Antibiotic susceptibility, serum response and surface properties of Klebsiella species

Altogether 130 clinical isolates of five Klebsiella species (K. pneumoniae, K. planticola, K. oxytoca, K. ornithinolytica and K. terrigena) were characterized, for their susceptibility to five antibiotics, for susceptibility to serum bactericidal activity and for their hydrophobic properties. All strains exhibited ampicillin resistance. Ampicillin/sulbactam, gentamicin and ofloxacin showed effectiveness in 63.1, 67.7 and 71.5% of the Klebsiella isolates. K. planticola manifested the highest level of resistance to these antibiotics. The majority of Klebsiella strains (93.9%) were susceptible to cefuroxime. Sixty-four strains (49.2%) were serum resistant and intermediate serum sensitivity was shown by 57 strains (43.8%). A high percentage of serum resistant strains (65%) was found in K. planticola. Moderately hydrophobic properties determined by adherence of bacteria to xylene were demonstrated in 25 strains (19.2%).     

Incidence of Klebsiella species in surface waters and their expression of virulence factors.

To investigate the occurrence of different Klebsiella spp. in aquatic environments, a total of 208 samples of natural surface waters was examined. From half (53%) of these samples, 123 Klebsiella strains were isolated, the most common species being Klebsiella pneumoniae. A comparison of these isolates to a group of 207 clinical K. pneumoniae isolates demonstrated that water isolates of K. pneumoniae, unlike those of K. oxytoca and K. planticola, are as capable as clinical isolates of expressing putative virulence factors such as serum resistance and capsular polysaccharides, pili, and siderophores.     

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90.6% of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.     

Klebsiella pneumoniae produces no histamine: Raoultella planticola and Raoultella ornithinolytica strains are histamine producers

Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiella pneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine.     

The Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae

A pathogenicity island termed high-pathogenicity island (HPI) is present in pathogenic Yersinia. This 35 to 45 kb island carries genes involved in synthesis, regulation and transport of the siderophore yersiniabactin. Recently, the HPI was also detected in various strains of Escherichia coli. In this study, the distribution of the HPI in the family Enterobacteriaceae was investigated. Among the 67 isolates pertaining to 18 genera and 52 species tested, nine (13.4%) harbored the island. These isolates were three E. coli, one Citrobacter diversus and five Klebsiella of various species (Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Klebsiella planticola, and Klebsiella oxytoca). As in Yersinia sp., all nine isolates synthesized the HPI-encoded iron-repressible proteins HMWP1 and HMWP2. In the K. oxytoca strain, the right-end portion of the HPI was deleted, whereas the entire core region of the island was present in the eight other enterobacteria strains analyzed. In most of these isolates, the HPI was bordered by an asn tRNA locus, as in Yersinia sp. This report thus demonstrates the spread of the HPI among various members of the family Enterobacteriaceae.     

Hydrophobic and hemagglutinating properties of Klebsiella pneumoniae and Klebsiella oxytoca

The aim of this study was to estimated hydrophobic and hemnagglutinating properties of Klebsiella pneumoniae and Klebsiella oxytoca rods. The hydrophobcity was evaluated according to the method of Rosenberg et al. The hemagglutinating properties were estimated by method of Blanco et al. Forty seven hydrophobic Klebsiella strains (30 K. pneumoniae strains and 17 K. oxytoca strains) were detected. Hemagglutinating properties were observed in 65 Klebsiella strains (45 K. pneumoniae strains and 20 K. oxytoca strains). Hemagglutination of sheep tannined erythrocytes the most frequently was observed. Inhibition of hemagglutination of erythrocytes by K. pneumoniae strains was most frequently observed in presence of D-glucose and D-mannose and by K. oxytoca strains in presence of D-glucose.     

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.     

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.     

Hemolytic activity of Klebsiella pneumoniae and Klebsiella oxytoca

We demonstrated that Klebsiella pneumoniae and Klebsiella oxytoca possess a selective haemolytic activity on rabbit erythrocytes. Thirty one Klebsiella strains (18 strains of K. pneumoniae and 13 strains of K. oxytoca) were isolated from hospitalized patients. The liquid (Trypcase-soy broth--TSB) and solid (Trypcase-soy agar--TSA) medium, containing the red cells were used for the tests. All the screened strains showed a haemolytic effect on rabbit erythrocytes, provided that the supernatants of the cultures were preincubated with beta-mercaptoethanol or calcium chloride. There was no human and sheep erythrocyte lysis.     

