gene combination being the combination of CTX-M- and OXA-48-like genes. In 69 samples with identified species of uropathogens, resistance profile to antimicrobial, determined by microbiological methods, correlated with detected resistance genetic determinants.Conclusion. Authors suggest that introduction of testing for the presence of genes associated with antibacterial resistance to general clinical practice would not only provide an opportunity to conduct epidemiological monitoring of the genetic determinants of antibiotic resistance, but also provide an opportunity to select the correct timely treatment of childhood bacteriuria caused by antibiotic-resistant infectious agents.

Profiles and genetic determinants of antimicrobial resistance in pathogens causing community-acquired urinary tract infections in reproductive-age women

Objective. To characterize the profiles and genetic determinants of antibiotic resistance in pathogens of community-acquired urinary tract infections (UTI) in women of reproductive age in St. Petersburg (Russia).Materials and methods. The study included strains of microorganisms obtained from 145 women of reproductive age with diagnosed with a UTI. Antibiotic susceptibility was evaluated by disk diffusion method; the interpretation of the results was carried out in accordance with the EUCAST criteria. Strains of uropathogenic enterobacteria were tested by PCR for the presence of beta-lactamase genes: beta-lactamase genes encoding enzymes of the AmpC group (MOX, CMY, LAT, BIL, DHA, ACC, MIR, ACT, FOX), TEM, SHV, OXA‑1, ESBL genes of the CTX–M group, genes of carbapenemases (KPC, OXA‑48) and metal-beta-lactamases (VIM, IMP, NDM).Results. Most cases (81 %) of UTIs in women in St. Petersburg were due to enterobacteria, with Escherichia coli highly prevailing (66 %). Fosfomycin, meropenem and nitrofurantoin had the highest in vitro activity against uropathogenic Enterobacteriaceae. High rates of resistance to betalactam antibiotics were found (from 16 % to cefotaxime to 28 % to amoxicillin/clavulanic acid). The genes of TEM-beta-lactamases were found in 31 isolates (26.7 %), SHV – in 17 (14.6 %), CTX–M type – in 15 (12.9 %), DHA – in 2 (1.7 %). The other beta-lactam resistance genes (MOX, CMY, LAT, BIL, ACC, MIR, ACT, FOX, KPC group, OXA-like group, VIM, IMP, NDM) were not detected.Conclusion. Microbiological and molecular analysis of the structure of beta-lactam resistance is important for the effective of epidemiological control of antimicrobial resistance in pathogens causing urinary tract infections.

Genome-Scale Metabolic Models and Machine Learning Reveal Genetic Determinants of Antibiotic Resistance in Escherichia coli and Unravel the Underlying Metabolic Adaptation Mechanisms.

ABSTRACTAntimicrobial resistance (AMR) is becoming one of the largest threats to public health worldwide, with the opportunistic pathogen Escherichia coli playing a major role in the AMR global health crisis. Unravelling the complex interplay between drug resistance and metabolic rewiring is key to understand the ability of bacteria to adapt to new treatments and to the development of new effective solutions to combat resistant infections. We developed a computational pipeline that combines machine learning with genome-scale metabolic models (GSMs) to elucidate the systemic relationships between genetic determinants of resistance and metabolism beyond annotated drug resistance genes. Our approach was used to identify genetic determinants of 12 AMR profiles for the opportunistic pathogenic bacterium E. coli. Then, to interpret the large number of identified genetic determinants, we applied a constraint-based approach using the GSM to predict the effects of genetic changes on growth, metabolite yields, and reaction fluxes. Our computational platform leads to multiple results. First, our approach corroborates 225 known AMR-conferring genes, 35 of which are known for the specific antibiotic. Second, integration with the GSM predicted 20 top-ranked genetic determinants (including accA, metK, fabD, fabG, murG, lptG, mraY, folP, and glmM) essential for growth, while a further 17 top-ranked genetic determinants linked AMR to auxotrophic behavior. Third, clusters of AMR-conferring genes affecting similar metabolic processes are revealed, which strongly suggested that metabolic adaptations in cell wall, energy, iron and nucleotide metabolism are associated with AMR. The computational solution can be used to study other human and animal pathogens.IMPORTANCE Escherichia coli is a major public health concern given its increasing level of antibiotic resistance worldwide and extraordinary capacity to acquire and spread resistance via horizontal gene transfer with surrounding species and via mutations in its existing genome. E. coli also exhibits a large amount of metabolic pathway redundancy, which promotes resistance via metabolic adaptability. In this study, we developed a computational approach that integrates machine learning with metabolic modeling to understand the correlation between AMR and metabolic adaptation mechanisms in this model bacterium. Using our approach, we identified AMR genetic determinants associated with cell wall modifications for increased permeability, virulence factor manipulation of host immunity, reduction of oxidative stress toxicity, and changes to energy metabolism. Unravelling the complex interplay between antibiotic resistance and metabolic rewiring may open new opportunities to understand the ability of E. coli, and potentially of other human and animal pathogens, to adapt to new treatments.