Genetic diversity of enteroaggregative Escherichia coli.

Studied 74 E. coli strains isolated from stool samples (60) and urine samples (14) of patients examined for clinical indications. Molecular methods included: PCR with electrophoretic detection of genes associated with diarrheal E. coli pathogroup EAgEC (aggR, aaf, aap, aatA, pet, ast, aai) and ExPEC pathogroup UPEC (pap, sfa, afa, kpsMT II, iutA, hlyA, cnf), MLST typing, whole genome sequencing. Strains isolated from stool samples were significantly more likely (88.3%, p > 0.05) to be typical EAgECaggR+ compared to atypical EAgECaggR-. Strains isolated from urine samples, significant differences between typical and atypical EAgEC were not detected (p > 0.05). Genes associated with ExPEC were present in all strains isolated from urine samples and in 45 strains (75%) isolated from stool samples. Coproisolates belonged to 10 serogroups and 13 serovars: O3:H2, O11:H10, O16:H48, O51:H30, O55:H21, O73:H18, O73:H33, O86:H2, O86:H10, O92:H33, O140:H2, O159:H10. Two strains had unique nucleotide sequences of genes encoding O-antigens that were missing from the SerotypeFinder database. 80% of EAgEC isolated from feces and urine was characterized by an enteroaggregative/uropathogenic genotype (EAgEC/UPEC). Most of the strains isolated from urine belonged to the virulent clone of high-risk epidemic spread ST 38 associated with hybrid strains of UPEC / EAgEC.

Approbation of russian test strips for antimicrobial susceptibility testing of microorganisms by gradient diffusion method (E-test).

The most reliable criterion for the microorganisms antibiotic susceptibility is the value of the minimum inhibitory concentration (MIC). The gradient diffusion method (epsilometric test, e-test), carried out using test strips impregnated with an antimicrobial agent, allows to obtain the quantitative value of MIC, bypassing the time-consuming steps of the traditional method of serial dilutions. We tested strips for the epsilometric test, developed at Saint-Petersburg Pasteur Institute. The quality control, carried by testing the reference strains of E. coli ATCC 25922 and S. aureus 29213, confirmed compliance of the manufacturer's declared antibiotic concentration ranges. The MIC values obtained in the study of the antibiotic susceptibility of 101 clinical isolates of microorganisms of the ESKAPE group, isolated from patients of various hospitals in St. Petersburg, were compared to 8 antimicrobial agents using tested test strips and the corresponding M.I.C. Evaluator of the Oxoid (UK). A high percentage of compliance of MIC values was demonstrated, as well as full compliance of susceptibility categories for all microorganism/antibiotic combinations. The resulting divergences in the MIC values did not exceed one step of a double serial dilution, which is permissible according to GOST R ISO 20776-1-2010. The study showed that the test strips developed at the Saint-Petersburg Pasteur Institute DNT are an acceptable alternative to import tests and, after the registration certificate will be completed, can be offered to determine the susceptibility of microorganisms to antimicrobial agents in bacteriological laboratories.

[Laboratory errors for identification of the escherichia coli strains of the serological groups O6 and O25 as acute intestinal infections.]

Were studied the genes encoding the virulence factors of 221 strains: E. coli O6:H1 (194) and E. coli O25:H4 (27), isolated in 2014-2018 from stool samples of children and adults examined according to epidemic indications. Molecular methods included PCR with hybridization-fluorescence and electrophoresis detection of amplified products. The strains did not have virulence genes for diarrheagenic E. coli (DEC) pathogroups EPEC, ETEC, EIEC, EHEC, EAggEC, and belonged to the phylogenetic group B2. They contained from four to eight genes encoding virulence factors of ExPEC: E. coli O6:H1 - pap (68,6%), sfa (87,6%), fimH (96,4%), hly (62,4%), cnf (74,7%), iutA (97,9%), fyuA (95,9%), chu (100%); E. coli O25:H4 - pap (66,7%), afa (22,2%), fimH (100%), hly (44,4%), cnf (44,4%), iutA (100%) , fyuA (100%), chu (100%). The antimicrobial susceptibility testing to 6 classes of antimicrobials (beta-lactams, fluoroquinolones, aminoglycosides, nitrofurantoin, sulfanilamide, trimethoprim / sulfamethoxazole) according the EUCAST. 60,3% of E. coli O6:H1 were sensitive to antibiotics, E. coli O25:H4 remained sensitive to carbapenems and nitrofurans. Extended-spectrum cephalosporins resistance was due to the production ESBL (CTX-M). The 57,1% resistant strains of E. coli O6:H1 and 100% of E. coli O25:H4 strains belonged to the MDR phenotype. The XDR phenotype had one in five MDR strains of E. coli O6:H1 and E. coli O25:H4. All strains of E. coli O25:H4 belonged to ST131. Given the important role of E. coli in human pathology, detection of virulence genes should be performed to confirm the etiological significance of the isolated strain.

