Isolation of Klebsielleae from within living wood.

Previous studies from this laboratory have documented the presence of coliform bacteria emanating from wooden reservoirs containing finished drinking water. Coliforms were identified as Klebsiella pneumoniae and Enterobacter spp. In the present report, evidence is presented which suggests that the origin of these coliforms is from the wood used to construct the reservoirs. In liquid expressed from freshly cut redwood, total bacterial counts in the range of 10(5) to 10(6)/ml were commonly observed. When present, coliform counts were over 10(3)/ml of expressed liquid. E. agglomerans was the most prevalent coliform present, but Klebsiella was isolated from freshly cut logs. Citrobacter freundii was also occasionally isolated. No fecal coliform-positive Klebsiella were obtained from any of the samples. Highest total bacteria and coliform counts were observed in sapwood specimens. Coliforms were present throughout sapwood as evidenced by contact plating serial sections of freshly cut wood. Scanning electron micrographs illustrate the presence of bacterial colonies within sapwood tracheids. Other wood species also contained coliform bacteria but in numbers lower than found in redwood.     

Klebsielleae in drinking water emanating from redwood tanks.

A survey was made of the bacteriological quality and chlorine content of 33 public and private water systems that utilize redwood storage tanks. Coliforms of the genera Klebsiella and Enterobacter were isolated from 9 of 10 private drinking water systems and from 11 of 23 water systems in state and federal parks. Total coliform counts in the private systems exceeded federal membrane filter guidelines by as much as 10-to 40-fold. Coliform counts were highest in the newer reservoirs. Factors contributing to poor water quality are: lack of automated chlorination equipment or an insufficient supply to maintain a residual, common inlet/outlet plumbing design, and lengthy average retention periods. The latter two factors contribute to improper mixing and stagnation of the water, whereas the former allows microbes to multiply on the water-soluble nutrients that leach from the wood. Wooden reservoirs exert a high chlorine demand, and 0.4 ppm of chlorine residual in the incoming tank water proves inadequate. It is suggested that specific water-soluble nutrients in redwood (and in numerous other types of botanical material) induce a natural nutritional selection for coliforms of the tribe Klebsielleae.     

Klebsielleae in drinking water emanating from redwood tanks.

A survey was made of the bacteriological quality and chlorine content of 33 public and private water systems that utilize redwood storage tanks. Coliforms of the genera Klebsiella and Enterobacter were isolated from 9 of 10 private drinking water systems and from 11 of 23 water systems in state and federal parks. Total coliform counts in the private systems exceeded federal membrane filter guidelines by as much as 10-to 40-fold. Coliform counts were highest in the newer reservoirs. Factors contributing to poor water quality are: lack of automated chlorination equipment or an insufficient supply to maintain a residual, common inlet/outlet plumbing design, and lengthy average retention periods. The latter two factors contribute to improper mixing and stagnation of the water, whereas the former allows microbes to multiply on the water-soluble nutrients that leach from the wood. Wooden reservoirs exert a high chlorine demand, and 0.4 ppm of chlorine residual in the incoming tank water proves inadequate. It is suggested that specific water-soluble nutrients in redwood (and in numerous other types of botanical material) induce a natural nutritional selection for coliforms of the tribe Klebsielleae.     

Cyclitol utilization associated with the presence of Klebsielleae in botanical environments.

Bacteria of the tribe Klebsielleae are capable of metabolizing the cyclitols myoinositol, sequoyitol, and pinitol, which are present in aqueous extracts of redwood. Of the combined Klebsiella isolates from clinical and environmental origins, 100% (138/138), 97% (34/35), and 86% (119/138) fermented inositol, sequoyitol, and pinitol, respectively. These compounds were also used as a sole source of carbon and energy by Klebsiella. Similar results were obtained with Enterobacter isolates, but most other enteric bacteria tested could not metabolize cyclitols. Strains of Klebsiella multiplied to levels exceeding 10(5)/ml in aqueous extracts of nonsterile redwood within 6 days. Most other enteric bacteria did not grow in these extracts. Cyclitol metabolism was found to correlate well with the ability to multiply in redwood extract in the presence of cyclitol-negative indigenous bacteria. The capacity to use cyclitols, which are present in a variety of plant material, might afford Klebsielleae of both environmental and clinical origins an advantage in competing for nutrients and colonizing botanical environments.     

