Etiology and microbiological diagnosis of nosocomial pneumonia in newborns]

A comparative analysis of the cases of ventilator-associated pneumonia (VAP) among premature infants in intensive care units and premature infant nurseries in 1994 (group I) and 1999 (group II) is presented. It was shown that the number of the cases of ventilator-associated pneumonia in the premature infants of group. I was 2,4 times higher than that in the group II (45.8 and 19.2 per cent respectively). A marked difference in the species pattern of the pathogens isolated from the endobronchial aspirate in 1994 and 1999 was observed. The species pattern of the isolates from the respiratory tract (Pseudomonas aeruginosa--40 per cent; Klebsiella pneumoniae--31 per cent; Staphylococcus epidermidis and Enterococcus--rare) showed that the pneumonia were nosocomial. The revealed similarity of the species patterns of the microflora in various parts of the respiratory tract and the throat posterior wall made it possible to consider the isolates of the throat posterior wall as a relative guide for confirming the etiological diagnosis of nosocomial pneumonia.     

Microflora of the mucous membrane of human tonsils in the normal state and in pathology

A total of 50 healthy persons and 50 chronic tonsillitis patients were examined with the use of the bacteriological method. The microflora of tonsillar mucous membrane was represented by 8 genera of facultative anaerobic microorganisms. In the biocenosis of the tonsillar mucosa of the healthy subjects the occurrence of Streptococcus was 1.5-fold greater than in that of the patients; microorganisms of the genus Staphylococcus prevailed on the tonsillar mucosa of chronic tonsillitis patients 1.3-fold prevailed more often and Klebsiella and Neisseria prevailed 4-fold more often. Among the microorganisms isolated from the patients, cultures simultaneously having two or more pathogenicity markers occurred twice as often as among the representatives of the microbiocenosis obtained from the healthy subjects. The microflora of the tonsillar mucosa of the chronic tonsillitis patients was characterized by resistance to penicillins, macrolides and aminoglygosides, but was simultaneously sensitive to lincosamides and glycopeptides.     

Administration of different Lactobacillus strains in fermented oatmeal soup: in vivo colonization of human intestinal mucosa and effect on the indigenous flora.

In vivo colonization by different Lactobacillus strains on human intestinal mucosa of healthy volunteers was studied together with the effect of Lactobacillus administration on different groups of indigenous bacteria. A total of 19 test strains were administered in fermented oatmeal soup containing 5 x 10(6) CFU of each strain per ml by using a dose of 100 ml of soup per day for 10 days. Biopsies were taken from both the upper jejunum and the rectum 1 day before administration was started and 1 and 11 days after administration was terminated. The administration significantly increased the Lactobacillus counts on the jejunum mucosa, and high levels remained 11 days after administration was terminated. The levels of streptococci increased by 10- to 100-fold in two persons, and the levels of sulfite-reducing clostridia in the jejunum decreased by 10- to 100-fold in three of the volunteers 1 day after administration was terminated. In recta, the anaerobic bacterium counts and the gram-negative anaerobic bacterium counts decreased significantly by the end of administration. Furthermore, a decrease in the number of members of the Enterobacteriaceae by 1,000-fold was observed on the rectal mucosa of two persons. Randomly picked Lactobacillus isolates were identified phenotypically by API 50CH tests and genotypically by the plasmid profiles of strains and by restriction endonuclease analysis of chromosomal DNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial colonization of an in vitro model of a tissue engineered human skin equivalent--a novel approach.

This was a preliminary investigation to define the conditions of colonization of a human skin equivalent (SE) model with cutaneous microorganisms. SEs of 24 mm diameter were constructed with a dermal matrix of fibrin containing fibroblasts and a stratified epidermis. Microbial colonization of the SEs was carried out in a dry environment, comparable to 'in vivo' skin, using a blotting technique to remove inoculation fluid. The microbial communities were sampled by scrub washing and viable cells enumerated on selective growth medium. Staphylococcus epidermidis, Propionibacterium acnes and Malassezia furfur (human skin commensals) and Staphylococcus aureus (transient pathogen) were colonized at inoculum densities of 10(2)-10(6) CFU SE(-1) on the surface of replicate SEs. Growth of all species was supported for upto 72-120 h, with recovery densities of between 10(4)-10(9) CFU SE(-1). A novel, real-time growth monitoring method was also developed, using S. aureus containing a lux cassette. Light output increased from 20 to 95 h, and colonization increased from 10(2) to 10(8) CFU SE(-1), as confirmed by conventional recovery. Thus, the SE model has potential to investigate interactions between resident and transient microbial communities with themselves and their habitat, and for testing treatments to control pathogen colonization of human skin.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Effects of antimicrobial agents fed to chickens on some gram-negative enteric bacilli.