[Phylogenetic groups and virulence genes of Escherichia coli strains isolated from the children gut microbiota.]

Escherichia coli is characterized by a wide intraspecific diversity. The species includes both commensals and pathogens that cause diarrhea and extra-intestinal diseases. Pathogenic strains differ from non-pathogenic ones by the presence of virulence factors and their genes. The phylogenetic structure of the species is represented by four main groups (A, B1, B2, D), which differ in their prevalence among residents of different geographical regions. Pathogenic members of the species have been studied in detail, while non-pathogenic strains have not received such attention. This report presents the results of a study of 511 E. coli strains isolated from the gut microbiota of children without diarrhea and urinary tract infections, aged from 1 month to 17 years, living in St. Petersburg. The main phylogenetic groups were determined by PCR, and E. coli virulence genes associated with diarrhea and extra-intestinal diseases were identified. Results: population structure of E. coli is represented by the following groups: A-33.3%, B1-6.7%, B2-34.0%, D-26%. In the studied population 2.5% of strains belonded to EPEC and 4.5% to EAggEC. EPEC virulence genes were more often detected in strains of phylogroup B1, and EAggEC virulence genes in isolates of phylogroup D. The prevalence of extra - intestinal virulence genes was as follows: pap - 29.5%; sfa - 19.8%; afa - 3.3%; hly - 20.9%; cnf - 17.4%; aer-20.0%. The pap, sfa, hly, and cnf genes were detected mostly in the B2 phylogenetic group. Obtained data shows the similarity of E. coli phylogenetic groups structure in St. Petersburg with E. coli populations isolated from residents of Paris and Sydney. Analysis of the virulence genes prevalence showed the dependence of their presence on the genetic background bacteria.

[Current trends in the development of resistance to clinically significant antibiotics in Salmonella (review of literature).]

Salmonella is one of the leading bacterial pathogens of acute diarrhea as well as foodborne outbreaks. Salmonellosis can occur as gastroenteritis with the development of complications and generalization of infection, also the extra intestinal diseases that require antibiotic therapy are often registered. Currently, the effectiveness of many antibiotics is reduced due to the development of resistance in Salmonella. National Salmonella surveillance systems monitor Salmonella resistance to «critically important for medicine¼ antibiotics (extended-spectrum cephalosporins and fluoroquinolones), as well as multidrug resistance. Quinoloneresistant Salmonella is considered as a high-priority resitant pathogen by the World Health Organization. The article describes the current situation on salmonellosis in the world. Foreign and Russian current data about the leading Salmonella serotypes in different regions of the world are presented. The prevalence of clinically significant resistance depending of the Salmonella serotypes in countries with state monitoring systems is shown. The authors described the leading molecular resistance mechanisms (chromosomal and plasmid mediated) and showed their prevalence in different Salmonella serotypes. The article gives the information about Salmonella successful international multidrug resistant clones with specific resistance phenotypes and genotypes. The authors describe the molecular methods for detection of resistance mechanisms, and show the necessity and significance of antimicrobial susceptibility monitoring in the epidemiological Salmonella surveillance.

[Antimicrobial resistance and clinical significant resistance mechanisms of Salmonella isolated in 2014-2018 in St.Petersburg, Russia.]

