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.     

How to identify Raoultella spp. including R. ornithinolytica isolates negative for ornithine decarboxylase? The reliability of the chromosomal bla gene

Although Raoultella planticola and Raoultella ornithinolytica were described more than 20 years ago, identifying them remains difficult. The reliability of the chromosomal bla gene for this identification was evaluated in comparison with that of the 16S rDNA and rpoB genes in 35 Raoultella strains from different origins. Of the 26 strains previously identified as R. planticola by biochemical tests alone or in association with molecular methods, 21 harboured a bla gene with 99.8% identity with the bla gene of two reference R. ornithinolytica strains (bla(ORN) gene) and 5 harboured a bla gene with 99.2% identity with the bla gene of two reference R. planticola strains (bla(PLA) gene). The 9 isolates previously identified as R. ornithinolytica harboured a bla(ORN) gene. The bla gene-based identification was confirmed by 16S rDNA and rpoB sequencing. The 21 isolates newly identified as R. ornithinolytica had a test negative for ornithine decarboxylase (ODC). Molecular experiments suggested one copy of ODC-encoding gene in both ODC-negative R. ornithinolytica and R. planticola strains and two copies in ODC-positive R. orninthinolytica strains. Analysis of the 35 bla genes allowed us (i) to confirm an identity of only 94% between the bla genes of the two Raoultella species while this identity was > 98% for rpoB and > 99% for 16S rDNA genes and (ii) to develop and successfully apply a bla PCR RFLP assay for Raoultella spp. identification. Overall, this study allowed us to discover ODC-negative R. ornithinolytica and to provide a reliable Raoultella identification method widely available as not requiring sequencing equipment.     

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%).     

Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.     

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%).     

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.     

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.     

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.     

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.     

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.     

Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.     

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.     

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.     

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.     

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.     

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.     

The use of purine compounds as sole sources of carbon and nitrogen by Klebsiella species

Sixty-one strains of Enterobacteriaceae were tested for purine assimilation, including twenty-five Klebsiella pneumoniae, seventeen K. oxytoca and nineteen others. Only K. pneumoniae and K. oxytoca were able to use guanosine triphosphate, xanthine, hypoxanthine, or uric acid as sole sources of carbon and nitrogen. When guanosine triphosphate was used as sole source of nitrogen and carbon, the lag phase was prolonged. The addition of glucose did not affect the maximum number of viable cells for K. pneumoniae ATCC 29665, but produced an increase for strain K. oxytoca ATCC 13030. In the case of uric acid, ATCC 29665 had a more distinct lag phase of growth than ATCC 13030. Apart from this, they appeared to be very similar. On solid chemically defined GTP medium, some strains of Klebsiella were able to produce a water-soluble brown pigment.     

Fermentation and evaluation of Klebsiella pneumoniae and K. oxytoca on the production of 2,3-butanediol

Klebsiella is one of the genera that has shown unbeatable production performance of 2,3-butanediol (2,3-BD), when compared to other microorganisms. In this study, two Klebsiella strains, K. pneumoniae (DSM 2026) and K. oxytoca (ATCC 43863), were selected and evaluated for 2,3-BD production by batch and fed-batch fermentations using glucose as a carbon source. Those strains' morphologies, particularly their capsular structures, were analyzed by scanning electron microscopy (SEM). The maximum titers of 2,3-BD by K. pneumoniae and K. oxytoca during 10 h batch fermentation were 17.6 and 10.9 g L(-1), respectively; in fed-batch cultivation, the strains showed the maximum titers of 50.9 and 34.1 g L(-1), respectively. Although K. pneumoniae showed higher productivity, SEM showed that it secreted large amounts of capsular polysaccharide, increasing pathogenicity and hindering the separation of cells from the fermentation broth during downstream processing.     

Histamine-producing bacteria in blue scad (Decapterus maruadsi) and their abilities to produce histamine and other biogenic amines

Using decarboxylation medium and 16S rDNA sequence analysis, histamine-producing bacteria (HPB) in blue scad (Decapterus maruadsi) were isolated and identified, and the histamine-producing abilities of the isolated HPB were determined. Nine mesophilic strains (H1–H9) isolated from the muscle of blue scad were identified as the genera of HPB, including Arthrobacter bergeri (H1), Pseudomonas sp. (H2, H5 and H6), Psychrobacter sp. (H3), Shewanella baltica (H4 and H7), and Aeromonas salmonicida (H8 and H9), respectively. Results showed that most of the HPB strains were weak on histamine formation (13.0–20.4 mg/l), except for the H8 strain with the ability of producing 115 mg of histamine/l in trypticase soy broth containing 1.0 % l-histidine. As the strongest HPB in blue scad, bacterial strain H8 also presented a strong ability to produce other biogenic amines, such as putrescine, cadaverine, spermidine, spermine, tyramine and tryptamine. Therefore, the H8 strain identified as the genus of A. salmonicida was the dominant mesophilic HPB strain for producing histamine and other biogenic amines in blue scad at room temperature.     

Histidine Decarboxylases and Their Role in Accumulation of Histamine in Tuna and Dried Saury

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.