Potential Pathogens in the Environment: Klebsiella pneumoniae, a Taxonomic and Ecological Enigma

A nitrogen-deficient medium and m-Endo agar were employed in the isolation of members of the tribe Klebsielleae from surfaces of vegetables and seeds. With m-Endo agar at an incubation temperature of 37 C, nearly 50% of the vegetables and seven out of seven seed samples yielded organisms which biochemically and serologically were identified as Klebsiella pneumoniae, Viable counts were generally in the range of 10(3) cells per g of vegetable peel or seed. Organisms classified as K. pneumoniae exhibited seven different IMViC patterns, with the --++, ++++, and -+++ patterns most common. Seven of the eleven K. pneumoniae serotypes encountered have previously been isolated from human urinary tract and other infections. Fifty percent of the 40 K. pneumoniae examined exhibited positive acetylene-reducing activity, i.e., they possessed the capability for fixing N(2). Vegetables containing K. pneumoniae may constitute a potential reservoir for human nosocomial genitourinary or other infections.     

Fermentation of polysaccharides by Klebsielleae and other facultative bacilli.

Fermentations of 10 polysaccharides by species of the family Enterobacteriaceae were examined. Algin, guar, karaya, xanthan, and xylan were not fermented by any of the strains tested. Most of the activity was found in the tribe Klebsielleae. Klebsiella oxytoca fermented amylopectin (97% of the strains studied), carrageenan (100%), inulin (68%), polypectate (100%), and tragacanth (100%). Klebsiella pneumoniae fermented amylopectin (91%), carrageenan (100%), and tragacanth (86%). Carrageenan was also fermented by Enterobacter aerogenes (100%), Enterobacter agglomerans (63%), Enterobacter cloacae (95%), and Pectobacterium (38%). Pectobacterium shared polypectate fermentation (100%) with K. oxytoca. With one exception, Serratia strains were negative on all polysaccharides. These results, along with other evidence, indicate that (i) the genus Klebsiella is biochemically the most versatile genus of the tribe, (ii) because of its distinct characteristics, K. oxytoca warrants species designation separate from K. pneumoniae, and (iii) some food additives generally considered indigestible can be metabolized by a few species of facultative bacilli, whereas others appear to be resistant.     

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.     

Hydrolytic and haemolytic activity of Klebsiella pneumoniae and Klebsiella oxytoca

Hydrolytic enzymes and haemolysins are important extracellular substances produced by many bacteria. We investigated 57 K. pneumoniae strains and 40 K. oxytoca strains isolated from clinical materials. We estimated the ability to produce: proteases hydrolyzing milk powder, caseinase, gelatinase, elastase, lecithinase, lipases, DNase and haemolysins on human, sheep and horse erythrocytes on TSA medium with or without 5% Egg Yolk. We detected that K. oxytoca strains produced proteases hydrolyzing milk powder (37.5%), caseinase (15.0%) and gelatinase (17.5%) more frequently than K. pneumoniae strains (respectively 21.0%, 5.3%, 8.9%). None of the analysed Klebsiella spp. strains produced elastase. Only K. pneumoniae strains produced lecithinase (5.3%). Lipases hydrolyzing Tween were produced from 3.5% (for Tween 60 and 80) to 7.0% (for Tween 20). Among K. oxytoca strains only one (2.5%) hydrolyzing Tween 20. DNase was produced by 38.6% of K. pneumoniae strains and by 27.5% K. oxytoca strains. Haemolytic properties on human erythrocytes were detected in 5.3% K. pneumoniae strains on TSA medium and 29,8% on medium with Egg Yolk. In K. oxytoca strains haemolytic properties on human erythrocytes were detected only on medium with Egg Yolk (12.5%). Haemolytic properties on sheep erythrocytes were detected respectively in 21.0% and 22.8% K. pneumoniae strains and in 7.5% K. oxytoca strains on each medium. Haemolytic properties on horse erythrocytes were detected respectively in 33.4% and 52.6% K. pneumoniae strains and in 15.0% and 20.0% K. oxytoca strains.     

Short and long courses of ofloxacin therapy of Klebsiella pneumoniae sepsis following irradiation.