Total and antimicrobial agent-resistant aerobic and facultative anaerobic gram-negative enteric bacilli in fecal samples of broiler chickens fed growth-promotional levels of antimicrobial agents were determined quantitatively. Two 8-week studies were conducted utilizing groups of chickens fed antimicrobial-supplemented rations; the second study involved feed "pasteurization" as a means of minimizing colonization from the feed. Dilution/spread-plating/replica-plating techniques on selective media were used to obtain counts of total organisms and those resistant to tetracycline, chloramphenicol, streptomycin, ampicillin, or kanamycin. The predominant aerobic and facultative anaerobic gram-negative organism was Escherichia coli, which was detected in all samples at levels ranging from 10(5) to over 10(10) CFU/g of feces. Less common were Proteus mirabilis, Klebsiella pneumoniae, and Pseudomonas sp., which varied in occurrence and levels from group to group (range, less than 10(3) to 10(8) CFU/g). Resistance to all antimicrobials (except chloramphenicol in E. coli) was commonly observed at incidences exceeding 10(3) CFU/g in the total populations. Colonization of the chickens' intestinal tracts by susceptible and resistant strains of E. coli, K. pneumoniae, and Pseudomonas sp. appeared to result from their presence in the environment of the newly hatched chickens. Ration pasteurization did affect P. mirabilis, which appeared to colonize from the feed. The results suggest that colonization by, and proliferation of, antimicrobial-resistant aerobic and facultative anaerobic gram-negative enteric bacilli in chicken intestinal tracts may be less dependent on selection through antimicrobial supplementation of the ration than on their prevalence in environments from which they can colonize newborns.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent.

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Effects of antimicrobial agents fed to chickens on some gram-negative enteric bacilli

Total and antimicrobial agent-resistant aerobic and facultative anaerobic gram-negative enteric bacilli in fecal samples of broiler chickens fed growth-promotional levels of antimicrobial agents were determined quantitatively. Two 8-week studies were conducted utilizing groups of chickens fed antimicrobial-supplemented rations; the second study involved feed "pasteurization" as a means of minimizing colonization from the feed. Dilution/spread-plating/replica-plating techniques on selective media were used to obtain counts of total organisms and those resistant to tetracycline, chloramphenicol, streptomycin, ampicillin, or kanamycin. The predominant aerobic and facultative anaerobic gram-negative organism was Escherichia coli, which was detected in all samples at levels ranging from 10(5) to over 10(10) CFU/g of feces. Less common were Proteus mirabilis, Klebsiella pneumoniae, and Pseudomonas sp., which varied in occurrence and levels from group to group (range, less than 10(3) to 10(8) CFU/g). Resistance to all antimicrobials (except chloramphenicol in E. coli) was commonly observed at incidences exceeding 10(3) CFU/g in the total populations. Colonization of the chickens' intestinal tracts by susceptible and resistant strains of E. coli, K. pneumoniae, and Pseudomonas sp. appeared to result from their presence in the environment of the newly hatched chickens. Ration pasteurization did affect P. mirabilis, which appeared to colonize from the feed. The results suggest that colonization by, and proliferation of, antimicrobial-resistant aerobic and facultative anaerobic gram-negative enteric bacilli in chicken intestinal tracts may be less dependent on selection through antimicrobial supplementation of the ration than on their prevalence in environments from which they can colonize newborns.     

Microbial community of the water column of the Selenga River-Lake Baikal biogeochemical barrier

The microbial communities of the estuarine zone and the mixing zone of river and lake waters in the Selenga River estuary were studied using the fluorescence in situ hybridization (FISH) method. The microorganisms belonging to the phylogenetic group Gammaproteobacteria were found to predominate in the river estuary, constituting up to 17% of the total bacterial community. Among cultivable microorganisms, organotrophic bacteria were predominant (2040 CFU/ml) in this zone, which results in high rates of microbial production (6.0 μg C/(l day). The microbial community structure changed with distance from the river estuary; representatives of the Alpha-, Beta-, and Gammaproteobacteria were present in equal proportions; psychrotolerant and oligotrophic bacteria were numerous. The rate of heterotrophic carbon dioxide assimilation decreased to 3.8 μg C/(l day). At 5–7 km from the river estuary, where the hydrologic, physical, and chemical conditions are similar to those of lake waters, members of the Betaproteobacteria, which are typical of the open waters of Lake Baikal, are the major representatives of planktonic microorganisms.     