Detection, prevalence, purification and characterization of lecithinase of Klebsiella pneumoniae

Lecithinase activity in Klebsiella is a rare trait as out of 208 strains of Klebsiella belonging to 3 species, viz. K. pneumoniae (168), K. planticola (29) and K. oxytoca (11), only 4 strains of K. pneumoniae produced lecithinase positive colonies on egg-yolk-agar. Although cell lysates of 16 K. pneumoniae yielded positive results for lecithinase assay on egg-yolk-agar, 19 strains were detected positive for lecithinase with ELISA using anti-lecithinase serum. Release of up to 52.12% cell-bound lecithinase could be achieved with polymyxin-B treatment at 100 micrograms/ml concentration. Purified lecithinase was determined to be a high molecular weight (70 kDa), crystalizable, anionic (pI, 3.5) protein. It possessed cytolytic, haemolytic and dermonecrotic activities but did not induce fluid accumulation in rabbit ileal loop or infant mouse guts. It was inactivated by boiling, trypsin and chymotrypsin treatment and alkaline pH. Serologically, it was related to lecithinase of Aeromonas caviae and phospholipase-C of Salmonella.     

Susceptibility of Klebsiella oxytoca to selected antibiotics

Fifty two clinical isolates of K. oxytoca were included. All of analysed strains were isolated from wound swabs. The aim of this study was to evaluate MIC value of amoxicillin with clavulanic acid, tigecycline and ciprofloxacin. The susceptibility to amoxicillin with clavulanic acid and tigecycline was tested by the Etest. The susceptibility to ciprofloxacine was tested by the agar dilution method. Among of analysed K. oxytoca strains 44 (84.6%) were susceptible to tigecycline, 27 (51.9%) to amoxicilline with clavulanic acid and 21 (40.4%) to ciprofloxacine. These data suggest that tigecycline, may be an effective therapeutic option for the treatment infections caused by K. oxytoca strains.     

Antibiotic susceptibility and molecular mechanisms of cephalosporins resistance in Klebsiella isolates from patients with hospital-acquired infections]

Antibiotic susceptibility of nosocomial Klebsiella isolates from inpatients of 30 medical centres in 15 various regions of Russia was studied. In total 212 strains were tested. The Klebsiella genus was represented by the following species: Klebsiella pmeumoniae ss. pneumoniae (182 isolates, 85.8%), Klebsiella pneumonia ss. ozaenae (1 isolate, 0.5%), Klebsiella oxytoca (29 isolates, 13.7%). The susceptibility was determined by the broth microdilution method. Carbapenems (imipenem and meropenem) remained to be the most active antibacterial agents. However, 1 imipenem resistant strain and 2 meropenem resistant strains were isolated. As for the 3rd generation cephalosporins, the lowest MICs were observed with the use of the inhibitor-protected agents, such as ceftazidime/clavulanic acid (MIC50 0.25 mcg/ml, MIC90 64 mcg/ml). 48.8%, 16.9%, 29.7% and only 10.5% of the isolates was susceptible to cefepime, cefotaxime, ceftazidime and cefoperazone respectively. Detecting of the beta-lactamase genes (TEM, SHV and CTX) was performed by PCR in 42 strains of Klebsiella pneumoniae ss. pneumoniae. Alone or in various combination the TEM type beta-lactamases were detected in 16 (38.1%) isolates. SHV and CTX were detected in 29 (69%) and 27 (64.3%) isolates respectively. Combinations of 2 and 3 different determinants of resistance to beta-lactams were revealed in 23.8% and 26.2% of the isolates respectively. No isolates producing class B MBL among the carbapenem resistant nosocomial Klebsiella strains were detected.     

Klebsiella oxytoca: opportunistic infections in laboratory rodents

Opportunistic pathogens have become increasingly relevant as the causative agents of clinical disease and pathological lesions in laboratory animals. This study was conducted to evaluate the role of Klebsiella oxytoca as an opportunistic pathogen in laboratory rodents. Therefore, K. oxytoca-induced lesions were studied from 2004 to early 2006 in naturally infected rodent colonies maintained at The Jackson Laboratory (TJL), Bar Harbor, USA, the Animal Research Centre (Tierforschungszentrum, TFZ) of the University of Ulm, Germany and the Central Animal Facility (ZTM) of the Hannover Medical School, Germany. K. oxytoca infections were observed in substrains of C3H/HeJ mice, which carry the Tlr4(Lps-d) allele; in LEW.1AR1-iddm rats, the latter being prone to diabetes mellitus; in immunodeficient NMRI-Foxn1(nu) mice; and in mole voles, Ellobius lutescens. The main lesions observed were severe suppurative otitis media, urogenital tract infections and pneumonia. Bacteriological examination revealed K. oxytoca as monocultures in all cases. Clonality analysis performed on strains isolated at the ZTM and TFZ (serotyping, pulse field gel electrophoresis [PFGE], enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction, sequencing of 16S rRNA and rpoB genes) revealed that the majority of bacteria belonged to two clones, one in each facility, expressing the capsule type K55 (ZTM) or K72 (TFZ). Two strains, one isolated at the ZTM and one at the TFZ, showed different PFGE and ERIC pattern than all other isolates and both expressed capsule type K35. In conclusion, K. oxytoca is an opportunistic pathogen capable of inducing pathological lesions in different rodent species.     

