A comparative analysis of the quality of the developed nutrient media, Baird-Parker dry agar base and Vogel-Johnson dry agar Base and foreign analogues, was done based on results of clinical trials. The tested media were qualified by the main biological parameters, such as sensitivity, growth rate, and differentiating and inhibiting properties. The evaluation of statistical reliability of the results of trials of clinical samples was evaluated taking into account the number of parallel studies and the number of matches of the results of studies conducted by different performers. 116 clinical samples of received by a laboratory of the testing laboratory center for research from hospital no.164 over the period of clinical trials were analyzed. 46 cultures of potential pathogens were isolated when culturing on test and control media: S. aureus -35; S. epidermidis-6; S. saprophyticus - 5. Lecithinase activity on the medium “Baird-Parker dry agar Base” and mannitol fermentation on the medium “Vogel-Johnson dry agar Base” in the preliminary phenotypic test allow the isolation and differentiation of clinical isolates of S. aureus from S. epidermidis, and S. saprophyticus.

This review presents various strategies to fight causative agents of infectious diseases. Species-specific programmable RNA-containing antibiotics open up new possibilities for creating next-generation of personalized drugs based on microbiome editing and can serve as a new tool for selective elimination of pathogenic bacterial species while keeping intact the rest of microbiota. Another promising approach in combating bacterial infections is genome editing using the CRISPR-Cas systems. Expanding knowledge on the molecular mechanisms of innate immunity has been actively used for developing new antimicrobials. However, obvious risks of using antibiotic adjuvants aimed at activation of the host immune system include development of the autoimmune response with subsequent organ damage. To avoid these risks, it is essential to elucidate action mechanisms of the specific ligands and signal molecules used as components of the hybrid antibiotics. Bacteriophage endolysins are also considered as effective antimicrobials against antibiotic-resistant bacteria, metabolically inactive persisters, and microbial biofilms. Despite significant advances in the design of implants with antibacterial properties, the problem of postoperative infections still remains. Different nanomodifications of the implant surface have been designed to reduce bacterial contamination. Here, we review bactericidal, fungicidal, and immunomodulating properties of compounds used for the implant surface nanomodifications, such as silver, boron nitride nanomaterials, nanofibers, and nanogalvanic materials.

Clinical strains of Staphylococcus aureus were tested for hemolytic activity on blood agar, in the PCR test and by analyzing the gene alleles of hemolytic toxins. The study analyzed the information content of the phenotypic determination of hemolytic activity to assess the pathogenic properties of S. aureus isolates.

ABSTRACT We report here the draft genome sequences of eight Staphylococcus aureus strains isolated during three large food poisoning outbreaks in the Russian Federation. The strains were collected from clinical specimens and various foodstuff samples.

Staphylococcus aureus clonal complex (СС) 30 are associated with hospital-acquired and community-associated invasive infections and may cause outbreaks of staphylococcal food-borne infections (SFI). In recent years, severe SFI outbreaks caused by S. aureus CC30 in cohorts not linked to high-risk groups have been detected in Russia.Aim: The aim of the study is to conduct a comparative genomic analysis of S. aureus strains B-7778 and B-7779 isolated during widespread SFI outbreak at the International Youth Forum Seliger in 2014, and S. aureus strains B-7738 and B-7739 isolated during widespread SFI outbreak among construction personnel in Saint Petersburg in 2013.Methods: Seliger-2014 S. aureus cultures were screened by PCR and sequence typing. S. aureus strains B-7778 and B-7779 were isolated from clinic material and from food handlers, respectively. Draft genome sequencing and phylogenetic analysis of S. aureus strains B-7778 and B-7779 were carried out. The production of enterotoxin A was determined by the enzyme immunoassay.Results: S. aureus strain B-7778 isolated from 38 patients and S. aureus strain B-7779 isolated from two food handlers at the Forum Seliger-2014 have identical nucleotide sequences, belong to spa-type t122 and sequence-type 30, and carry a set of toxin genes being responsible for SFI manifestations. The core-genome SNP typing has shown that S. aureus B-7738/ B-7739 (St. Petersburg, 2013) and S. aureus B-7778/ B-7779 (Seliger, 2014) belong to different clusters of S. aureus СС30 clade 3. S. aureus B-7778/ B-7779 not closely related with major clusters of S. aureus СС30. The production of enterotoxin A, SFI etiological factor, by S. aureus strains B-7738, B-7739, B-7778, and B-7779 has been confirmed.Conclusion: The genomic analysis of SFI-associated S. aureus strains isolated in Russia has been conducted for the first time. Two different genetic clones of S. aureus СС30 which are able to cause severe SFI outbreaks in cohorts not linked to high-risk groups have been identified and characterized. SNP typing of Seliger-2014 S. aureus genomes has revealed their genetic specificity among known strains of S. aureus CC30. Identified genome sequences of SFI-associated strains will be used for further studies S. aureus clones circulating through the food chain in Russia.