The microflora of ulzerous zone mucosa in patients with duodenal ulcer

Bacteriological study of the biopsies taken from gastric and duodenal mucosa of 10 healthy volunteers and 74 patients with duodenal ulcer, was carried out. In the gastroduodenal zone of healthy subjects microorganisms of 6 genera (Streptococcus, Candida, Staphylococcus, Bacillus, Helicobacter and Lactobacillus) were detected. H. pylori was isolated in 20% of cases only in biopsy specimens taken from the antral section of the stomach of healthy as monoculture or in combination with C. albicans. In patients with duodenal ulcer activation of opportunistic microflora was observed in the periulcerous zone. More often H. pylori occurred in associations with fungi of the genus Candida, streptococci, staphylococci, enterobacteria, Pseudomonas and other microorganisms (of more than 30 genera). Quantitatively the dominating microorganisms (3.8-5.7 lg CFU/g) were H. pylori, fungi of the genus Candida, bacteria of the genera Streptococcus, Peptostreptococcus, Bacteroides, Gemella, Prevotella, Veillonella, Peptococcus, Bacillus, different species of opportunistic enterobacteria, as well as bacteria of the genera Staphylococcus, Micrococcus, Corynebacterium, Neisseria, Pseudomonas, etc. Opportunistic bacteria detected in the ulcerous zone, as a rule, expressed hemolytic, lecithinase, RNAase, caseinolytic, catalase and urease activity. Sonicated filtrates of such cultures produced a cytotoxic effect on cells HEp-2. Ulcer is an infected wound that needs sanitation.     

Microbial colonization and succession on the faucial mucosa in newborn infants rooming in with their mothers in a maternity hospital]

The formation of microflora on the laryngeal mucosa in newborn infants during the first 5 days of their life was studied in one of the maternity hospitals of Moscow. In this work modern methods of the isolation and identification of aerobic and anaerobic microorganisms were used, and the results thus obtained were computer-processed. In the maternity hospital of the "mother-child" type the microbial colonization of the laryngeal mucosa by normal and opportunistic microorganisms was noted in newborn infants. A wave-like course of the formation of laryngeal microflora, indicative of microbial succession occurring in the child, was revealed. The attempt to establish the cases of microbial interference between the species colonizing the laryngeal mucosa revealed that it was very rarely observed in 5-day-old newborns. This feature was seemingly the cause of low resistance of the larynx to colonization in newborn infants, which determined frequent colonization of their laryngeal mucosa with Staphylococcus aureus and Klebsiella.     

Fecal microflora in healthy young people

The study of the intestinal microflora in 119 young adults was carried out. A high content of anaerobic representatives of the intestinal microflora (bifido- and lactobacteria) and extremely wide fluctuations in the number of E. coli (1-5 million to 700-800 million cells per g of feces) were shown. The species composition of the facultative group was found to be variegated. Staphylococci, yeast, fungi, opportunistic enterobacteria, as well as Escherichia and cocci with changed characteristics were detected. 23.5% of the subjects showed a high content of E. coli (greater than 200 million cells per g of feces) accompanied by the increased occurrence of Klebsiella and Escherichia with changed properties. These persons can be regarded as a high risk group with a higher incidence of acute intestinal diseases with unknown etiology.     

Effects of water depth and livelihood activities on plant species composition and diversity in Nyando floodplain wetland,Kenya

The influence of water levels and livelihood activities on plant species composition, diversity and structuring of wetland ecosystem is a concern as wetlands undergo human exploitation and the increasing threat from climate change. To evaluate the effect of seasonal changes in water depth and human activities, plant density and species composition were assessed in wet and dry seasons and in natural and converted wetland zones of the Nyando papyrus wetland, Kenya. Three transects with different water regimes and livelihood activities were identified. Overall, 30 plant species were identified. In the less disturbed zone, differences between transects were small and 79 % (dry season) to 99 % (wet season) of the plant density consisted of obligate and facultative wetland plants. These groups were dominated by Cyperus papyrus and Vossia cuspidata. In the converted zone, facultative, facultative upland and upland plants became more important with 36 % of the plant density. The seasonally inundated zone had species diversity and species richness increased under dry conditions with more facultative upland species. In the converted zone, disturbance caused by vegetation removal, cropping and other livelihood activities lead to lower soil moisture and more colonization opportunity for facultative and upland species that are more adapted to dry conditions.     

