Specific Interaction of Novel Friunavirus Phages Encoding Tailspike Depolymerases with Corresponding Acinetobacter baumannii Capsular Types.

Acinetobacter baumannii is one of the most clinically important nosocomial pathogens. The World Health Organisation refers it to its «critical priority¼ category to develop new strategies for effective therapy. This microorganism is capable of producing structurally diverse capsular polysaccharides (CPSs), which serve as primary receptors for A. baumannii bacteriophages carrying polysaccharide-depolymerasing enzymes. In this study, eight novel bacterial viruses that specifically infect A. baumannii strains belonging to K2/K93, K32, K37, K44, K48, K87, K89 and K116 capsular types were isolated and characterized. The overall genomic architecture demonstrated that these viruses are representatives of the Friunavirus genus of the family Autographiviridae The linear double-stranded DNA phage genomes of 41,105-42,402 bp share high nucleotide sequence identity, except for genes encoding structural depolymerases or tailspikes which determine the host specificity. Deletion mutants lacking N-terminal domains of tailspike proteins were cloned, expressed and purified. The structurally defined CPSs of the phage bacterial hosts were cleaved with the specific recombinant depolymerases, and the resultant oligosaccharides that corresponded to monomers or/and dimers of the CPS repeats (K-units) were isolated. Structures of the derived oligosaccharides were established by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The data obtained showed that all depolymerases studied were glycosidases that cleave specifically the A. baumannii CPSs by the hydrolytic mechanism, in most cases, by the linkage between the K-units.IMPORTANCE Acinetobacter baumannii, a nonfermentative, Gram-negative, aerobic bacterium, is one of the most significant nosocomial pathogens. The pathogenicity of A. baumannii is based on the cooperative action of many factors, one of them being the production of capsular polysaccharides (CPSs) that surround bacterial cells with a thick protective layer. Polymorphism of the chromosomal capsule loci is responsible for the observed high structural diversity of the CPSs. In this study, we describe eight novel lytic phages which have different tailspike depolymerases (TSDs) determining the interaction of the viruses with corresponding A. baumannii capsular types (K-types). Moreover, we elucidate the structures of oligosaccharide products obtained by cleavage of the CPSs by the recombinant depolymerases. We believe that as the TSDs determine phage specificity, the diversity of their structures should be taken into consideration as selection criteria for inclusion of certain phage candidate to the cocktail designed to control A. baumannii with different K-types.

[Etiology of severe community - acquired pneumonia in adults: results of the first Russian multicenter study].

AIM: to study the etiology of severe community - acquired pneumonia (SCAP) in adults in Russian Federation. SCAP is distinguished by high mortality and socio - economic burden. Both etiology and antimicrobial resistance are essential for appropriate antibiotic choice. MATERIALS AND METHODS: A prospective cohort study recruited adults with confirmed diagnosis of SCAP admitted to multi - word hospitals of six Russian cities in 2014-2018. Etiology was confirmed by routine culture of blood, respiratory (sputum, endotracheal aspirate or bronchoalveolar lavage) and when appropriate, autopsy samples, urinary antigen tests (L. pneumophila serogroup 1, S. pneumoniae); real - time PCR for identification of "atypical" bacterial pathogens (M. pneumoniae, C. pneumoniae, L. pneumophila) and respiratory viruses (influenza viruses A and B, parainfluenza, human metapneumovirus, etc.) was applied. RESULTS: Altogether 109 patients (60.6% male; mean age 50.8±18.0 years old) with SCAP were enrolled. Etiological agent was identified in 65.1% of patients, S. pneumoniae, rhinovirus, S. aureus and K. pneumoniae were the most commonly isolated pathogens (found in 43.7, 15.5, 14.1 and 11.3% of patients with positive results of microbiological investigations, respectively). Bacteriemia was seen in 14.6% of patients and most commonly associated with S. pneumoniae. Co - infection with 2 or more causative agents was revealed in 36.6% of cases. Combination of bacterial pathogens (mainly S. pneumoniae with S. aureus or/and Enterobacterales) prevailed - 57.7% of cases; associations of bacteria and viruses were identified in 38.5% of patients, different viruses - in one case. CONCLUSION: S. pneumoniae was the most common pathogen in adults with SCAP. A high rate of respiratory viruses (mainly rhinovirus and influenza viruses) identification both as mixt infection with bacteria and mono - infection should be taken into account.