ABSTRACT Staphylococcus aureus clonal complex 8 (CC8) has not been associated with staphylococcal scalded-skin syndrome (SSSS) in newborns and exfoliative toxin genes. Here, we report the draft genome sequences of exfoliative toxin A-producing B-7772, B-7777 (both CC8), and B-7774 (CC15) strains associated with SSSS in newborns.

Increases in the prevalence of antibiotic-resistant strains of Staphylococcus aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twort endolysin (PlyTW) harbors three domains, a cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), an amidase-2 domain and a SH3b-5 cell wall binding domain (CBD). Our results indicate that the CHAP domain alone is necessary and sufficient for lysis of live S. aureus, while the amidase-2 domain is insufficient for cell lysis when provided alone. Loss of the CBD results in ∼10X reduction of enzymatic activity in both turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW Δ172-373) with lytic activity that exceeded the activity of the full length construct in both the turbidity reduction and plate lysis assays. Addition of Ca(2+) enhanced the turbidity reduction activity of both the full length protein and truncation constructs harboring the CHAP domain. Chelation by addition of EDTA or the addition of zinc inhibited the activity of all PlyTW constructs.

BACKGROUND:Staphylococcus aureus is one of the most important human pathogens and causes over 100 nosologicalforms of diseases. The lack of data on the spread of S. aureus genetic types specific for different forms of staphylococcal infections in Russia makes it difficult to timely identify and control strains of this epidemiologically dangerous bacterial pathogen.OBJECTIVE:The aim of the study was to carry out a molecular genetic research of S. aureus isolates obtained during a widespread foodborne illness outbreak among builders at the Pulkovo airport in St. Petersburg in 2013.METHODS:The ability of the isolates to produce staphylococcal enterotoxins was studied by immunoenzyme techniques. Gene typing was carried out by sequence-specific primer-based PCR, as well as by sequencing genomic nucleotide sequences of two independent isolates of the pathogen.RESULTS:An enterotoxin A gene in genomes of S. aureus isolates etiologically associated with the outbreak was identified. The production of enterotoxin A by the isolates was shown. According to the complex analysis all isolates producing staphylococcal enterotoxins were identical and constituted the S. aureus strain, sequence-type ST30 and spa-type t2509. The genome of the identified S. aureus strain carried a set of various staphylococcal toxins. The full genome sequence among other techniques revealed high levels of similarity between genomes of the strain under study and well-known reference strain S aureus MRSA 252.CONCLUSION:The complete molecular genetic study of the S. aureus strain involved into the widespread foodborne illness outbreak was first carried out in Russia, allowing of further using the strain as a Russian reference strain to study potential epidemic outbreaks in the Russian Federation.

ABSTRACT The novel β-lactamase gene bla CTX-M-116 was identified in a Proteus mirabilis nosocomial isolate recovered from the urine of a patient in Moscow in 2005. DNA sequence analysis showed bla CTX-M-116 to be a hybrid gene consisting of 5′ bla CTX-M-23 (nucleotides 1 to 278) and 3′ bla CTX-M-22 (nucleotides 286 to 876) moieties separated by an intervening putative site of recombination (GTTAAAT). A retrospective analysis of available bla CTX-M genes in the GenBank database revealed 19 bla CTX-M genes that display the same hybrid structure.

Staphylococcus aureus is a notorious pathogen highly successful at developing resistance to virtually all antibiotics to which it is exposed. Staphylococcal phage 2638A endolysin is a peptidoglycan hydrolase that is lytic for S. aureus when exposed externally, making it a new candidate antimicrobial. It shares a common protein organization with more than 40 other reported staphylococcal peptidoglycan hydrolases. There is an N-terminal M23 peptidase domain, a mid-protein amidase 2 domain ( N -acetylmuramoyl-L-alanine amidase), and a C-terminal SH3b cell wall-binding domain. It is the first phage endolysin reported with a secondary translational start site in the inter-lytic-domain region between the peptidase and amidase domains. Deletion analysis indicates that the amidase domain confers most of the lytic activity and requires the full SH3b domain for maximal activity. Although it is common for one domain to demonstrate a dominant activity over the other, the 2638A endolysin is the first in this class of proteins to have a high-activity amidase domain (dominant over the N-terminal peptidase domain). The high activity amidase domain is an important finding in the quest for high-activity staphylolytic domains targeting novel peptidoglycan bonds.

Staphylococcus aureus is an important pathogen, with methicillin-resistant (MRSA) and multi-drug resistant strains becoming increasingly prevalent in both human and veterinary clinics. S. aureus causing bovine mastitis yields high annual losses to the dairy industry. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a promising new source of antimicrobials. The endolysin of prophage ΦSH2 of Staphylococcus haemolyticus strain JCSC1435 (ΦSH2 lysin) is a peptidoglycan hydrolase consisting of two catalytic domains (CHAP and amidase) and an SH3b cell wall binding domain. In this work, we demonstrated its lytic activity against live staphylococcal cells and investigated the contribution of each functional module to bacterial lysis by testing a series of deletion constructs in zymograms and turbidity reduction assays. The CHAP domain exhibited three-fold higher activity than the full length protein and optimum activity in physiological saline. This activity was further enhanced by the presence of bivalent calcium ions. The SH3b domain was shown to be required for full activity of the complete ΦSH2 lysin. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and CoNS.

