Ul Mira 14, Saint Petersburg 197101, Russia 6 St. Petersburg Hospital No. 31, Pr. Dinamo 3, Saint Petersburg

The spread of carbapenemase-producing Enterobacteriaceae is a global problem; however, no exact data on the epidemiology of carbapenemase in the Baltic countries and St. Petersburg area is available. We aimed to evaluate the epidemiology of carbapenemaseproducing Escherichia coli and Klebsiella pneumoniae in the Baltic States and St. Petersburg, Russia, and to compare the different methods for carbapenemase detection. From January to May 2012, all K. pneumoniae ( = 1983) and E. coli ( = 7774) clinical isolates from 20 institutions in Estonia, Latvia, Lithuania, and St. Petersburg, Russia were screened for carbapenem susceptibility. The IMP, VIM, GIM, NDM, KPC, and OXA-48 genes were detected using real-time PCR and the ability to hydrolyze ertapenem was determined using MALDI-TOF MS. Seventy-seven strains were found to be carbapenem nonsusceptible. From these, 15 K. pneumoniae strains hydrolyzed ertapenem and carried the NDM gene. All of these strains carried integron 1 and most carried integron 3 as well as genes of the CTX-M-1 group. No carbapenemase-producing E. coli or K. pneumoniae strains were found in Estonia, Latvia, or Lithuania; however, NDM-positive K. pneumoniae was present in the hospital in St. Petersburg, Russia. A MALDI-TOF MS-based assay is a suitable and cost-effective method for the initial confirmation of carbapenemase production

Soole piimhappebakterite iseloomustus bakterioloogiliste, biokeemiliste ja molekulaarsete meetoditega