Cyclitol utilization associated with the presence of Klebsielleae in botanical environments.

Bacteria of the tribe Klebsielleae are capable of metabolizing the cyclitols myoinositol, sequoyitol, and pinitol, which are present in aqueous extracts of redwood. Of the combined Klebsiella isolates from clinical and environmental origins, 100% (138/138), 97% (34/35), and 86% (119/138) fermented inositol, sequoyitol, and pinitol, respectively. These compounds were also used as a sole source of carbon and energy by Klebsiella. Similar results were obtained with Enterobacter isolates, but most other enteric bacteria tested could not metabolize cyclitols. Strains of Klebsiella multiplied to levels exceeding 10(5)/ml in aqueous extracts of nonsterile redwood within 6 days. Most other enteric bacteria did not grow in these extracts. Cyclitol metabolism was found to correlate well with the ability to multiply in redwood extract in the presence of cyclitol-negative indigenous bacteria. The capacity to use cyclitols, which are present in a variety of plant material, might afford Klebsielleae of both environmental and clinical origins an advantage in competing for nutrients and colonizing botanical environments.     

Occurrence of Potential Pathogens in Water Containing Ornamental Fishes

The bacterial population of the water supplied with ornamental fish purchased from retail outlets was examined qualitatively and quantitatively. As many as 10(9) viable aerobic organisms per 100 ml were present, with fecal coliform counts as high as 10(5) per 100 ml. Citrobacter, Escherichia, Pseudomonas, and Vibrio were isolated from 75% or more of the samples, whereas Aeromonas, Alcaligenes, Enterobacter, Flavobacterium, and Streptococcus were isolated from 45 to 65% of the samples. Pseudomonas aeruginosa, Edwardsiella tarda, and Klebsiella pneumoniae were also isolated.     

Colonization of the botanical environment by Klebsiella isolates of pathogenic origin.

Growth, survival, and pathogenicity of Klebsiella growing in and on environmental foci were examined. Total coliforms present in raw wastes from pulp mills were in excess of 10(5)/ml, and 60 to 80% were Klebsiella. Fecal coliform counts ranged from 10(1) to 10(5)/ml. Klebsiella isolates from industrial effluents and a variety of human and bovine mastitis origins multiplied in pulp waste and commonly exceeded 10(6) cells per ml. Pathogenic isolates also multiplied in dilute aqueous extracts of sawdust to comparable levels. Klebsiella strains from vegetable surfaces and human infections grew rapidly on the surfaces of potatoes and lettuce and exceeded 10(3) organisms per g of surface peel and leaf after a 24h incubation at room temperature. After 7 weeks on potatoes stored at 5 degrees C, some 10 to 30% of the day 1 Klebsiella counts were recoverable. Three Klebsiella isolates of pathogenic origin were passed 45 times through sterile pulp effluent (270 generations), and mean lethal dose levels in mice were periodically monitored. In two instances, a significant decrease in virulence was noted after 15 to 26 passes (90 to 156 generations). The third culture, of bovine mastitis origin, retained its original mean lethal dose value. Botanical milieu provided suitable habitats for the multiplication and colonization of Klebsiella isolates of disease origins in the same manner as indigenous isolates. Aquatic environments polluted with botanical material served as potential reservoirs for perpetuating the growth and spread of opportunistic Klebsiella pathogens that may ultimately colonize animals, humans, and aquatic organisms.     