Exposure to whole-body irradiation is associated with fatal gram-negative sepsis. The optimal length of therapy of such infection is not established. The effect of short and long courses of oral therapy with the quinolone ofloxacin for orally acquired Klebsiella pneumoniae infection was tested in B6D2F1 mice exposed to 8.0 Gy of bilateral radiation from 60Co. A dose of 10(8) organisms was given orally 4 days after irradiation, and therapy was started 1 day later. Cultures of the ileum 7 days after irradiation showed the recovery of K. pneumoniae in 7 of 10 untreated mice and in 3 of 20 treated with ofloxacin. However, 14 days after irradiation K. pneumoniae was isolated in 5 of 6 untreated mice, in 7 of 9 that received the short course of therapy, and in one of those that received the long course of therapy (P less than 0.05). At Day 7, K. pneumoniae was isolated from the livers of 6 of 10 untreated mice, and from none of those receiving ofloxacin (P less than 0.05). At 14 days, K. pneumoniae was isolated in 4 of 6 untreated animals, in 4 of 9 that received the short course of therapy, and in none of the mice that received the long course of therapy (P less than 0.05). Only 3 of 20 (15%) untreated mice survived for 30 days as compared to 11 of 20 (55%) mice treated for 7 days with ofloxacin and 18 of 20 (90%) mice treated for 21 days with ofloxacin (P less than 0.05). These survival data illustrate the efficacy of a 21-day course over a 7-day course of ofloxacin therapy for orally acquired K. pneumoniae infection in irradiated hosts.     

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.     

Cloning and expression of Klebsiella pneumoniae genes coding for citrate transport and fermentation.

Three Escherichia coli clones (DH1/Cit1, DH1/Cit2 and DH1/Cit3) capable of utilizing citrate as a sole carbon source were isolated from a cosmid bank of Klebsiella pneumoniae wild-type DNA. Two of these clones (DH1/Cit1 and DH1/Cit2) only grew aerobically on citrate minimal medium, the third clone (DH1/Cit3) could also be cultured under fermentative conditions. The aerobic as well as the anaerobic generation times of the three clones were from 4.5 to 7 h. Whereas clone DH1/Cit3 showed a pronounced lag phase on citrate when the cells were pre-grown in medium without citrate, clone DH1/Cit1 immediately started growth, while with clone DH1/Cit2 a short lag phase could be observed upon transfer to citrate minimal medium. Restriction analyses of the three plasmids showed that no common fragments had been cloned. The length of the inserts were 13 and 6 kb for the aerobic Cit+ clones and 27 kb (10 kb) for the anaerobic one. Cultures of the anaerobic Cit+ clone were analyzed by immunoblotting techniques and shown to contain oxaloacetate decarboxylase, which confers citrate utilization under anaerobic conditions to K. pneumoniae. Enzyme assays demonstrated the active state of this biotin-containing membrane protein. The specific activity in vesicle preparations from the E. coli clone was 30% of the wild-type K. pneumoniae vesicles. Citrate acts as an inducer of enzyme protein synthesis in the E. coli clone as it does in K. pneumoniae.     

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.     

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.     

Profiles of the utilization of 20 amino acids as the only source of nitrogen and carbon in bacteria of the genera Klebsiella, Enterobacter, Serratia, Escherichia

The profiles of the utilization of 20 protein amino acids in 118 Klebsiella pneumoniae sub- sp. pneumoniae, K. oxytoca, K. planticola, K. mobilis, Enterobacter cloacae, Serratia marscescens, S. liquefaciens, Escherichia coli strains isolated from clinical material were studied. The utilization of amino acids was determined on minimal saline agar containing amino acid as the only source of nitrogen and carbon; the results were evaluated after 72-hour incubation at 37 degrees C. 17 profiles of amino-acid utilization were thus determined, most of them genus-specific in enterobacteria: Klebsiella (profiles No. 1--6, 9, 10), Enterobacter (No. 11--13), Serratia (No. 14--16), Escherichia (No. 17). The full coincidence of amino-acid utilization profiles in bacteria of K. mobilis (No. 1, 6) and K. pneumoniae subsp. pneumoniae with out of such profiles in bacteria of the genera Enterobacter, Serratia, Escherichia was established, which confirmed that K. mobilis (formerly Enterobacter aerogenes) belonged to the genus Klebsiella.     

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.     