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Piimhappebakterid (lactic acid bacteria, LAB), sealhulgas Lactobacillus spp. ja Bifidobacterium spp., kuuluvad inimese kasuliku mikrobioota hulka, säilitades seedekulgla mikroobikoosluste tasakaalu. Samal ajal kutsuvad nad oma rakkude ja ainevahetusproduktidega organismis esile üldefekte, mis võivad mõjustada inimese tervist. Seedekulgla piimhappebakterite liigilist jaotumist ja metaboliite pole erinevatesse eagruppidesse kuuluvatel isikutel, erinevates geograafilistes piirkondades elavatel lastel ning allergilistel ja mitteallergilistel lastel uuritud. Bakterite metaboolset aktiivsust hinnatakse tavaliselt bakterioloogiliste ja biokeemiliste metoodikate abil. Seevastu molekulaarsete uurimismeetoditega saadakse täpsemaid tulemusi. Senini pole aga rakendatud kombineeritud lähenemist, et mõista mikroobide mõju inimese ainevahetusele. Uurimistöö ülesandeks oli iseloomustada bakterioloogiliste, biokeemiliste ja molekulaarsete meetodite abil soolestiku piimhappebakterite liigilist koostist ja metaboolset toimet erineva vanuse, elukoha ja tervisliku seisundiga isikutel. Uurimisalusteks olid 79 Eesti ja 65 Rootsi 1–2 aastast last, 40 last vanuses 5aastat (20 allergilist ja 20 mitteallergilist), 24 tervet täiskasvanut ja 37 eakat isikut. Kokku uuriti 133 laktobatsilli tüve: 35 juhuslikult valitud seedetrakti tüve , 76 Eesti ja Rootsi laste seedetraktist isoleeritud tüve, 21 referentstüve ja üht L. plantarum Inducia DSM 21379 tüve. Isoleeritud laktobatsillide tüved samastati liigi tasemel feno- ja genotüübiliste meetoditega (API 50CHL ja ITS-PCR). Laktobatsillide metaboliite määrati gaaskromatograafiaga: orgaanilisi rasvhappeid (äädik-, piim-, ja merevaikhape) ja etanooli erinevates keskkonnatingimustes ning polü-ja biogeenseid amiine erinevate aminohapete keskkonnas, inimese uriinis ja probiootilises juustus. Inimeste rooja analüüsides määrati laktobatsillide olemasolu PCR meetodiga ja hulk FISH ning real-time PCR meetoditega. Allergiliste ja mitteallergiliste laste bifidobakterite liikide määramiseks kasutati liigi-spetsiifilist PCR-DGGE meetodit koos järgneva kloneerimise ja sekveneerimisega. L. plantarum Inducia DSM 21379 tüve iseloomustati fenotüübilise laktobatsillide API 50CHL samastamiskitiga, molekulaarselt ITS-PCR ja 16S rRNA sekveneerimise ning metaboliite gaaskromatograafiaga. Täiskasvanute ja eakate kehamassi indeks arvutati kehakaalu (kg) ja pikkuse (cm) ruudu suhte alusel. Vereplasma glükoosisisaldus (mmol/L) määrati laboratoorsete standardmeetoditega. Selgus, et erinevates biotehnoloogilistes rakendustes kasutatavate laktobatsillide omaduste kirjeldamiseks sobivad fenotüübilised meetodid, mis põhinevad bakterite ainevahetusel, nagu biokeemilise profiili kindlakstegemine API 50CHL-ga ja metaboliitide määramine gaaskromatograafiaga. Laktobatsillide liikide, eelkõige Lactobacillus acidophilus gruppi kuuluvate bakterite määramiseks on molekulaarsed meetodid nagu ITS-PCR ja 16S rRNA sekveneerimine võrreldes API 50CHLga selgelt eelistatavamad. Bifidobacterium spp. hulga määramiseks imikutel on FISH meetod märkimisväärselt kõrgema tundlikkusega kui bakterioloogiline meetod. Kuid Lactobacillus spp. hulga määramisel nii lastel kui täiskasvanutel ühtib bakterioloogilise ja FISH meetodi kõrge tundlikkus. Erinevates vanuserühmades on laktobatsillide hulgad FISH ja real-time PCR meetoditega määramisel kõrgemad võrreldes bakterioloogiliste meetoditega. Erinevate inimeste soole indigeensete laktobatsillide hulkade ja liikide mitmekesisus on seotud erinevate geograafiliste piirkondadega ja inimese vanusega. Bakterioloogiliselt määratud Lactobacillus spp. kõrgemad hulgad seedekulglas seostuvad täiskasvanutel ja eakatel isikutel kõrgema kehamassi indeksi ja vere glükoosi sisaldusega, kusjuures erinevad laktobatsillide liigid seostuvad erinevalt glükoosi ainevahetusega. Molekulaarsete metoodikatega kinnitusid 5 aastastel lastel eelnevate bakterioloogiliste uuringute tulemused soole bifidobakterite levimuse ja allergia seoste kohta allergilistel ja mitteallergilistel imikutel. Allergilistel lastel prevaleerib seedekulgla bifidobakterite perekonnas B. adolescentis, samas kui mitte-allergilistel lastel on sagedasem B. catenulatum/pseudocatenulatum. Probiootikumina patenteeritud tüvi Lactobacillus plantarum Inducia DSM 21379 samastati fenotüpeerimis- (API 50CHL) ja genotüpeerimismeetoditega (ITS-PCR, PFGE), millele lisandus 16S rRNA sekveneerimine. Need uurimismeetodid on heaks võimaluseks laktobatsillide samastamisel ja tüpiseerimisel bioloogilisest materjalist ja probiootilistest toodetest. Putrestsiini märkimisväärne hulk söötmes ja Inducia juustus on vastavuses laktobatsilli tüve Inducia poolse putrestsiini metabolismiga tarbija organismis. Saadud tulemused ja kogutud laktobatsillide tüved pakuvad edaspidiseks võimalusi uute probiootiliste toodete väljatöötamiseks.Lactic acid bacteria (LAB), included Lactobacillus spp. and Bifidobacterium spp., are considered to be members of beneficial microbiota that is important in maintaining a healthy, balanced microbial community in the GI-tract. LAB is proposed to induce systemic effects that may improve host’s health by their cells and metabolites. The species composition of LAB and metabolites of lower parts of GI tract are still not well elaborated in different age groups and some environmental bound diseases like allergy. Usually, metabolic impact has been assessed by phenotypic and biochemical analysis. However, molecular analysis provides more precise data. There are still a few data on the combined approach for understanding the impact of microbes on host metabolism. The aim of present thesis was to apply different methods for estimation of phenotypic and genetic properties of LAB intestinal isolates for characterization of the intestinal LAB microbiota of humans at different age, geographical location and health status. Altogether 79 Estonian and 65 Swedish infants, 40 children of 5-years age (20 allergic, 20 non-allergic), 24 healthy adults and 37 elderly persons were recruited in this study. The number of Lactobacillus sp. strains investigated was 133, incl. 35 randomly collected intestinal strains, 76 intestinal strains of Estonian and Swedish children 1-2yrs , 21 reference strains and 1 strain L. plantarum Inducia DSM 21379. Lactobacilli isolates have been typed using API CHL50 kit and ITS-PCR with species-specific primers. LAB metabolites were assessed by gas chromatography (GC): SCFA (acetic, lactic, succinic acids) and ethanol in MRS medium in different environments; poly- and biogenic amines in media with different precursor amino acids (arginine; glutamine; lysine; ornitine; histidine); in human urine samples and in the probiotic cheese.The counts of LAB in human fecal samples were detected by bacteriological (specific media) and molecular (FISH, real-time PCR) methods. The Bifidobacterium genus-specific PCR in combination with DGGE, cloning and sequencing was used to detect bifidobacterial communities in allergic and non-allergic children. L. plantarum Inducia DSM 21379 has been characterized by using ITS-PCR, PFGE, 16S rRNA sequencing and the metabolites by GC methods. Body mass index (BMI) in adults and elderly was calculated as weight (kg)/height square meters. Plasma glucose (mmol/L) was determined by standard laboratory methods. It was assessed that the phenotypic methods used including culture on artifical media with subsequent biochemical profiling by API 50CHL and GC estimation of organic acids and ethanol are appropriate for estimation of the comparative Lactobacillus species composition for biotechnological applications. The molecular methods such as ITS-PCR, controlled by 16S rRNA sequencing are clearly superior to API 50CHL in species identification, particularly for the Lactobacillus acidophilus group. For quantitative estimation of Bifidobacterium spp. of infants the FISH method has remarkably higher sensitivity in comparison with bacteriological methods. At the same time, for Lactobacillus spp. the concordance between bacteriology and FISH methods is high both in infants and adults. In comparison with bacteriological data, the higher counts of lactobacilli can be detected in different age groups using FISH and RT-PCR. The composition of intestinal indigenous lactobacilli is characterised with large individual variety both in counts, number and species composition depending on the geographical location and age. The faecal microbiota of elderly people in comparison with adults express higher total counts of lactobacilli. Both in adults and the elderly the higher counts of cultivable intestinal Lactobacillus spp. can be associated with higher BMI and blood glucose content while particular species. are differentially associated with glucose metabolism. The previously bacteriologically assessed association between the shifts of prevalence of culturable bifidobacteria in infants and the development of allergy was confirmed with molecular methods in children at 5 years of age. Particularly, in allergic children, B. adolescentis is prevalent among intestinal Bifidobacterium spp., whereas in non-allergic children B. catenulatum/pseudocatenulatum is prevalent. Elaborated into probiotics and patented, the L. plantarum Inducia DSM 21379 strain was identified using phenotypic (API 50CHL) and genotypic (ITSPCR, PFGE) methods followed by 16S rRNA sequencing. These methods serve as useful tools for strain identification and typing of lactobacilli from biological material and probiotic products. The concordance between the high proportions of putrescine amounts produced by strain L. plantarum Inducia in decarboxylation media, Inducia cheese and urine of its consumer was detected. The obtained knowledge and collected large pool of well characterized Lactobacillus sp. strains can open new possibilities to elaborate novel probiotics in future