Characterization of Klebsiella Isolates from Natural Receiving Waters and Comparison with Human Isolates

Two hundred sixty-six strains of Klebsiella pneumoniae isolated from natural water sources in geographically diverse areas (Florida, Massachusetts, and Oregon) were analyzed to determine the serotype, biochemical, virulence, and antimicrobial susceptibility differences between these natural strains and human Klebsiella isolates. Sixty of 72 defined serotypes were found among 210 typable strains. Geographic patterns were present, but in general were not pronounced among serotypes. Reactions with 28 biochemical tests showed percentage responses which were very similar to the summaries of primarily human Klebsiella isolates (as reported by Edwards and Ewing, 1972) and that represented diverse geographic sampling. Virulence studies in representative strains showed no geographic variability and little difference from comparable hospital patient-obtained isolates. In contrast to human hospital isolates, strains demonstrated 90% or greater susceptibility to all antibiotics except ampicillin and carbenicillin; and in further contrast, there was little multiple antibiotic resistance beyond that with ampicillin and carbenicillin.     

Taxonomic position of Klebsiella atlantae and Klebsiella edwardsii.

The taxonomic position of two species of the genus Klebsiella (K. atlantae and K. edwardsii) is being introduced. 211 strains of different origin were studied: 80 strains of K. pneumoniae, 46 strains of K. oxytoca, 50 strains of K. atlantae and 35 strains of K. edwardsii. Sixty biochemical characteristics were determined 40 of these pertaining to carbohydrate metabolism. It was discussed whether both are to be considered species or biotypes of another Klebsiella species, however, by determining citrate as carbon source, by MR test and by tests on malonate, gluconate, methyl-xyloside, 1 (--) sorbose, inulin, amylose, methyl-d-mannoside, glycogen, melezitose, VP test, amygdalin, d-tartrate and gas from glucose, we arrived at the conclusion that both could be considered species of the genus. These conclusions were confirmed by the determination of biotypes of both (according to C. Richard). Later, we expect to study their participation in human infective processes and their sensitivity by antibiotic therapy.     

Role of microbiological surveillance in the choice of antibacterial therapy for intestinal Klebsiella infection in infants

Data on the frequency of intestinal infections caused by Klebsiella spp. in infants at the age of 1 month to 1 year and antibiotic resistance of K. pneumoniae and K. oxytoca isolates from newborns and infants aged 1 month to 1 year are presented. The frequency of the Klebsiella isolates from the newborns and infants at the age of 1 month to 1 year with acute intestinal infections amounted to 17.7 +/- 1.4 and 42.5 +/- 1.4 per cent, respectively. The majority of the clinical strains had a multiple resistance to 7-9 drugs. The overwhelming majority of the strains were sensitive to gentamicin, monomycin, kanamycin, neomycin and polymyxin B.     

Multiplex-PCR assay for identification of Klebsiella pneumoniae isolates carrying the cps loci for K1 and K2 capsule biosynthesis.

Multiplex-PCR assay for identification of Klebsiella pneumoniae isolates carrying gene clusters for biosynthesis of capsular polysaccharide (CPS) types K1 and K2 was developed. Genes wzc and orf10 of the cps cluster were applied as K1 and K2 specific markers respectively. The assay specificity was confirmed using 147 isolates of Klebsiella spp. including 77 K-antigen reference strains. The multiplex-PCR assay was found simple and cost-effective tool for identification of K. pneumoniae clinical isolates of K1 and K2 geno-serotypes.     

Identification of the 4-hydroxycinnamate decarboxylase (PAD) gene of Klebsiella oxytoca

A 7.1-kbp DNA fragment isolated from a wild strain of Klebsiella oxytoca was sequenced, leading to the identification of 10 open-reading frames (ORFs), including a 504-bp Pad gene. The Pad gene of the Gram-negative bacterium was subsequently expressed in Escherichia coli as a chimeric Pad. The deduced amino acid (AA) sequence of the Pad gene from wild-type K. oxytoca showed approximately 50% homology to those of other bacterial PADs from Gram-positive bacilli plus a coccus. These data and a genomic library search of some gamma-proteobacteria, including E. coli and Vibrio sp., indicated that PAD of K. oxytoca is a member of the bacterial PAD family characteristic of Gram-negative bacteria. Using Pad-specific PCR primers designed from the Gram-negative bacterial Pad of K. oxytoca, Pad genes of two further strains of K. oxytoca, another wild isolate and JCM 1665 and two PAD-positive Enterobacter spp. were successfully amplified for specific Pad detection.     

Combined Biochemical and Serological Typing of Clinical Isolates of Klebsiella

In a series of 640 strains of Klebsiella isolated from clinical specimens over a 7-month period, there were sufficient biochemical differences between strains to allow a biochemical typing system to be established. Biochemical tests were done in solid media inoculated with a modified Steers inocula replicator. Biotypes were designated by a numerical coding system; 29 distinct biotypes were found among the 640 strains of Klebsiella. Serotyping of 270 of the strains was done by the Quellung reaction, and 40 capsular types were identified. Numerical biotypes and serotypes of strains appeared to vary independently. When used in conjunction, the two methods subdivided the strains into many more distinct types than either used alone. With the combined method over 100 types of Klebsiella were distinguished among the 270 isolates.     

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.