The article presents the results of antimicrobial resistance monitoring of Salmonella isolated from children and adults with diarrhea in St. Petersburg in 2014-2018. In 746 isolates of 42 serovars more than 90,0% belonged to three: S. enteritidis (79,6%), S. typhimurium (6,8%) and S. infantis (3,8%). The antimicrobial susceptibility testing (according the EUCAST) to 7 classes of antimicrobials revealed the resistance in 78,6% of Salmonella. Low-level quinolone resistance (MIC of ciprofloxacin 0,12-0,5 mg/l) was detected in 63,3% isolates (S. enteritidis -71,0%, S. typhimurium - 15,7%, S. infantis - 89,3%) and was due to five kinds of single nucleotide substitutions in gyrA: Asp87Tyr - 36,1% of studied isolates (only S. infantis); Ser83Phe - 22,2% (only S. enteritidis); Asp87Asn - 19,4% (S. enteritidis, S. typhimurium, S. hadar, S. newport); Ser83Tyr -11,1% (S. enteritidis and S. infantis) and Asp87Gly - 8,3% (only S. enteritidis). Only in one S. kentucky isolate with high-level fluoroquinolone resistance (MIC of ciprofloxacin > 8,0 mg/l) two substitutions (Ser83Phe and Asp87Asn) were detected. Two Salmonella isolates (S. typhimurium and S. corvallis) had plasmid-mediated quinolone resistance (qnrS). Extended-spectrum cephalosporin resistance was found in 6 Salmonella serovars (1,6%). The bla-genes were detected: of genetic group CTX-M1 - in 10 isolates (serovars S. typhimurium, S. enteritidis, S. abony, S. coeln and S. virchow), CTX-M2 - in 2 S. typhimurium isolates, CTX-M9 - in three S. enteritidis isolates. In one S. typhimurium CTX-M1 and CTX-M2 were detected. The gene of CMY-2 (molecular class C cephalosporinase) was revealed in two isolates (S. newport and S. enteritidis). Our study showed that Salmonella (the main bacterial pathogen of acute diarrhea in children and adults) isolated in Saint-Petersburg had antimicrobial resistance to drugs of choice for salmonellosis treatment.

[Methodical aspects of antimicrobial susceptibility testing of Salmonella (review of literature).]

Salmonella is one of the leading food-borne infection pathogen: annually in the Russian Federation about 50 thousand cases of salmonellosis are registered. Antimicrobial therapy is necessary in the case of severe infection in children under 6 years and persons over 50 years, in patients with severe accompanying disease, as well as in the case of generalization of the infection. Beta-lactam antibiotics, quinolones and azithromycin are included in the list of drugs recommended for antimicrobial therapy of salmonellosis, including typhoid fever. The effectiveness of therapy largely depends on the appropriate antimicrobial susceptibility testing: the choice of testing method, indicator antibiotics and result interpretation. Salmonella belong to the Enterobacteriacae family and are characterized by common mechanisms of resistance to quinolones and beta-lactams, but antimicrobial susceptibility testing of Salmonella to these groups of antibiotics has a number of features. The article presents current data on the susceptibility of Salmonella, including S. Typhi, to antibiotics and leading clinically significant resistance mechanisms. The methodical aspects of Salmonella antimicrobial susceptibility testing of the drugs used for the treatment of salmonellosis (quinolones, beta-lactams and azithromycin) are described in detail. Interpretation of Salmonella testing results according the modern international and Russian recommendations are presented. The authors propose the algorithms for Salmonella antimicrobial susceptibility testing of quinolones, cephalosporins and carbapenems, as well as criteria for result interpretation, allowing the detection of clinically significant mechanisms of resistance to beta-lactams (production of beta-lactamases of different molecular classes) and quinolones (chromosomal mutations and acquired resistance genes).

[Molecular-genetic characterization of shiga-toxin producing Escherichia coli isolated during a food-borne outbreak in St. Petersburg in 2013].

UNLABELLED: Shiga toxin-producing Escherichia coli (STEC) food-borne infections are reported worldwide and represent a serious problem for public healthcare. In the Russian Federation there is little information on epidemiology and etiology of STEC-infections as well as on molecular-genetic peculiarities of STEC pathogens. OBJECTIVE: Our aim was to describe a food-borne outbreak as hemorrhagic colitis (HC) along with hemolytic uremic syndrome (HUS), enterocolitis, and acute gastroenteritis in children in St. Petersburg in 2013. METHODS: Epidemiological, microbiological, molecular-genetic and bioinformatic methods were applied. RESULTS: Objects to study were clinical specimens, milk and food samples, as well as STEC strains isolated during the outbreak. The outbreak of food-borne infection was found to be caused by STEC-contaminated raw milk as confirmed by epidemiological analysis, detection of STEC DNA and isolation of relevant pathogens in milk and sick children fecal specimens. The whole-genome sequencing revealed two groups ofpathogens, E. coli O157:H7 and E. coli O101:H33 among collected strains. Group I strains were attributed to the previously known sequence type ST24, while group II strains belonged to the previously non-described sequence type ST145. In strain genomes of both groups there were identified nucleotide sequences of VT2-like prophage carrying stx2c gene, plasmid enterohemolysin gene, and gene of the STEC main adhesion factor intimin. Gene of intimin gamma was identified in E. coli O157:H7 strains and intimin iota 2 in E. coli O101:H33 strains. The latter previously was identified only in enteropathogenic E. coli (EPEC) strains. CONCLUSION: The additional knowledge of epidemiology and biology of STEC pathogens would assist clinicians and epidemiologists in diagnosing, treating and preventing hemorrhagic colitis.