Gas chromatographic analysis of in situ cyclitol utilization by Klebsielleae growing in redwood extracts

Gas chromatographic analysis was employed to demonstrate in situ cyclitol utilization in aqueous extracts of redwood by isolates of Klebsiella, Enterobacter, and several other genera of gram-negative bacteria. In aqueous redwood extracts, all but one of the Klebsiella and Enterobacter isolates tested reached densities exceeding 5.0 x 10(6) cells/ml within 4 days, and all utilized pinitol and sequoyitol. Other enteric bacteria did not utilize cyclitols in this extract. A defined minimal medium, containing the carbohydrates and cyclitols (including myo-inositol) in redwood, was used to determine which carbon sources are preferentially utilized by Klebsielleae and other bacteria. It was found that D-glucose and L-arabinose were consumed by Klebsiella before the three cyclitols were utilized. Pinitol utilization proceeded in more slowly than that of sequoyitol and myo-inositol. Cyclitol utilization in the defined medium was also observed for Yersinia, Erwinia, and Salmonella. Escherichia coli isolates did not utilize cyclitol compounds. The ability to use cyclitols as a sole source of carbon can explain the high cell densities of Klebsielleae in redwood water reservoirs and in redwood lumber.     

Gas chromatographic analysis of in situ cyclitol utilization by Klebsielleae growing in redwood extracts.

Gas chromatographic analysis was employed to demonstrate in situ cyclitol utilization in aqueous extracts of redwood by isolates of Klebsiella, Enterobacter, and several other genera of gram-negative bacteria. In aqueous redwood extracts, all but one of the Klebsiella and Enterobacter isolates tested reached densities exceeding 5.0 x 10(6) cells/ml within 4 days, and all utilized pinitol and sequoyitol. Other enteric bacteria did not utilize cyclitols in this extract. A defined minimal medium, containing the carbohydrates and cyclitols (including myo-inositol) in redwood, was used to determine which carbon sources are preferentially utilized by Klebsielleae and other bacteria. It was found that D-glucose and L-arabinose were consumed by Klebsiella before the three cyclitols were utilized. Pinitol utilization proceeded in more slowly than that of sequoyitol and myo-inositol. Cyclitol utilization in the defined medium was also observed for Yersinia, Erwinia, and Salmonella. Escherichia coli isolates did not utilize cyclitol compounds. The ability to use cyclitols as a sole source of carbon can explain the high cell densities of Klebsielleae in redwood water reservoirs and in redwood lumber.     

Enterobacteriaceae, coliforms, faecal coliforms and salmonellas in raw ewes' milk

Thirty-nine samples of freshly drawn ewes' milk collected at 13 farms, and 120 samples of raw ewes' milk collected on arrival at a dairy were examined. Farm samples had geometric mean counts of 4.4 X 10(2) Enterobacteriaceae/ml, 3.9 X 10(2) coliforms/ml and 2.0 X 10(2) faecal coliforms/ml, whereas the respective mean counts were 6.2 X 10(3)/ml, 5.4 X 10(3)/ml and 1.3 X 10(3)/ml for dairy samples. Salmonellas were not detected by enrichment procedures in any of the 159 samples examined. Escherichia coli (47.5% strains), Enterobacter cloacae (17.7%), Ent. agglomerans (11.3%), Hafnia alvei (6.5%) and Klebsiella oxytoca (6.0%) were the predominant species in 434 Enterobacteriaceae strains isolated from farm samples. Levels and species of Enterobacteriaceae found in the present work in raw ewes' milk imply a considerable risk of early blowing in cheese-making from unpasteurized milk.     

Impact of Mount St. Helens Eruption on Bacteriology of Lakes in the Blast Zone

Lakes lying within the blast zone of Mount St. Helens showed dramatic increases in heterotrophic bacterial numbers after the eruption of 18 May 1980. The total microscopic counts of bacteria in some of the most severely affected lakes were more than 10⁷ cells per ml, an order of magnitude above the counts in outlying control lakes. Likewise, the numbers of viable bacteria reached levels of more than 10⁶ cells per ml, compated with fewer than 10⁴ cells per ml in control lakes. The CPS medium used for enumeration provided growth of up to 81.5% of the bacteria during sampling of one of the blast zone lakes. The high numbers of bacteria and the efficacy of the viable enumeration procedure are evidence that the lakes have been transformed rapidly from oligotrophy to eutrophy due to the eruption and its aftermath. Organic material leached from the devastated forest vegetation is thought to be responsible for the enrichment of heterotrophs. Total coliform bacteria were found in all of the blast zone lakes, and some lakes contained fecal coliform bacteria. Klebsiella pneumoniae was the predominant total coliform and was also identified as one of the fecal coliform bacteria, although Escherichia coli was the predominant species in that category. Our data indicate that bacterial populations peaked in the outer blast zone lakes in the summer of 1980 and in most of the inner lakes during the summer of 1981.     