Detection of Klebsiella pneumoniae antibodies in Aotus l. lemurinus (Panamanian owl monkey) using an enzyme linked immunosorbent assay (ELISA) test

An enzyme linked immunosorbent assay (ELISA), was adapted to detect antibodies against Klebsiella pneumoniae in Aotus l. lemurinus monkeys. It was used to define the prevalence of infection and the immunogenicity of an Al(OH)3 bacterin in a population of laboratory born A. l. lemurinus monkeys. This represents a preliminary step to reduce K. pneumoniae produced mortality. A striking finding during a cross-sectional prevalence study was that none of the babies of less than 2 months old had detectable levels of antibody. The antibody prevalence gradually increased in all other age groups reaching 87.5% in the 8-10-month-old group. These results indicate that infection with K. pneumoniae occurred sometime between 2 and 6 months of age, probably as a result of oral-faecal contamination and a change in the feeding and grooming behaviour. To determine whether infants had maternal antibodies or if they were asymptomatic carriers of the bacterium, a cross-sectional study was done in 15 infants less than 4 months old and their mothers. K. pneumoniae antibodies were detected in 11/15 mothers with serum titers ranging from 1:4 to greater than 1:256 and the bacterium was isolated from 3 babies and one mother and her baby. Results showed that no maternal antibodies remained in babies older than 3 weeks old. A prospective study indicated a reduction in mortality from 20% for the previous 3 years to 3.7% (3/79) in AL(OH)3 K. pneumoniae bacterin vaccinated infants born during 1988-89.     

Quinolone therapy of Klebsiella pneumoniae sepsis following irradiation: comparison of pefloxacin, ciprofloxacin, and ofloxacin

Exposure to whole-body irradiation is associated with fatal gram-negative sepsis. The effect of oral therapy with three quinolones, pefloxacin, ciprofloxacin, and ofloxacin, for orally acquired Klebsiella pneumoniae infection was tested in B6D2F1 mice exposed to 8.0 Gy whole-body irradiation from bilaterally positioned 60Co sources. A dose of 10(8) organisms was given orally 2 days after irradiation, and therapy was started 1 day later. Quinolones reduced colonization of the ileum with K. pneumoniae: 16 of 28 (57%) untreated mice harbored the organisms, compared to only 12 of 90 (13%) mice treated with quinolones (P less than 0.005). K. pneumoniae was isolated from the livers of 6 of 28 untreated mice, compared to only 1 of 90 treated mice (P less than 0.001). Only 5 of 20 (25%) untreated mice survived for at least 30 days compared with 17 of 20 (85%) mice treated with ofloxacin, 15 of 20 (75%) mice treated with pefloxacin, and 14 of 20 (70%) treated with ciprofloxacin (P less than 0.05). These data illustrate the efficacy of quinolones for oral therapy of orally acquired K. pneumoniae infection in irradiated hosts.     

Comparative characteristics of the adhesive properties of microorganisms isolated from the urine of children with chronic obstructive pyelonephritis

The adhesive properties of 215 cultures, including 215 Escherichia coli strains, 43 Klebsiella pneumoniae strains and 60 Pseudomonas aeruginosa strains isolated from the urine of 124 children with chronic obstructive pyelonephritis were studied in the direct hemagglutination test simultaneously with those of 30 E. coli strains and 20 K. pneumoniae strains isolated from the feces of 50 healthy children, as well as 60 P. aeruginosa strains isolated from children with parenteral infections of other localization. E. coli and K. pneumoniae strains isolated from the urine of children with chronic obstructive pyelonephritis were found to have D-mannose-resistant hemagglutinins (68% and 37.2%) and a combination of mannose-sensitive and mannose-resistant adhesins (44.6% and 13.3% respectively). P. aeruginosa strains isolated from the urine of urological patients in the postoperative period showed the presence of mannose-resistant hemagglutinins to a greater extent (76.6%) than those isolated from children with parenteral infections of other localization (45%).     

Identification of species and capsular types of Klebsiella clinical isolates, with special reference to Klebsiella planticola

In the 77 reference strains for Klebsiella K types, there are 17 strains (22.1%) of Klebsiella planticola, 6 strains (7.8%) of Klebsiella oxytoca, 1 strain (1.3%) of Klebsiella terrigena, and 53 strains (68.8%) of Klebsiella pneumoniae. The species K. planticola, which was originally isolated from botanical and aquatic environments and hence thus named, was also identified at high incidence (81 strains, 18.5%) among the 439 recent clinical isolates of Klebsiella species. Among these K. planticola strains of hospital origin, 52 (64%) were isolated from sputum, 17 (21%) from urine, and the remaining 12 (15%) from other sources. The capsular types of these isolates were determined by the gel precipitation reaction. Seventy of 81 K. planticola isolates (86.4%) were typable by antisera to Klebsiella reference strains for K types and the K types of the clinical isolates distributed to 35 kinds of K types. The proportion of typable strains among clinical isolates of K. planticola was very similar to those in K. pneumoniae (87.5%) and K. oxytoca (86.0%).     

