Hetero-Pathogenic O181:H4 EAHEC Strain of Sequence Type ST678 Associated with Hemolytic-Uremic Syndrome in Schoolchildren in Russia.

BACKGROUND: In the last decade, the importance of hetero-pathogenic enteroaggregative Shiga-toxin-producing E. coli for public health has increased. Recently, we described the genetic background of the EAHEC O181:H4 strain of ST678 carrying the stx2 gene in prophage and five plasmids, including the plasmid-carrying aggR and aaiC genes. Here, we present the morphological and enzymatic characteristics of this strain, as well as susceptibility to antimicrobials, biofilm formation, etc. Methods: Bacterial morphology was studied using an electron microscope. Susceptibility to antimicrobials was determined using the microdilution method. Cytotoxicity was estimated in Vero cells. Virulence was studied on mice. RESULTS: The morphological and enzymatic properties of the hetero-pathogenic EAHEC strain were typical for E. coli; electron microscopy revealed the specific flagella. The strain was susceptible to most antibiotics and disinfectants but resistant to ampicillin and ciprofloxacin and showed a high degree of biofilm formation. Cytotoxicity towards Vero cells was estimated as 80%. CONCLUSIONS: The emergence of a new O181:H4 EAHEC strain poses a potential threat to humans because of the virulence potential that must be taken into account in the epidemiological analysis of outbreaks and sporadic cases of foodborne infections associated with hemolytic-uremic syndrome.

Genomic Analysis of a Hybrid Enteroaggregative Hemorrhagic Escherichia coli O181:H4 Strain Causing Colitis with Hemolytic-Uremic Syndrome.

Hybrid diarrheagenic E. coli strains combining genetic markers belonging to different pathotypes have emerged worldwide and have been reported as a public health concern. The most well-known hybrid strain of enteroaggregative hemorrhagic E. coli is E. coli O104:H4 strain, which was an agent of a serious outbreak of acute gastroenteritis and hemolytic uremic syndrome (HUS) in Germany in 2011. A case of intestinal infection with HUS in St. Petersburg (Russian Federation) occurred in July 2018. E. coli strain SCPM-O-B-9427 was obtained from the rectal swab of the patient with HUS. It was determined as O181:H4-, stx2-, and aggR-positive and belonged to the phylogenetic group B2. The complete genome assembly of the strain SCPM-O-B-9427 contained one chromosome and five plasmids, including the plasmid coding an aggregative adherence fimbriae I. MLST analysis showed that the strain SCPM-O-B-9427 belonged to ST678, and like E. coli O104:H4 strains, 2011C-3493 caused the German outbreak in 2011, and 2009EL-2050 was isolated in the Republic of Georgia in 2009. Comparison of three strains showed almost the same structure of their chromosomes: the plasmids pAA and the stx2a phages are very similar, but they have distinct sets of the plasmids and some unique regions in the chromosomes.

Draft Genome Sequence of Enterococcus mundtii SCPM-O-B-8398 (E28), Isolated from Fermented Milk in the Moscow Region, Russian Federation.

We report the draft genome sequence of the bacteriocin-producing Enterococcus mundtii strain SCPM-O-B-8398 (E28), which was isolated from fermented milk in the Moscow region, Russian Federation.

Identification of IS1R and IS10R elements inserted into ompk36 porin gene of two multidrug-resistant Klebsiella pneumoniae hospital strains.

Hospital Klebsiella pneumoniae strains (n = 196) were collected in 2012-16 from the patients of a Moscow neurosurgical intensive care unit. Klebsiella pneumoniae strains were multidrug-resistant and carried beta-lactamase genes blaSHV (97.4% of strains), blaCTX-M (84.7%), blaTEM (56.1%), blaOXA-48-like (49.0%) and blaNDM-1 (one strain), class 1 integrons (43.4% of strains) and porin protein ompK36 gene (100% of strains). The ompK36 porin protein gene disruption by insertion sequence (IS) elements and OmpK36 production loss in two strains were detected in this study. Outer membrane proteins were isolated according to Carlone et al. (Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 1986;24:330-2). The IS10R element belonging to the IS4 family, IS10 group was detected at the position of the 41st nucleotide of the ompK36 gene in K. pneumoniae strain KPB-2304K/15 (the first report for a certain IS element in K. pneumoniae). The IS1R element belonging to the IS1 family was identified at the position of the 86th nucleotide of the ompK36 gene in the K. pneumoniae strain KPB-367K/15 (novel insertion site for IS1 element into ompK36 gene). DNA transfer of the intact ompK36 gene into the strain KPB-367K/15 by vector plasmid restored OmpK36 porin protein production and resulted in a decrease of imipenem minimal inhibitory concentration. Such data confirm the importance of IS elements in ongoing multidrug-resistant evolution in hospital Klebsiella.