[Characteristics of enterohemorrhagic Escherichia coli O145:H28 isolated from a patient with hemolytic-uremic syndrome].

AIM: Determine etiologic significance of clinical strains of E. coli O145:H28 isolated from feces of a patient with hemolytic-uremic syndrome (HUS). MATERIALS AND METHODS: 20 E. coli strains isolated from feces of a patient with HUS that developed after an acute intestine infection were studied. Antigenic structure of strains was determined by sequencing of rib and fliC genes; presence of virulence genes (pap, aaf, sfa, afa, eaeA, bfpA, ial, hly, cnf, stx1, stx2, LT, ST and aer)--in PCR; ESBL production --by double disk method, ESBL genes--in PCR. RESULTS: The strains contained rfb gene coding O145, fliC gene coding H7. Genes coding synthesis of stx2-toxin and intimin (eaeA) were detected. The strains were resistant to beta-lactams due to production of CTX-M class ESBL. CONCLUSION: A causative agent E. coli O145:H28 was isolated from a patient with HUS that produces stx2-toxin and CTX-M class ESBL and has not been previously registered in Russian Federation.

[The characteristics of biological properties of E. coli O104:H4--the causative agent of large-scale alimentary ictus in Germany may 2001].

The review presents the characteristics of E. coli O104:H4, the causative agent of large-scale alimentary ictus in Germany in spring time 2011. The antigenic characteristics and factors of E. coli pathogenicity are taken into account. The causative agent has a combination of pathogenic factors of two groups of diarrheigenic Escherichia: shigella similar toxin, specific for entero-hemorrhagic E. coli and adhesins of enteroaggregative E. coli.

[Biodiversity and evolution of circulating bacteria and virus populations. Novel problems of medical microbiology].

Biodiversity and evolution of circulating bacteria and virus populations is a serious scientific problem, solving this problem is necessary for effective prophylaxis of infectious diseases. Principal trends of development in this field of science are described. Results of studies that were carried out and investigated biodiversity of principal pathogens in Russia and St. Petersburg in particular are presented. Risk of infectious security of society caused by increasing diversity of pathogenic microorganisms is described, and priority trends of research development in this field are specified.

[Virulence markers of Escherichia coli O1 strains].

AIM: To detect virulence genes in clinical isolates of Escherichia coli O1 using polymerase chain reaction (PCR). MATERIALS AND METHODS: One hundred and twenty strains of E.coli O1 strains isolated from faeces of patients with acute diarrhea (n = 45) and healthy persons (n = 75) were studied. PCR with primers for rfb and fliC genes, which control synthesis of O- and H- antigens respectively, was used. Fourteen virulence genes (pap, aaf, sfa, afa, eaeA, bfpA, ial, hly, cnf, stx1, stx2, lt, st, and aer) were detected by PCR primers. K1-antigen was determined by Pastorex Meningo B/E. coli O1 kit (Bio-Rad). RESULTS: rfb gene controlling O-antigen synthesis in serogroup O1 as well as fliC gene controlling synthesis of H7 and K1 antigens were detected in all strains. Thus all E. coli strains had antigenic structure O1:K1 :H-:F7. Virulence genes aafl, sfa, afa, eaeA, bfpA, ial, hly, cnf, stx1, stx2, lt, and st were not detected. All strains owned pap and aer genes regardless of the presence of acute diarrhea symptoms. CONCLUSION: It was shown that E. coli O1:KI:H-:F7 strains do not have virulence genes which are characteristic for diarrhea-causing Escherichia. In accordance with the presence of pap and aer genes they could be attributed to uropathogenic Escherichia (UPEC) or avian-pathogenic Escherichia (APEC). It is necessary to detect virulence factors in order to determine E. coli as a cause of intestinal infection.