[Current state of antibiotic resistance of pathogens causing community-acquired urinary tract infections in Russia, Belarus and Kazakhstan: results of the international multicenter study Darmis-2018].

OBJECTIVE: To study in vitro activity of antimicrobials against clinical isolates from patients with community-acquired urinary tract infections (UTIs) in different regions of Russia, Belarus and Kazakhstan in 2017-2018. MATERIALS AND METHODS: A total of 1260 isolates collectedin the Russian Federation, Belarus and Kazakhstanas a part of the international multicenter prospective epidemiological study of the dynamics of antimicrobial resistance of uropathogens causing community-acquired urinary tract infections in different subsets of patients ("DARMIS-2018") were included in the analysis. 1124 strains represented the Enterobacterales order. Uropathogenswere isolated from children and adults of both sexes of all age groups with acute (and recurrences of chronic) community-acquired UTIs including pregnant women with asymptomatic bacteriuriain 34 centers of 26 cities of Russia, Kazakhstan and Belarus in 2017-2018. RESULTS: Enterobacterales jointly comprised a total of 89,2% of all isolated bacterial pathogens (88,9% in the adult subset; 89,3% in the subset of pregnant women and 91,4% in the subset of children and adolescents under 18). The most prevalent species were Escherichia coli (69,4% in the adult subset; 73,6% in the subset of pregnant women and 77,1% in the subset of children and adolescents under 18) and Klebsiella pneumoniae (11,5% in the subset of adults; 10,4% in the subset of pregnant women and 7,1% in the subset of children and adolescents under 18 years of age). The maximum activity against E. coli among oral drugs demonstrated fosfomycin (97,9% in the adult subset; 95,9% in the subset of pregnant women and 99,1% in the subset of children and adolescents under 18) and nitrofurantoin (97,9% in adult subset; 100% in the subset of pregnant women and 96,3% in the subset of children and adolescents under 18). Out of the parenteral drugs meropenem (out of carbapenems) and amikacin showed the highest activity (99,5% and 97,7% in the adult subset; 99,5% and 99,1% in the subset of pregnant women; 100% and 97,2% in the subset of children and adolescents under 18 years of age, respectively). Ampicillin, amoxicillin/clavulanic acid and trimethoprim/sulfamethoxazole demonstrated the lowest in vitro activity against Escherichia coli (less than 80% for everypatient subset). The susceptibility of E. colito ciprofloxacin was 60,4% in the adult subset; 80,0% in the subset of pregnant women and 80,6% in the subset of children and adolescents under 18. The rate of production of extended spectrum beta-lactamases among E. coli isolates based on the results of phenotypic tests was 21,9%. CONCLUSIONS: Results of this study indicate the increase of resistance of community-acquired isolates of Enterobacterales and in particular E. coli to the most of antimicrobials in Russia, Belarus and Kazakhstan.

C-reactive protein evaluation in communityacquired pneumonia with comorbid chronic heart failure as criterion of antibiotic prescription.