连续12年产超广谱β-内酰胺酶肺炎克雷伯菌感染分布及耐药性分析

目的了解产超广谱β-内酰胺酶（ESBLs）肺炎克雷伯菌（KPN）的临床分布及耐药性,为临床合理选用抗菌药物提供依据。方法用常规方法,结合法国梅里埃公司的自动化细菌鉴定分析仪分离鉴定细菌;用K-B纸片扩散法进行药敏试验;用双纸片协同筛选法和酶抑制剂增强纸片法检测ESBLs。对中国医科大学附属盛京医院1999年1月至2010年12月期间临床分离的产ESBLs和非产ESBLs KPN菌株的临床分布及其耐药趋势进行回顾性分析。结果 KPN的ESBLs检出率分别从1999年的19.82%上升至2010年的49.34%;KPN主要分离于痰占52.4%,其次是血液占17.74%,主要来源于外科病区和新生儿病区;产ESBLs菌和非产ESBLs菌对阿米卡星、左氧氟沙星等非β-内酰胺酶类抗菌药物的耐药率均显著增高,差异具有统计学意义（P〈0.01）,对亚胺培南和美罗培南都保持高度敏感,耐药率均低于2%。结论 12年间ESBLs检出率总体呈上升趋势,产ESBLs肺炎克雷伯菌表达对包括非β-内酰胺类在内的抗菌药物多重耐药,对碳青霉烯类抗菌药物仍保持高度敏感性。     

Comparative study of immunobiological properties of Klebsiella planticola and Klebsiella pneumoniae strains

The present investigation was based on information on the frequent isolation of K. planticola, inhabiting soil and water ecosystems, from respiratory tract secretions, urine, wound surface and other clinical material in infectious pathology of humans. The comparative study of the main immunobiological properties of K. planticola strain TCXA 91 and K. pneumoniae strain 204 was carried out. The strains were equally virulent for white mice, the infective dose being about 100x10(6) when introduced intraperitoneally in 0.4% agar. These strains exhibited protective cross activity in the animals challenged with K. pneumoniae virulent strain K16. The survival rates of mice for K. planticola TCXA 91 and K. pneumoniae 204 were, respectively, 60% and 70%, the death rate of the control animals being 100%. For the first time K. pneumoniae 204 was found capable of stimulating the growth of lawn grasses in the presence of sodium chloride. The results were evaluated by the germination of seeds, the length of the plantlet and the root. The data obtained in this study are indicative of the similarity of the immunobiological properties of K. planticola TCXA 91 and K. pneumoniae 204.     

Protease activity of Klebsiella pneumoniae of different virulence

The study of substrate specificity and activity of proteolytic enzymes secreted by K. pneumoniae strains with different virulence was carried out. The strains were cultivated in a liquid semi-synthetic medium. The biomass was inactivated, and the supernatant fluid was separated from microbial cells by centrifuging. In the supernatant thus obtained and in the fractions isolated by gel filtration with the subsequent purification on DEAE Sepharose elastase-like, trypsin-like and chemotrypsin-like proteolytic activity was determined. In K. pneumoniae strains with different virulence only a single proteolytic enzyme--elastase with a mol. wt. of 21 kD--was detected. The protease activity of the supernatant culture fluid did not depend on the virulence of the strain and was equal to 5,416-7,476 I.U./ml. The activity of the purified enzyme was 100% of the elastase-like activity of the supernatant culture fluid. The most virulent K. pneumoniae strain K2, whose LD50 for white mice was less than 10 microbial cells, was characterized by lower elastase-like activity. The absence of correlation between protease activity and K. pneumoniae virulence may be explained by the fact that surface glycoproteins of eukaryotic cells are glycosilated and thus slightly accessible for proteases.     

Histamine production by Klebsiella pneumoniae and an incident of scombroid fish poisoning.

A histamine-producing strain of Klebsiella pneumoniae was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning. None of the other nine gram-negative bacterial strains isolated from the tuna sashimi was capable of equivalent histamine production. Bacterial histamine production was monitored in a tuna fish infusion broth (TFIB), and the implicated K. pneumoniae was capable of producing 442 mg of histamine per 100 g of tuna in TFIB in 7 h under controlled incubation conditions. Only 12 of 50 other K. pneumoniae strains, representing 5 distinct biochemical types, which had been originally isolated from foods, were able to produce such levels of histamine in TFIB. No correlation was found between histamine production and other biochemical characteristics or antibiotic resistance. Of the 12 histamine-producing strains, 11 belonged to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the type 2 strains examined produced high levels of histamine in TFIB. Additionally, the implicated K. pneumoniae strain and one other strain belonged to type 1, which is characterized by positive reactions in the indole, urea, and Voges-Proskauer tests.