An algorithm for step-by-step identification of bla CTX-M genes determining the resistance to cephalosporins III–IV in gram-negative bacteria is developed. The algorithm allows identification of 49 genes out of 96, which are already present in the GenBank database. The remaining 47 genes are identified in several small groups of 2 to 6 genes, excluding the bla CTX-M-14-like subgroup, which comprises 13 genes. The bla CTX-M genes’ identification is carried out with two-step restriction fragment length polymorphism analysis (PCR-RFLP) of a 544-bp-long PCR-product.

The study showed that bla(CTX-M) genes were present in the genomes of 71% of cephalosporin resistant Enterobacteriaceae nosocomial isolates (n=833) collected in Russian hospitals within 2003-2007, including 91% of E.coli, 90% of Klebsiella spp., 38% of Enterobacter spp., 31% of Citrobacter spp. (n=9), and 36% of the other Enterobacteriaceae species. The genes belonging to the following subtypes (clusters) were identified: bla(CTX-M-1) (529 bla(CTX-M-15) genes; 25 bla(CTX-M-3) genes; 1 bla(CTX-M-22) gene, 1 bla(CTX-M-23) gene, and 1 bla(CTX-M-34) gene); bla(CTX-M-2) (1 bla(CTX-M-2) gene, and 4 bla(CTX-M-5) genes), and bla(CTX-M-9) (2 bla(CTX-M-9) genes, and 28 bla(CTX-M-14) genes). It was shown that bla(CTX-M) genes were located on high-molecular weight (60-160 bp) conjugative plasmids belonging mainly to the incompatibility groups IncF, IncL/M and IncA/C (bla(CTX-M-15) gene); IncL/M(bla(CTX-M-3) gene); and IncF, IncL/Mand IncI1-ly (CTX-M-14 gene). The gene environments of bla(CTX-M) genes were shown specific for the subtype of the genes. A mobile genetic element ISEcp1 (in some cases deleted or inserted by IS26, IS1, IS10, resTn2, or resTn3 sequences, in direct or reverse position) were detected upstream of bla(CTX-M-3), bla(CTX-M-14), and bla(CTX-M-15) genes. A special characteristic was the sequence between ISEcp1 and bla(CTX-M) gene: 48 bp for bla(CTX-M-15) (except 1 E.coli isolate having such a sequence deleted by 3 bp); 127 bp for bla(CTX-M-3); 42 bp for bla(CTX-M-14). Downstream of bla(CTX-M) and bla(CTX-M-15) genes in the major bacterial isolates orf477 mucA and Delta orf477-Delta mucA sequences were detected respectively. Two isolates had additional Delta orf3 insertion inside of Delta orf477-Delta mucA sequence. Insertion sequence IS903 (intact or deleted) was detected downstream of bla(CTX-M-14) gene. Unlike the others, bla(CTX-M-2) and bla(CTX-M-9) genes were located inside of ISCR1 mobile element, downstream of class 1 integron and orf513 sequence.

Nosocomial bacterial isolates collected within 2003-2004 (n=411) and 2005-2007 (n=422) were highly resistant to cephalosporins III-IV and antibacterials of other groups (aminoglycosides, fluoroquinolons, chloramphenicol, and co-trimoxazole). Genes encoding TEM, SHV, CTX-M, OXA-2, and AmpC types of beta-lactamases (BLs) in the E. coli, Klebsiella spp., and Enterobacter spp. isolates were detected using polymerase chain reaction (PCR). Prevalent CTX-M-type BLs were detected in 85% of the E. coli, 87% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the first strain collection and in 94% of the E. coli, 91% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the second one. Genes belonging to three subtypes of blacTx-M genes were identified: bla(CTX-M-1) (228 bla(CTX-M-15) and six bla(CTX-M-3) of the first strain collection; 275 bla(CTX-M-15), three bla(CTX-M-3), and one bla(CTX-M-22) of the second one), bla(CTX-M-2) (one bla(CTX-M-5) of the first strain collection and one bla(CTX-M-2) of the second one), bla(CTX-M-9) (17 bla(CTX-M-14) and one bla(CTX-M-9) of the first strain collection; seven bla(CTX-M-14) and one bla(CTX-M-9) of the second one). Three isolates of the first strain collection and one isolate of the second one carried two genes belonging to two different subtypes, i.e., bla(CTX-M-15) and bla(CTX-M-14) simultaneously. The bacterial isolates had high levels of associative resistance to ciprofloxacin, co-trimoxazole, gentamicin, amikacin, and chloramphenicol associated with the resistance gene cassettes aadA1, aadA2, aadA5, aadB, aacA4, aac(6')Ib; dfrA1, dfrA5, dfrA12, dfrA17, cmlA1, ereA2, and catB8 in the class 1 integrons and the resistance gene cassettes dfrA1, sat1, and aadA1 in the class 2 integrons.