AIM: To prove that diagnostic algorithm based on additional measurement of serum C-reactive protein (CRP) for administration of systemic antibacterial therapy (ABT) to patients with suspected community-acquired pneumonia (CAP) and concomitant chronic heart failure (CHF) does not influence outcomes of disease. MATERIALS AND METHODS: This open, single-center, randomized, prospective, noninferiority study included 160 adult patients with documented functional class II-IV CHF who had been admitted with a preliminary diagnosis of non-severe CAP. Patients were randomized at 1:1 to two groups; group 1 - with additional measurement of CRP (n=80) and group 2 - with the use of routine diagnostic methods (n=80). In group 1, systemic ABT was administered only when serum CRP was >28.5 mg / l (threshold level of the biomarker calculated at the previous stage of the study); group 2 received a standard treatment. Noninferiority test result for both algorithms was evaluated by the number of patients with clinical success on days 12-14 (primary endpoint). Non-inferiority margin was δ=-13.5 %. In addition secondary endpoints (early clinical response on days 3-5; early in-hospital adverse events (development of complications; admission to intensive care unit (ICU); death), death, recurrent CAP or CHF worsening with readmission at 28 day; mortality at 90 and 180 days) were estimated. Standard statistical tools were used for all intergroup comparisons. RESULTS: 76 patients of each group reached the primary endpoint. Systemic ABT was administered to 51 (67.1 %) patients in group 1 and 76 (100 %) patients in group 2 (p<0.05). Both groups were comparable (p>0.05) regarding all endpoints: clinical success, 70 (92.1 %) vs. 69 (90.8 %), Δ=1.3 % (one-sided 97.5 % CI: - 8.25 % for non-inferiority margin δ=-13.5 %); early clinical response, 66 (86.8 %) vs. 68 (89.5 %); admission to ICU, 1 (1.3 %) vs. 1 (1.3 %); development of complications, 20 (26.3 %) vs. 22 (28.9 %); readmission, 5 (6.6 %) vs. 6 (7.9 %); in-hospital mortality, 2 (2.6 %) vs. 1 (1.3 %), mortality at 28 day, 3 (3.9 %) vs. 2 (2.6 %), at 90 day, 5 (6.6 %) vs. 4 (5.3 %), at 180 day, 8 (10.5 %) vs. 9 (11.8 %) cases, respectively. CONCLUSION: additional measurement of serum CRP in patients with CHF and suspected non-severe CAP was able to reduce rate of systemic ABT administration without outcomes and prognosis worsening.

[Surgical Site Infections after Open Appendectomy and Effectiveness of Complex Approach to Their Prevention].

AIM: To assess an incidence rate of surgical site infections (SSI) after open appendectomy and effectiveness of combined preventive measures (CPM). MATERIAL AND METHODS: This study was performed at three surgical departments of Smolensk hospitals. A total of 150 consecutive patients (50 at each department) hospitalized since January 2012 were included into the retrospective observation (period I). In order to perform prospective evaluation of CPM, a total of 66 consecutive patients (randomized 1:1) hospitalized since December 2012 (period II) were followed up at each of the departments. Antibiotic prophylaxis (AP) with IV amoxicillin/clavulanate (1.2 g) was planned for all patients from period II. The study group (group 1) included patients with surgical wound closure with triclosan-coated polyglactin 910 and additionally with a skin 2-octylcyanoacrylate-based adhesive. The control group (group 2) included patients with surgical wound closure with non-triclosan-coated polyglactin 910. Each patient from the period II was assigned to an "Individual SSI Prevention Package" (IPP), which included an antibiotic, sutures, skin adhesive (only in a package for CPM) and label "AP" for patients' medical records. Patients' medical records were reviewed by one expert. Exclusion criteria were: age <14 years; transition to midline laparotomy; drainage of the abdominal cavity through the surgical wound; simultaneous interventions; secondary appendicitis; refusal to use of sutures from the IPP. In order to determine signs of SSI presence/absence within 30 days after surgery, attempts to contact with patients by phone were made. The data obtained was recorded into case report forms and then entered into the study database. RESULTS: A total of 322 patients were included into the final analysis (mean age: 34.8±17.1 years). The mean length of hospital stay was 8.2±2.5 days. The mean duration of hospital stay with or without SSI was 7.9±1.8 and 14.2±4.0 days, respectively (p<0.001). The AP during the periods I and II was performed in 56.1% (83/148) and 97.7% (170/174) of patients, respectively (p<0.00001). Cephalosporins I-IV were the most frequently used antibiotics during the period I (85.6%). During the period II, amoxicillin/clavulanate from IPP was used in 98.2% of patients. Percentage of IV antibiotic administration in different time periods was 57.3% and 98.2%, respectively (p<0.0001); frequency of the first administration before skin incision was 53.6% and 97.1%, respectively (p<0.0001). The telephone contact with patient was successful in 74.8% (both periods), 56.8% (period I) and 90.2% (period II) of cases, respectively. SSI was recorded only once per patient with the following priority: SSI was documented in the patient's medical record; patient developed SSI that was not documented (in the expert's opinion) in the patient's medical record; SSI signs were determined during the telephone contact or reported by the patient. The incidence of SSI in both study periods, period I and period II was 14.9%, 15.5% and 14.4%, respectively (p>0.05 for all comparisons). In the patient subgroup with successful telephone contact, the incidence of SSI in both study periods, period I and period II was 17.4%, 21.4% and 15.3%, respectively; the incidence of SSI in group 1 and group 2 of the period II was 12.0% and 18.9%, respectively (p>0.05 for all comparisons). CONCLUSION: SSI after an open appendectomy remains an important problem. In order to determine a true incidence of SSI, it is necessary to improve the national nosocomial infection surveillance system. The CMP used in the study have showed a trend to significant SSI risk reduction and may be recommended to maximize patient protection. Further large studies are needed to confirm effectiveness of the proposed CMP.