The Prevalence of Helicobacter pylori in Estonian Bariatric Surgery Patients

Helicobacter pylori (Hp) is one of the most important human pathogens that can cause duodenal and gastric ulcers, gastritis and stomach cancer. Hp infection is considered to be a cause of limiting access to bariatric surgery. The aim of this study was to determine the prevalence of Hp in patients with obesity going into bariatric surgery and to reveal the relationship between Hp and clinical data. The study group was formed of 68 preoperative bariatric surgery patients (body mass index (BMI) 44.7 ± 4.8). Gastric biopsies (antrum and corpus) were used for histological and molecular (caqA and glmM genes) examinations. The PCR method revealed Hp infection in 64.7% of obese patients that is higher in comparison with histological analysis (55.9%). The prevalence of cagA and glmM genes in antrum mucosa was 45.6% and 47.0% while in the corpus it was 41.2% and 38.3%, respectively. The coincidence of both cagA and glmM virulence genes in the antrum and corpus mucosa was 33.8% and 22.1%, respectively. Either of the genes was found in 58.8% of antrum and 57.3% of corpus mucosa. Presence of caqA and glmM genes was in association with active and atrophic chronic gastritis. In conclusion, our study demonstrated that two thirds of morbidly obese patients undergoing bariatric surgery are infected with Hp and have a high prevalence of cagA and glmM virulence genes that points out the necessity for diagnostics and treatment of this infection before surgery