Survival of coliform bacteria in sewage sludge applied to a forest clearcut and potential movement into groundwater.

Anaerobically digested dewatered sludge (10 to 15 cm thick) was applied to a forest clearcut as a fertilizer source in northwest Washington on gravelly glacial outwash soil. This sludge is not microbiologically sterile and may contain pathogenic organisms. Fecal coliform bacterial counts in sludge applied in summer (July) fell from 1.08 X 10(5) to 358/g in 204 days and to 0/g in 267 days. Dieoff appeared more rapid in winter (January)-applied sludge, when colnts fell from 1.2 X 10(5) to 20/g in 162 days. Initial death rates were related to sludge temperature, moisture, pH, physical composition, and microbial competition. Aftergrowth of fecal coliforms occurred in warm summer and fall months, but counts were of similar magnitude to background levels in forest soils, where a maximum count of 54/g was recorded. Total coliform counts in fresh sludge ranged from 1.4 X 10(4) to 1.9 X 10(6)/g. Numbers stabilized at 10(3) to 10(4)/g in spring, fall, and summer, with lower numbers in winter. Both total and fecal bacteria moved from the sludge to the soil beneath, but few penetrated past the first 5 cm. The soil acts as an effective biological filter. Few fecal coliform bacteria were recorded in the groundwater, generally being less than 5/100 ml and mostly 0/100 ml. A maximum count of 52/100 ml was recorded. Groundwater contamination from vertical movement of potential pathogens appears unlikely, but hazards from surface runoff and direct handling in the first year may arise.     

Survival of coliform bacteria in sewage sludge applied to a forest clearcut and potential movement into groundwater.

Anaerobically digested dewatered sludge (10 to 15 cm thick) was applied to a forest clearcut as a fertilizer source in northwest Washington on gravelly glacial outwash soil. This sludge is not microbiologically sterile and may contain pathogenic organisms. Fecal coliform bacterial counts in sludge applied in summer (July) fell from 1.08 X 10(5) to 358/g in 204 days and to 0/g in 267 days. Dieoff appeared more rapid in winter (January)-applied sludge, when colnts fell from 1.2 X 10(5) to 20/g in 162 days. Initial death rates were related to sludge temperature, moisture, pH, physical composition, and microbial competition. Aftergrowth of fecal coliforms occurred in warm summer and fall months, but counts were of similar magnitude to background levels in forest soils, where a maximum count of 54/g was recorded. Total coliform counts in fresh sludge ranged from 1.4 X 10(4) to 1.9 X 10(6)/g. Numbers stabilized at 10(3) to 10(4)/g in spring, fall, and summer, with lower numbers in winter. Both total and fecal bacteria moved from the sludge to the soil beneath, but few penetrated past the first 5 cm. The soil acts as an effective biological filter. Few fecal coliform bacteria were recorded in the groundwater, generally being less than 5/100 ml and mostly 0/100 ml. A maximum count of 52/100 ml was recorded. Groundwater contamination from vertical movement of potential pathogens appears unlikely, but hazards from surface runoff and direct handling in the first year may arise.     

A microbiological study of Sobia: a fermented beverage in the Western province of Saudi Arabia