The spread of bla OXA-48 and bla OXA-244 carbapenemase genes among Klebsiella pneumoniae, Proteus mirabilis and Enterobacter spp. isolated in Moscow, Russia.

BACKGROUND: The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a great problem of healthcare worldwide. Study of the spread for bla OXA-48-like genes coding epidemically significant carbapenemases among hospital pathogens is important for the regional and global epidemiology of antimicrobial resistance. METHODS: Antibacterial resistant isolates of Klebsiella pneumoniae (n = 95) from 54 patients, P. mirabilis (n = 32) from 20 patients, Enterobacter aerogenes (n = 6) from four patients, and Enterobacter cloacae (n = 4) from four patients were collected from January, 2013 to October, 2014 in neurosurgical intensive care unit (ICU) of the Burdenko Neurosurgery Institute, Moscow. Characteristics of the isolates were done using susceptibility tests, PCR detection of the resistance genes, genotyping, conjugation, DNA sequencing, and bioinformatic analysis. RESULTS: Major strains under study were multi drug resistant (MDR), resistant to three or more functional classes of drugs simultaneously-98.9 % K. pneumoniae, 100 % P. mirabilis, one E. aerogenes isolate, and one E. cloacae isolate. Molecular-genetic mechanism of MDR in K. pneumoniae and P. mirabilis isolates were based on carrying of epidemic extended-spectrum beta-lactamase bla CTX-M-15 gene (87.2 and 90.6 % accordingly), carbapenemase bla OXA-48-like gene (55.3 and 23.3 % accordingly), and class 1 (54.8 and 31.3 % accordingly) and class 2 (90.6 % P. mirabilis) integrons. The bla OXA-48-like-positive K. pneumoniae were collected during whole two-year surveillance period, while P. mirabilis and Enterobacter spp. carrying bla OXA-48-like genes were detected only after four and 18 months after the research start, respectively. The bla OXA-48-like gene acquisition was shown for P. mirabilis isolates collected from five patients and for E. cloacae isolate collected from one patient during their stay in the ICU, presumably from bla OXA-48-like-positive K. pneumoniae. The source of the bla OXA-244 gene acquired by E. aerogenes isolates and the time of this event were not recognized. CONCLUSIONS: The expanding of CPE in the surveyed ICU was associated with the spread of bla OXA-48 and bla OXA-244 carbapenemase genes documented not only among K. pneumoniae, well-known bacterial host for such genes, but among P. mirabilis, E. aerogenes, and E. cloacae.

Molecular Characterization of Enterotoxin-Producing Escherichia coli Collected in 2011-2012, Russia.

Enterotoxin-producing Escherichia coli (ETEC) are one of the main causative agents of diarrhea in children especially in developing countries and travel diarrhoea in adults. Pathogenic properties of ETEC associated with their ability to produce a heat-stable (ST) and/or heat-labile (LT) enterotoxins, as well as adhesins providing bacterial adhesion to intestinal epithelial cells. This study presents the molecular characterization of the ETEC isolates collected from the Central and Far-Eastern regions of Russia in 2011-2012. It was shown that all ETEC under study (n=18) had the heat-labile enterotoxin-coding operon elt, and had no the genes of the heat-stable enterotoxin operon est. DNA sequencing revealed two types of nucleotide exchanges in the eltB gene coding subunit B of LT in isolates collected from Cherepovets city (Central region, Russia) and Vladivostok city (Far-East region, Russia). Only one ETEC strain carried genes cfaA, cfaB, cfaC and cfaD coding adhesion factor CFA/I. Expression of LT in four ETEC isolates in the agglutination reaction was detected using a latex test-system. The isolates were assigned to serogroups O142 (n = 6), О6 (n = 4), О25 (n = 5), О26 (n = 2), and O115 (n = 1). Genotyping showed that they belonged to an earlier described sequence-type ST4 (n = 3) as well as to 11 novel sequence-types ST1043, ST1312, ST3697, ST3707, ST3708, ST3709, ST3710, ST3755, ST3756, ST3757 and ST4509. The ETEC isolates displayed different levels of antimicrobial resistance. Eight isolates were resistant to only one drug, three isolates-to two drugs, one isolate-to three drugs, two isolates-to four antibacterials, and only one isolate to each of the five, six and ten antibacterials simultaneously. Genetic determinants of the resistance to beta-lactams and other classes of antibacterials on the ETEC genomes were identified. There are blaTEM (n = 10), blaCTX-M-15 (n = 1), class 1 integron (n = 3) carrying resistance cassettes to aminoglycosides and sulphonamides dfrA17-aadA5 and dfrA12-orfF-aadA2. One isolate ETEC_Ef-6 was found to be a multidrug-resistant (MDR) pathogen that carried both the beta-lactamase gene and class 1 integron. These data suggest the circulation of ETEC in Russia. Further investigations are necessary to study the spread of the revealed ETEC sequence types (STs) and serotypes. Their role in the etiology of diarrhea should be also estimated.