[Verotoxin as a major laboratory criterion for Escherichia coli O157-induced enteric infection complicated by hemolytic uremic syndrome in infants in the Omsk region].

The paper presents the results of etiological interpretation of the outbreak of Escherichia coli O157-induced enteric infection complicated by hemolytic uremic syndrome in Omsk in spring 2009. Seventeen infants aged 5 months to 2.5 years were examined; of them 9 patients were on hemodialysis. The diagnosis was verified in 7 children, by isolating verotoxin after fecal enrichment on the concentrating nutrient RIDA Enrichment Bouillon medium containing bile salts and mitomycin C, E. coli O157:H7 cultures on the sorbitol medium and/or by identifying E. coli O157 DNA. Six (4 having verotoxin) infants were found to have rotavirus and noro-virus antigens or DNA/RNA, which may make a contribution into the development of mixed infection and increase the risk of complications.

[Epidemiological data on salmonellosis due to Salmonella enteritidis in some areas of the Russian Federation]. / Epidemiologicheskie dannye o sal'monelleze, obuslovlennom Salmonella enteritidis, na nekotorykh territoriiakh Rossiiskoi Federatsii.

A rise in morbidity caused by S. enteritidis at individual territories of the Russian Federation in the second half of 1980s was due to the consumption of insufficiently heated infected chicken eggs and the nonobservance of sanitary and hygienic rules in the preparation of food from chicken meat. The spread of S. enteritidis in the Russian Federation occurred mainly at the territories supplied with incubator eggs from the same poultry-breeding enterprise. S. enteritidis strains isolated from infected patients, chicken eggs, follicles and chicken-meat products belonged to biovar Jena (as a rule, to phagovar 1), had plasmid with a mol. wt. of 38 MD, produced no aerobactin, and their overwhelming majority was resistant to antibiotics.

Dynamics of non-specific antibacterial activity of the peritoneal cells of mice induced with Coxiella burnetii antigen.

It was found that primary immunization with Coxiella burnetii antigen increased mouse resistance to Salmonella typhimurium infection as evidenced by acceleration of bacterial elimination from the peritoneal cavity and a decrease in lethality of experimental animals. The existence of two rises of bactericidal activity of mouse peritoneal cells was ascertained: the "early" on days 1 and 2, and the "late" on day 14 after C. burnetii administration. The first rise was accompanied by some increase in the number of peritoneal cells as well as by some change of their qualitative representation. The second increase of antibacterial activity was detected during the pronounced cellular and humoral immune responses to C. burnetii.

[The role of day-old chicks in the transmission of Salmonella enteritidis]. / O roli odnodnevnykh tsypliat v peredache Salmonella enteritidis.

The epidemiological analysis of an outbreak of salmonellosis, registered in May 1988 on the territory of two regions of the Karelian ASSR and covering 10 villages, was carried out. Altogether 112 persons were affected. One-day old chickens sold to the population by the local poultry plant were the source of infection which was transmitted through everyday contacts: those persons who had direct contact with chickens were affected. S. enteritidis with similar biological characteristics were isolated from salmonellosis patients, from persons having had contacts with chickens and from chickens. Chickens were probably infected by oral route.

[Effect of plasmids for multiple antibiotic resistance on the change in Salmonella typhimurium phagovar]. / Vliianie plazmid mnozhestvennoi ustoichivosti k antibiotikam na izmenenie fagovara Salmonella typhimurium.

The data on the influence of acquired plasmid resistance to antibiotics on S. typhimurium phagovar. Plasmid R was transferred from S. typhimurium strain, isolated from the focus of hospital salmonellosis and resistant to the lytic action of phages, to Escherichia coli K12 and then to antibiotic-sensitive S. typhimurium strains of different phagovars, isolated from patients with alimentary toxicoinfections. The influence of plasmid R on the phagovar of recipient strains, most pronounced in strains of phagovar I, was revealed. The transconjugates of this phagovar considerably changed sensitivity to phages and in some cases acquired resistance to the lytic action of typing phages, thus becoming identical in this feature to the donor of the plasmid.