Coryneform bacteria in human semen: inter-assay variability in species composition detection and biofilm production ability

Background: Coryneform bacteria constitute an important segment of male urogenital microbiota. They have been generally considered as saprophytes, although some species have been associated with prostatitis as well. At the same time, biofilm infections have been suspected as a cause of prostatitis. Objective: To identify a set of coryneform bacteria isolated from semen of either healthy men or prostatitis patients applying different methods to reveal inter-assay variability and to determine their ability of adhesion and biofilm production. Design: Coryneform bacteria were identified by API Coryne 2.0 biochemical identification system and 16S rDNA sequencing using different primer sets. Quantitative assessment of biofilm production was performed using crystal violet binding assay method. Results: The most common species were Corynebacterium seminale, C. minutissimum, and Dermabacter hominis. Altogether 14 species and related genera were found. We observed the best inter-assay agreement when identifying C. seminale. Biofilm was observed in 7 out of 24 strains. The biofilm-producing strains belonged to Arthrobacter cumminsii, Dermabacter hominis, C. minutissimum, and Actinomyces neuii. No differences were found between the strains originating from prostatitis patients and healthy men. Dermabacter hominis strains were more potent biofilm producers than C. seminale strains (p=0.048). Conclusions: We can conclude that a wide variety of coryneform bacteria can be found from the male genital tract, although their exact identification is problematic due to insufficient representation in databases. Nearly one third of the strains are able to form biofilm that may give them an advantage for surviving several host- and treatment-related conditions

Different Patterns of Virulence Genes in Streptococcus mutans and Streptococcus sobrinus Originating from Estonian Toddlers—Mothers Cohort

Aims: Mutans streptococci include Streptococcus mutans and Streptococcus sobrinus, which can cause tooth decay. The current study aimed to compare their virulence genes with each other and to correlate them with the clinical data of patients. Materials and methods: Altogether 21 S. mutans and 19 S. sobrinus strains were investigated, originating from 24 children (age 2.7 ± 0.4 years) and 13 mothers (27.3 ± 3.7). The PCR method was applied to detect 11 virulence genes. Caries indices (dmf, decayed/missing/filled; DMFT, decayed/missing/filled teeth) and SM score (Mutans streptococci amount in saliva) were recorded. Results: Most of the S. mutans strains harbored all the virulence genes studied, while S. sobrinus had significantly fewer genes. The genes gbpA, gbpB, wapA and ftf were present in all isolates of S. sobrinus, the spaP, gtfB, vicR, SMU.1037c and SMU.105 genes were present in 41–88% of the isolates, while gtfD and SMU.104 genes were absent in S. sobrinus strains studied. A positive correlation appeared between the biofilm-related vicR and polysaccharide-production-related gtfD genes. In contrast, another polysaccharide-production-related gtfB gene was present in some cases in strains lacking the vicR or gtfD gene. Positive association was found between the presence of adhesion-related spaP gene in pediatric-derived S. sobrinus strains and an increase in SM score. Conclusions: Differences exist between the two common species of mutans streptococci: strains of S. mutans have more virulence genes than that of S. sobrinus, both crucial and virulence enhancing. Deeper research is needed to clarify the mechanisms behind the increased cariogenicity in cohabitation

Different Patterns of Virulence Genes in <i>Streptococcus mutans</i> and <i>Streptococcus sobrinus</i> Originating from Estonian Toddlers—Mothers Cohort

Aims: Mutans streptococci include Streptococcus mutans and Streptococcus sobrinus, which can cause tooth decay. The current study aimed to compare their virulence genes with each other and to correlate them with the clinical data of patients. Materials and methods: Altogether 21 S. mutans and 19 S. sobrinus strains were investigated, originating from 24 children (age 2.7 ± 0.4 years) and 13 mothers (27.3 ± 3.7). The PCR method was applied to detect 11 virulence genes. Caries indices (dmf, decayed/missing/filled; DMFT, decayed/missing/filled teeth) and SM score (Mutans streptococci amount in saliva) were recorded. Results: Most of the S. mutans strains harbored all the virulence genes studied, while S. sobrinus had significantly fewer genes. The genes gbpA, gbpB, wapA and ftf were present in all isolates of S. sobrinus, the spaP, gtfB, vicR, SMU.1037c and SMU.105 genes were present in 41–88% of the isolates, while gtfD and SMU.104 genes were absent in S. sobrinus strains studied. A positive correlation appeared between the biofilm-related vicR and polysaccharide-production-related gtfD genes. In contrast, another polysaccharide-production-related gtfB gene was present in some cases in strains lacking the vicR or gtfD gene. Positive association was found between the presence of adhesion-related spaP gene in pediatric-derived S. sobrinus strains and an increase in SM score. Conclusions: Differences exist between the two common species of mutans streptococci: strains of S. mutans have more virulence genes than that of S. sobrinus, both crucial and virulence enhancing. Deeper research is needed to clarify the mechanisms behind the increased cariogenicity in cohabitation