[Systemic Antimicrobials Consumption and Expenditures in Departments of Surgery of Multi-Profile Hospitals in the Russian Federation and the Republic of Belarus: Results of Multicentre Pharmacoepidemiological Study].

The results of the systemic antimicrobials (AM) consumption and expenditures assessment in the departments of surgery of multi-profile hospitals in different regions of the Russian Federation and the Republic of Belarus in 2009-2010 based on retrospective collection and analysis of the data from the hospital expenditure notes using ATC/DDD methodology are presented. The average AM consumption and expenditure rates in the above mentioned departments varied from 24.9 DDD/100 bed-days to 61.7 DDD/100 bed-days depending on the department profile, with beta-lactams (cephalosporins and penicillins) share in the consumption being as high as 70-90%, followed by fluoroquinolones and aminoglycosides. Only 55-70% of the consumed AM belonged to the drugs of choice, whereas the improper AM consumption and expenditure rates amounted up to 10-18%. The study outputs can be used for the budget allocation and AM distribution improvement in the departments of surgery, as well as for the development and efficacy control of the local antimicrobial stewardship programs.

The microbiome of the maxillary sinus and middle nasal meatus in chronic rhinosinusitis.

AIM: This multicenter study was focused on the identification of the microorganisms inhabiting the maxillary sinus and middle nasal meatus in chronic rhinosinusitis. METHODOLOGY: 112 middle meatus swabs and 112 maxillary sinus aspirates from 103 patients were available for culture. RESULTS: A total of 244 strains of microorganisms representing more than 50 families were identified in the maxillary sinus and middle nasal meatus (164 and 80, respectively). These included 154 (63.0%) strains of aerobic bacteria from 32 species and 90 (37.0%) strains of anaerobic bacteria from 23 species. Aerobes were more common than anaerobes in both the nasal cavity (78.7% vs. 21.3%) and in the maxillary sinus (55.2% vs. 44.8%). Species of Streptococci (28.8%) and Prevotella (17.8%) were the most common findings in the maxillary sinus aspirates. S. pneumonia, H. influenza, and S. aureus were relatively rare, and found in only 6.7%, 5.4%, and 8.9% of the samples, respectively. CONCLUSIONS: The results obtained suggest that common upper airway pathogens do not play a major role in the pathogenesis of chronic rhinosinusitis. The microbiome of inflamed sinonasal mucosa is extremely diverse and involves exotic species of bacteria that, to date, have not been considered as potential inhabitants of the paranasal sinuses.

[Choice of antibacterial drugs in urinary infection].

A rise in efficacy of the treatment of acute infection affecting the lower urinary tract (LUTI) and prolongation of recurrence-free interval in chronic LUTI can be achieved only by an optimal antibacterial treatment. The study was made of 987 community-acquired strains of uropathogens from the patients living in 20 cities of the Russian Federation, Belarus and Kazakhstan (of them, 903 strains were from Russia). Enterobacteriaceae comprised 83.5%. E. coli infection of LUTI was found in 63.5% patients. The incidence of this infection was about the same both in uncomplicated and complicated cases (64.6 and 62.1%, respectively). Most active oral drugs against E.coli were phosphomycin (98.4%), furasidin (95.7%), nitrofurantoin (94.1%) and oralcefalosporins of the third generation (ceftibuten and cefixim). As to Enterobacteriaceae, only phosphomycin had activity against these bacteria above 90%, i.e. 91.5%. Furasidin and nitrofurantoin activity was 86.3 and 76.8%, respectively. From parenteral drugs, most active against E. coli were carbapenems (ertapenem, meropenem, imipenem. Strains resistant to them were not isolated. High in vitro activity was demonstrated also by cefoperason/sulbactam (97.4%), piperacillin/tasobactam (95.7%), cefalosporins of the third/fourth generation and amikacin (98.9%). Carbapenems were also highly active against Enterobacteroaceae. Empiric treatment of uncomplicated urinary infection should be performed with medicines which are not used for other indications.