Bioluminescent assay of total bacterial contamination of raw milk]

A rapid (30-35 min) bioluminescence assay of total bacterial contamination (TBC) of raw milk was optimized. This method includes incubation of milk samples in the presence of Neonol-10 and medical purity grade pancreatin with further removal of nonbacterial ATP by filtration through a membrane filter, cell disruption by treatment with dimethyl sulfoxide, and measurement of ATP concentration in a reaction with the bioluminescent reagent Immolum. The TBC detection threshold is 0.5 x 10(5) colony-forming units (CFU) per ml milk. Coefficients of correlation between the standard plate count method and bioluminescence assay (R) and residual standard deviations (Sxy) in raw milk samples (n = 140) were 0.83 and 0.54, respectively. In sterilized milk samples artificially contaminated with pure cultures of the main representatives of milk microflora (coli-forms, Staphylococcus aureus, Streptococcus thermophilus, and Streptococcus group D), these values were 0.89-0.99 and 0.09-0.29, respectively. The specific content of ATP was found to be (0.8 +/- 0.1) x 10(-18) mol/CFU in coli-forms; (12.0 +/- 8.1) x 10(-18) mol/CFU in S. aureus; (35.2 +/- 16.9) x 10(-18) mol/CFU in S. thermophilus; and (42.5 +/- 1.3) x 10(-18) in Streptococcus group D.     

Antimicrobial activity of new beta-lactams and aminoglycosides in vitro under conditions of anaerobiosis

Sensitivity of 135 strains of aerobic, facultative anaerobic and anaerobic asporogenous bacteria was tested in vitro with respect to 7 beta-lactam antibiotics and 4 aminoglycosides. It was shown that anaerobiosis influenced the MICs of the drugs for the majority of the strains. Under such conditions sensitivity of the aerobic and facultative anaerobic organisms to the beta-lactams increased 2-8 times. On the contrary, the MICs of the aminoglycosides for 74.6-85.1 per cent of the strains increased 2-16 times. The asporogenous anaerobic bacteria of clinical origin were highly sensitive to the beta-lactam antibiotics such as cefoxitin, cefotaxime, mezlocillin and carbenicillin whose MICs did not exceed 16-31.2 micrograms/ml.     

Bacterial colonization and endotoxin content of a new renal dialysis water system composed of acrylonitrile butadiene styrene

We measured endotoxin and bacterial levels in tap water, in water purified by reverse osmosis, and in dialysate samples over a 4-month period in a new 10-bed renal dialysis unit. Water treated by reverse osmosis is conducted to the 10 stations through 111 m of piping composed of acrylonitrile butadiene styrene (ABS). All determinations were made prior to the opening of the unit and after the system was purged for 35 h with all bedside station taps open. Formaldehyde disinfection of the piping system was attempted with a recommended protocol after 11 weeks by feeding 2.5 liters of 37% formaldehyde (0.85%, vol/vol) into the delivery system. Prior to water purging, 24 ng of endotoxin per ml was detected. This level decreased to 2.0 ng of endotoxin after the purging. Levels of endotoxin remained below 1.0 ng of endotoxin per ml throughout the duration of the study. In contrast, the level of viable microorganisms recovered from the treated water was approximately 3.5 X 10(4) CFU/100 ml. Even after disinfection of the system, there was no significant decrease in culturable bacteria from the water even though endotoxin levels were lower. Species isolated from the renal dialysis system were predominately pseudomonads, whereas species isolated from the tap water were Bacillus and Flavobacterium species. ABS provides a surface suitable for long-term colonization and growth of bacteria. Currently recommended decontamination protocols are ineffective in removing potentially pathogenic bacteria from ABS pipes and thus constitute an increased risk to patients undergoing dialysis.     

Bacterial colonization and endotoxin content of a new renal dialysis water system composed of acrylonitrile butadiene styrene.