Effect of early directed implementation of family-integrated care measures on colonisation with Enterobacteriaceae in preterm neonates in NICU

Background Hospital-acquired strains (HASs) and multiresistant strains in neonatal intensive care unit often harbour virulence and resistance mechanisms, carrying the risk of invasive infections. We describe colonisation with Enterobacteriaceae in neonates receiving early directed versus routine family-integrated care (FIC) within the first month of life.Methods A prospective cohort study included neonates with a gestational age below 34 weeks. During the first period, neonates were admitted to an open bay unit with transfer to the single-family room if available; feeding with the mother’s own breast milk (MOBM) was introduced within 24 hours, and skin-to-skin contact (SSC) within 5 days of life (the routine care group). During the second period, following a wash-in of 2 months, care in a single-family room within 48 hours, the introduction of MOBM within two and SSC in 48 hours were applied (the intervention group). Enterobacteriaceae isolated from neonatal stool, breast milk and parental skin swabs were genotyped, Simpson’s Index of Diversity (SID) calculated, and extended-spectrum beta-lactamases (ESBL) detected.Results In 64 neonate-parents’ groups, 176 Enterobacteriaceae, 87 in routine care and 89 in the intervention group were isolated; 26 vs 18 were HAS and one vs three ESBL positive, respectively. In the intervention group compared with the routine care group, SSC and MOBM feeding was started significantly earlier (p&lt;0.001); during the first week of life, time spent in SSC was longer (median hours per day 4.8 (4–5.1) vs 1.9 (1.4–2.6), p&lt;0.001) and the proportion of MOBM in enteral feeds was higher (median (IQR) 97.8% (95.1–100) vs 95.1% (87.2–97.4), p=0.011). Compared with the routine care group, the intervention group had higher SID and a reduction of HAS by 33.1% (95% CI 24.4% to 42.4%) in time series analysis.Conclusions Early implementation of FIC measures may hold the potential to increase diversity and reduce colonisation with HAS Enterobacteriaceae

Quantification of Clostridium difficile in Antibiotic-Associated-Diarrhea Patients▿

Comparing culture- and non-culture-based methods for quantifying Clostridium difficile in antibiotic-associated-diarrhea patients, we found that the real-time PCR method correlated well with quantitative culture and was more sensitive. A positive association between the population levels of C. difficile and the presence of its toxins was found

Detection of Carbapenemase-Producing Enterobacteriaceae in the Baltic Countries and St. Petersburg Area

The spread of carbapenemase-producing Enterobacteriaceae is a global problem; however, no exact data on the epidemiology of carbapenemase in the Baltic countries and St. Petersburg area is available. We aimed to evaluate the epidemiology of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Baltic States and St. Petersburg, Russia, and to compare the different methods for carbapenemase detection. From January to May 2012, all K. pneumoniae  n=1983 and E. coli  n=7774 clinical isolates from 20 institutions in Estonia, Latvia, Lithuania, and St. Petersburg, Russia were screened for carbapenem susceptibility. The IMP, VIM, GIM, NDM, KPC, and OXA-48 genes were detected using real-time PCR and the ability to hydrolyze ertapenem was determined using MALDI-TOF MS. Seventy-seven strains were found to be carbapenem nonsusceptible. From these, 15 K. pneumoniae strains hydrolyzed ertapenem and carried the blaNDM gene. All of these strains carried integron 1 and most carried integron 3 as well as genes of the CTX-M-1 group. No carbapenemase-producing E. coli or K. pneumoniae strains were found in Estonia, Latvia, or Lithuania; however, NDM-positive K. pneumoniae was present in the hospital in St. Petersburg, Russia. A MALDI-TOF MS-based assay is a suitable and cost-effective method for the initial confirmation of carbapenemase production