[Russian results of the international epidemiological study of clinical aspects and resistance to antimicrobial medicines of cystitis uropathogens in females (ARESC): significance of empiric therapy].

A total of 656 female patients from Russia aged between 18 to 65 years with uncomplicated cystitis entered the international ARESC trial, of them 647 were eligible for final analysis. Positive cultural urine tests ( > 10(4) CFU/ml) were in 419 (64.7%) patients, 393 (93.8%) patients had monoinfection. The central laboratory of Genoa (to which the isolated samples were sent) has tested 416 uropathogens from 399 patients. The following pathogens were isolated: E. coli (72.6%), enterococcus (7.0%), Klebsiella pneumoniae (4.6%), Staphylococcus saprophyticus (3.6%), Proteus mirabilis (2.4%) and Staphylococcus aureus (1.7%). E. coli was most sensitive to phosphomycin (99.3%), mecillinam (97.3%), nitrofurantoin (94.7%), ciprofloxacin (87.4%). The lowest sensitivity was to ampicillin (42.1%) and cotrimoxasol (69.4%). As to the whole bacterial spectrum, the highest sensitivity was found to phosphomycin (96.5%), nitrofurantoin (85.6%) and citrofloxacin (82.8%), the less sensitivity--to ampicyllin (44.3%) and co-trimoxasol (70.1%). Phosphomycin, mecillinam (not registered in Russia) and nitrofurantoin showed activity in vitro and can be considered as drugs of choice for empiric therapy of cystitis. Because of high resistance of pathogens, co-trimoxasol (trimetoprim) and fluoroquinalones are not recommended as first-line treatment for uncomplicated cystitis in females.

Antimicrobial resistance of nasopharyngeal pneumococci from children from day-care centres and orphanages in Russia: results of a unique prospective multicentre study.

This study assessed the antimicrobial resistance of nasopharyngeal pneumococci isolated from children aged < 5 years in day-care centres and orphanages throughout Russia during 2001-2002. Swabs were collected from 2484 children in 43 day-care centres and eight orphanages in 11 cities of European Russia, and from 1669 children in 37 day-care centres and three orphanages in eight cities of Asian Russia, with a total of 1144 and 912 Streptococcus pneumoniae isolates being recovered in European and Asian Russia, respectively. All macrolide-non-susceptible (MICs 0.5-128 mg/L) and fluoroquinolone-non-susceptible (ciprofloxacin MICs > or = 4 mg/L) isolates were tested for resistance mechanisms and clonal relatedness. Non-susceptibility rates, by CLSI criteria, were 19.3%, 0.9% and 0.4% for penicillin G, cefotaxime and amoxycillin-clavulanate, respectively. Resistance to macrolides and lincosamides was also relatively low, i.e., < 7% for clindamycin and 14- and 15-membered macrolides. The highest rates of non-susceptibility were for tetracycline and co-trimoxazole (52.0% and 64.5%, respectively). No clones resistant to ciprofloxacin (MICs > or = 8 mg/L) were found, but 1.7% of isolates were non-susceptible (MIC 4 mg/L). No resistance was found to levofloxacin, gemifloxacin, telithromycin or vancomycin. Pulsed-field gel electrophoresis analysis showed no relationship between ciprofloxacin- and macrolide-non-susceptible isolates in European and Asian Russia. Resistance among macrolide-resistant isolates resulted mostly from the presence of erm(B) and mef(A), and from changes in L4; additionally, L22 mutations were common in isolates from Asian Russia. Non-susceptibility to quinolones was associated with mutations in parC and parE among European isolates. Asian Russian isolates had mutations in parC and gyrA, and alterations in parE were more common. There were substantial differences in non-susceptibility and mechanisms of resistance between pneumococci from Asian and European Russia, with orphanages appearing to be 'hot-spots' of resistance.

Immunochromatographic strip test for rapid detection of diphtheria toxin: description and multicenter evaluation in areas of low and high prevalence of diphtheria.