We measured endotoxin and bacterial levels in tap water, in water purified by reverse osmosis, and in dialysate samples over a 4-month period in a new 10-bed renal dialysis unit. Water treated by reverse osmosis is conducted to the 10 stations through 111 m of piping composed of acrylonitrile butadiene styrene (ABS). All determinations were made prior to the opening of the unit and after the system was purged for 35 h with all bedside station taps open. Formaldehyde disinfection of the piping system was attempted with a recommended protocol after 11 weeks by feeding 2.5 liters of 37% formaldehyde (0.85%, vol/vol) into the delivery system. Prior to water purging, 24 ng of endotoxin per ml was detected. This level decreased to 2.0 ng of endotoxin after the purging. Levels of endotoxin remained below 1.0 ng of endotoxin per ml throughout the duration of the study. In contrast, the level of viable microorganisms recovered from the treated water was approximately 3.5 X 10(4) CFU/100 ml. Even after disinfection of the system, there was no significant decrease in culturable bacteria from the water even though endotoxin levels were lower. Species isolated from the renal dialysis system were predominately pseudomonads, whereas species isolated from the tap water were Bacillus and Flavobacterium species. ABS provides a surface suitable for long-term colonization and growth of bacteria. Currently recommended decontamination protocols are ineffective in removing potentially pathogenic bacteria from ABS pipes and thus constitute an increased risk to patients undergoing dialysis.     

Genomic identification of microbial species adhering to maxillofacial prostheses and susceptibility to different hygiene protocols

This study investigated the microbial colonization of maxillofacial prostheses and support tissues using the Checkerboard DNA–DNA hybridization method, and the efficacy of 0.12% chlorhexidine gluconate, 10% Ricinus communis solutions, or brushing, on colony forming unit (CFU) reduction in monospecies biofilms (Candida glabrata, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa) formed on two silicones (MDX 4-4210 and Bio-Skin). Biofilm was harvested from 43 maxillofacial prosthesis wearers for detection of 38 species of microorganisms. The CFU counts of the six above mentioned species were recorded after using the hygiene protocols. All 38 investigated species were identified in prostheses and tissues, with a higher prevalence in the prostheses. 0.12% chlorhexidine gluconate immersion showed the greatest antimicrobial effectiveness, followed by mechanical brushing protocols. MDX 4-4210 silicone produced lower CFU counts than Bio-Skin.     

Action of polyene antibiotics on actinomycetes with a varying lipid makeup]

The effect of polyenic antibiotics, such as nystatin, levorin, amphotericin B and mycoheptin on 93 representatives of various species of the ray fungi was studied. It was shown that resistance of the actinomycetes to the polyens was connected with the absence or insufficient content of sterols (0.001--0.008 per cent in the dry mycelium). On addition of cholesterol to the nutrient media (100 microgram/ml) it was included into the membranes of some cultures and their sensitivity increased 2--60 times. Resistance of Actinomyces sp. LIA 0775 grown on the media with fats differing in their composition decreased 2--4 times. In these cases the culture lipids were characterized by lower content of phospholipids (35--45 per cent from the total lipids as compared to 70--80 per cent when grown on the control medium without fats) and significantly increased content of unsaturated fatty acids (3--4 times).     

Mesophilic and psychrotrophic aerobe sporulating microorganisms in raw cow’s milk

We studied the occurrence of mesophilic and psychrotrophic aerobic sporulating microorganisms (MPAS) in raw cow’s milk and their relations to microflora in milk. We took 294 samples of raw cow’s milk from 14 farms during one year. Briefly the method for MPAS assessment is to inactivate the milk sample by heating it to 80-82°C for 30 minutes. Mesophilic aerobic sporulates are incubated at 30°C for 3 days-, and psychrotrophic aerobic sporulates at 7°C for 10 days. Results of studied microbiological parameters characterize the sampled milk as complying with requirements of the EU regulation 92/46 and standard STN 57 0529. MPAS count was within the span 2.5–340 CFU/ml. The average value ofMPAS was 59.4 CFU/ml, with variation coefficient 93.1%. Counts up to 50 CFU/ml were in 55.4% samples, the value was not higher than 100 in 85%, and in 3.1% of the samples the MPAS count was higher than 200. MPAS do not show correlation with any of the studied microbiological parameters; marked influences of season were not observed either. On the basis of obtained results, it is possible to support the proposal of an initial limit of maximum 200 CFU/ml for the introduction of a MPAS parameter. MPAS count found in the same dishes at incubation for mesophilic and subsequently strictly psychrophilic microorganisms was 56.9 CFU/ml on average. This represents 95.8% of total CFU sums of individual dishes at two temperatures. The correlation coefficient of these two types of results, r = 0.99,gives evidence of close dependence expressed by the linear regression equation. Use of two incubation temperatures, one after another with an identical set of dishes, enables us to exclude overestimation of results due to sporulates able to grow at both incubation temperatures.