Sobia is a traditional fermented beverage prepared from wheat and malt flours in the Western province of Saudi Arabia. Fourteen samples of sobia were collected from Makkah Al-Mukarrmah (Western province) and from Riyadh (Central province). Samples were examined microbiologically for total bacterial counts, lactic acid bacteria, yeasts and moulds as well as coliforms. Titratable acidity and pH were also analysed. The results revealed that sobia samples had high total bacterial and lactic acid bacteria counts that ranged from 4.17 to 8.09 log c.f.u./ml and from 4.01 to 8.19 log c.f.u./ml respectively. Yeasts and moulds counts ranged from 3.96 to 5.87 log c.f.u./ml. The coliform count was from 0.67 to 3.84 log c.f.u./ml. Acidity expressed as percent lactic acid ranged from 0.04 to 0.30%, while the pH of the samples ranged from 3.37 to 5.53. Identification of microorganisms in sobia revealed the presence of lactic acid bacteria, coliforms, yeasts and moulds. Lactic acid bacteria consisted of Lactobacillus cellobiosus (26.8%), L. buchneri (17.9%), L. plantarum (8.9%), L. brevis (23.2%), L. delbrueckii. delbrueckii (8.9%), Leuconostoc lactis (8.9%), and Pediococcus pentosaceus (5.4%). Coliforms consisted of Klebsiella pneumoniae (25.8%), Enterobacter aerogenes (6.1%), E. sakazakii (33.3%), E. cloacae (19.7%), and Serratia liquefaciens (3%). Yeasts comprised Saccharomyces cerevisiae (27.6%), Candida tropicalis (26.3%), C. ciferrii (13.1%), C. guilliermondii (13.1%), C. lipolytica (6.58%), Kloeckera japonica (13.1%), and Rhodotorula rubra (1.3%). All moulds were Penicillium spp.     

Microbiological and virological analysis of water from two water filtration plants and their distribution systems

The microbial flora of the water produced by two water filtration plants and their drinking water distribution system were evaluated: the Pont-Viau (PV) and the Repentigny (RE) water filtration plants. Untreated water entering the plants contained 3.6 (PV) and 16.8 most probable number of infectious units (mpniu)/L (RE) enteric viruses and total coliform bacteria counts were 300,000 (PV) and 500,000 cfu/L (RE). Treated water leaving the plant was essentially free of all the bacterial indicators measured (total, stressed, and fecal coliforms; Aeromonas hydrophila; Pseudomonas aeruginosa; Clostridium perfringens; enterococci) as well as of human enteric viruses. Heterotrophic plate counts at 20 and 35 degrees C were low in the freshly treated water leaving the plants, but bacterial regrowth was observed in both distribution systems at all sampling sites. Average counts for the heterotrophic plate count (20 degrees C) were between 10(6) and 10(7) cfu/L and counts were clearly increased with the distance from the plant. The most numerous bacterial genera encountered were Bacillus, Flavobacterium, and Pseudomonas (nonaeruginosa).     

R factors in coliform-fecal coliform sewage flora of the prairies and Northwest Territories of Canada.

Coliform and fecal coliform populations found in the raw sewages and final sewage effluents of the prairie provinces and the Northwest Territories were examined for antibiotic resistance and the possession of R factors. It was determined that 8.91% of the total coliform and 10.80% of the fecal coliform populations carried R factors. The following numbers of combinations of R determinants were found: 39 in the Escherichia coli population, 6 in the Citrobacter population, 20 in the Enterobacter populations, 10 in the Klebsiella populations, and 11 in the Aeromonas populations. The maximum number of R determinants transferable simultaneously was seven; organisms with R factors containing determinants for chloramphenicol usually contained determinants for ampicillin. Of the coliform and fecal coliform populations, 2 to 4% were resistant to chloramphenicol in some provinces, and from 17 to 30% of the populations were resistant to three or more antibiotics. It was calculated that coliforms containing R factors in the raw sewage reached population levels of 1.5 X 10(7)/100 ml, and fecal coliforms containing R factors reached population levels of 8.6 X 10(5) ml. Final effluent discharges to the receiving environment contained R factor-containing coliform and fecal coliform populations of 3.1 X 10(4)/100 ml and 5.8 X 10(2)/100 ml, respectively. The incidence of bacteria containing R factors in sewage appears to be increasing with time, and their removal from sewage before discharge to the receiving environment is desirable. Consideration of data on bacteria with R factors should be made in future water quality deliberations and in discharge regulations.     

Source and extent of Klebsiella pneumoniae in the paper industry.