An immunochromatographic strip (ICS) test was developed for the detection of diphtheria toxin by using an equine polyclonal antibody as the capture antibody and colloidal gold-labeled monoclonal antibodies specific for fragment A of the diphtheria toxin molecule as the detection antibody. The ICS test has been fully optimized for the detection of toxin from bacterial cultures; the limit of detection was approximately 0.5 ng of diphtheria toxin per ml within 10 min. In a comparative study with 915 pure clinical isolates of Corynebacterium spp., the results of the ICS test were in complete agreement with those of the conventional Elek test. The ICS test was also evaluated for its ability to detect toxigenicity from clinical specimens (throat swabs) in two field studies conducted within areas of the former USSR where diphtheria is epidemic. Eight hundred fifty throat swabs were examined by conventional culture and by use of directly inoculated broth cultures for the ICS test. The results showed 99% concordance (848 of 850 specimens), and the sensitivity and specificity of the ICS test were 98% (95% confidence interval, 91 to 99%) and 99% (95% confidence interval, 99 to 100%), respectively.

Antimicrobial resistance of Streptococcus pneumoniae isolated from healthy children in day-care centers: results of a multicenter study in Russia.

BACKGROUND: It has been previously shown that study of susceptibility of nasopharyngeal isolates in healthy carriers can predict resistance in clinical isolates. The purpose of this multicenter study was to determine the carriage rate of Streptococcus pneumoniae in healthy children attending day-care centers in Moscow, Smolensk and Yartsevo, Russia, and in vitro activity of penicillin G, amoxicillin/clavulanate, cefaclor, erythromycin, roxithromycin, clarithromycin and trimethoprim-sulfamethoxazole (TMP-SMX) against representative isolates. METHODS: Included in this study were 305 pneumococcal isolates from 733 children attending 9 day-care centers in Moscow, Smolensk and Yartsevo. All children enrolled in this study were <7 years of age. MICs of selected antimicrobials were determined by Etest. Serotyping of selected pneumococcal isolates was done with pool and type antisera. RESULTS: The carriage rate of S. pneumoniae in the 3 centers varied from 44.9% to 66.0% (mean, 55.9%). Susceptibility testing was performed with 305 (74.4%) of 410 isolates. Only 23 (7.5%) of 305 pneumococcal isolates were penicillin-intermediate (range, 2.8 to 12.8%) with no penicillin-resistant strains. All tested pneumococci were susceptible to amoxicillin/clavulanate. Macrolides possessed comparable activity against S. pneumoniae, at 4.6% resistant strains for both erythromycin (range, 1.1 to 17.1%) and clarithromycin (range, 1.7 to 17.1%). The highest level of resistance was observed with TMP-SMX, 53.4% (range, 43.8 to 70.9%). Of 23 strains 20 (87.0%) with intermediate resistance to penicillin were serotyped. The most prevalent serotype was 14 (5 isolates), followed by serogroups 19 (4) and 23 (4). CONCLUSIONS: Resistance to penicillin, other beta-lactams and macrolides does not seem to be a problem for Russia now. The high level of resistance to TMP-SMX considerably restricts its usage for the treatment of pneumococcal infections.

[Comparison of the results of determining antibiotic sensitivity on AGV medium and on Mueller-Hinton and isosensitest agars]. / Sravnenie rezul'tatov opredeleniia chuvstvitel'nosti k antibiotikam na srede AGV i agarakh Miuller-Khinton i izosensitest.

To evaluate the adequacy of AGV agar for antimicrobial susceptibility testing, the susceptibility of a range of bacteria to 10 antimicrobials on AGV, Mueller-Hinton and isoSensitest agars, all supplemented with 5 per cent lyzed horse blood was determined. Disc tests were used. In general, AGV agar gave identical susceptibility results to Mueller-Hinton and isoSensitest agars for common gram positive and gram negative bacteria with most of the tested microbials excluding sulphonamides and trimethoprim. With those latter antimicrobials inhibition zones for susceptible organisms were not formed on AGV agar whereas large zones were present on Mueller-Hinton and isoSensitest agars. This discrepancy probably can be explained by the presence of high levels of thymidine in AGV agar; too high to be corrected even by the addition of 5 per cent lysed horse blood. AGV agar is possible to use for susceptibility testing with most of the microbials excluding trimethoprim and sulphonamides.