Three pulp and paper mill processing plants were evaluated for fecal coliform and Klebsiella pneumoniae bacterial concentrations. Freshwater consumed by paper industries contained minimum detectable levels of K. pneumoniae, less than 10 organisms per 100 ml. Elevated concentrations of K. pneumoniae could be traced from early pulping stages to water processing reuse systems. Concentrations of K. pneumoniae (thermotolerant and thermointolerant) ranged from 40,000 organisms per 100 ml to an estimated 3 x 10(6) organisms per 100 ml. K. pneumoniae biotyping provided evidence for the selective growth and persistence of K. pneumoniae from the initial wood washing stages through to the final effluent discharge. Wastewater treatment had limited effects in reducing K. pneumoniae concentrations. K. pneumoniae levels ranged from 40 organisms per 100 ml to an estimated 10(6) organisms per 100 ml. The presence of K. pneumoniae in water indicates degraded water quality, and its significance with regard to human health effects has yet to be examined.     

Bacteria in bivalve shellfish with special reference to the oyster

The bacterial flora of the Pacific oyster Crassostrea gigas, the sea mussel Perna viridis and the arkshell clam Scapharca cornea differed considerably from that of seawater in both numbers and generic composition. The numbers of heterotrophic bacteria in the bivalve shellfish, including the anaerobes and spore-forming bacteria, were greater than that in the surrounding water. Pseudomonas spp. were the dominant organisms, comprising over one third of the 321 strains characterized after isolation from the bivalves and seawater. Other bacteria isolated from the shellfish included Vibrio, Acinetobacter, and Aeromonas spp., whereas the seawater flora consisted mainly of coliform organisms, coryneform bacteria and Flavobacterium/Cytophaga spp. Bacteria associated with the deposit-feeding clams were higher in density and more distinct in generic composition as compared with those in the suspension-feeding oysters and mussels. Over 90% of the coliform and heterotrophic bacteria in oysters were found in organs associated with the digestive tract. Coliforms were mainly found in the stomach while heterotrophs were present in both stomach and the lower intestine. The results suggest that the stomach flora of oysters are mainly derived from the external environment and, through a process of selection and multiplication, that it may be gradually replaced by a more indigenous population which dominates the lower digestive tract.     

Phenotypic characteristics of coliform and noncoliform bacteria from a public water supply compared with regional and national clinical species.

During the summer and fall of 1984, elevated total coliform counts were observed in the distribution system of a public water supply serving 350,000 people in south central Connecticut. As part of an investigation of possible health risks associated with the presence of bacteria in the water supply, bacterial isolates from the distribution system were compared with bacterial isolates of the same species obtained from a large regional teaching hospital and from a national compendium of clinical isolates. Characteristics analyzed included phenotypic metabolic activity, antimicrobial susceptibilities to clinically utilized antibiotics, temperature tolerance at 44.5 degrees C, and beta-glucuronidase activity in single-test form and on a selective medium. Environmental isolates lacked known plasmid-mediated characteristics, with the exception of one Escherichia coli isolate which showed some antibiotic resistance. Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter agglomerans from all sources were temperature tolerant and yielded positive fecal coliform tests. Only E. coli showed beta-glucuronidase activity (both in a single biochemical test and on a selective medium). No single characteristic analyzed was sufficient to establish an organism as either environmental or clinical in origin.     

Phenotypic characteristics of coliform and noncoliform bacteria from a public water supply compared with regional and national clinical species

During the summer and fall of 1984, elevated total coliform counts were observed in the distribution system of a public water supply serving 350,000 people in south central Connecticut. As part of an investigation of possible health risks associated with the presence of bacteria in the water supply, bacterial isolates from the distribution system were compared with bacterial isolates of the same species obtained from a large regional teaching hospital and from a national compendium of clinical isolates. Characteristics analyzed included phenotypic metabolic activity, antimicrobial susceptibilities to clinically utilized antibiotics, temperature tolerance at 44.5 degrees C, and beta-glucuronidase activity in single-test form and on a selective medium. Environmental isolates lacked known plasmid-mediated characteristics, with the exception of one Escherichia coli isolate which showed some antibiotic resistance. Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter agglomerans from all sources were temperature tolerant and yielded positive fecal coliform tests. Only E. coli showed beta-glucuronidase activity (both in a single biochemical test and on a selective medium). No single characteristic analyzed was sufficient to establish an organism as either environmental or clinical in origin.