Strains Of Plesiomonas Shigelloides Research Articles

Characterisation of Plesiomonas shigelloides strains that share type-specific antigen with Shigella flexneri 6 and common group 1 antigen with Shigella flexneri spp. and Shigella dysenteriae 1

Three strains of Plesiomonas shigelloides isolated from patients with diarrhoea were agglutinated with Shigella flexneri 6 antiserum in slide and tube tests. All the strains were also agglutinated with a monoclonal antibody to the common group 1 antigen shared between S. flexneri serotypes and S. dysenteriae type 1. Further studies with one strain also showed sharing of antigenicity in an enzyme-linked immunosorbent assay. The results suggest that the strains share type-specific antigen with S. flexneri 6 and the common group 1 antigen with S. flexneri serotypes and S. dysenteriae 1. The sharing of antigens may have implications for cross-protection. One strain adhered to HEp-2 cell monolayers. None of the strains contained high mol. wt plasmids and there was no sequence homology with the invasiveness plasmid of Shigella spp. in DNA probe hybridisation. They were susceptible to the commonly used antibiotics. However, they possessed four other virulence-associated properties of Shigella spp. that included Congo-red binding, hydrophobicity, toxicity to HeLa cells and HEp-2 cell invasiveness (although they gave negative results in the Sereny test for invasiveness). These data suggest that the three unique strains might be considered pathogenic. Studies in animal models and human volunteers would be necessary to establish their pathogenic potential.

Expression of hemolytic activity by Plesiomonas shigelloides.

More than 90% of the Plesiomonas shigelloides strains that we tested produced a beta-hemolysin, as judged by the results of agar overlay and contact-dependent hemolysis assays. The hemolysin was cell associated, was active against the erythrocytes of various animal species, and was synthesized at both 25 and 35 degrees C. Activity was lost after thermal or proteolytic treatments or after preincubation in the presence of gentamicin; hemolytic activity did not appear to correlate with the previously established 50% lethal doses for seven of these strains. The hemolysin may play a role in iron acquisition in vivo via the lysis of erythrocytes, liberating hemoglobin, or, alternatively, may be involved in gastrointestinal disease.

Enzymatic profile of Plesiomonas shigelloides

The enzymatic activities of thirteen strains of Plesiomonas shigelloides were studied using the API enzyme research kit. This system uses chromogenic substrates to detect the presence or absence of 97 enzymes. Enzyme classes assayed include aminopeptidases, glycosidases, esterases, lipases, phosphatases and phosphoamidase. These strains were positive for 48 enzymes: 38 arylamidases, 7 esterases, alkaline phosphatase, acid phosphatase and N- acetyl-β- d- glucosaminidase . In contrast, 31 enzyme tests were negative in all strains. 18 enzymatic reactions were different in one or more strains. The results of tests for glycosidases were very heterogenous.

New Czechoslovak Serovars of Plesiomonas shigelloides

Summary Ten new Czechoslovak O and four H antigens of Plesiomonas shigelloides are described and compared with fifteen new Japanese O and ten H serovars. The Czechoslovak O serovars are as follows: “O35”-11440, “O36”- 13101, “O37”- 24827, “O38”- 27159, “O39”- 27492, “O40”- 27919, “O41 -28384, “O42”- 28720, “O43”- 29021 and “O44”- 29470, H antigens t, u, v and w. Two of the new Czechoslovak O antigens were identical with the Japanese ones: “O36”- 13101 with O64 and “O39”- 27492 with O66, but the latter Czechoslovak O serovar possessed a different H antigen t. The new Japanese serovar O61 was identical with the previously reported Czechoslovak serovar O4. No relationship was found between the four new Czechoslovak H antigens and the ten new Japanese ones. Three of the Czechoslovak serovars occurred in strains of previously unknown antigenic structure: “O35”- 11440, “O37”- 24827 and “O39”- 27492. Two recently described Japanese serovars O56 : H18 and O62 but with a distinct Czechoslovak H antigen d, and three H antigens H9, H22 and H28, were found in the Czechoslovak Plesiomonas shigelloides strains.

Optimal growth temperature for the isolation of Plesiomonas shigelloides, using various selective and differential agars

The growth characteristics of known strains of Plesiomonas shigelloides were compared with those of Aeromonas species (the major competing species in environmental waters) on plesiomonas differential agar, inositol brilliant green bile salt, and modified salmonella-shigella agar at incubation temperatures of 37, 42, and 44 degrees C. Using local isolates from clinical and environmental sources, optimal growth conditions, as determined by colony counts and the colony characteristics, plesiomonas differential agar proved to be ideal when incubated at 44 degrees C. Contrary to earlier recommendations for 48 h incubation, the colonies could be recognized readily after an incubation of 24 h.

Iron uptake in Plesiomonas shigelloides: cloning of the genes for the heme-iron uptake system

The iron uptake systems of Plesiomonas shigelloides strains were determined. Siderophore production was not detected by chemical or biological assays, and the strains tested were unable to use enterobactin, aerobactin, or vibriobactin for growth in low-iron media. Both hemin and hemoglobin supported full growth of the bacteria in media lacking other iron sources, but neither transferrin nor lactoferrin served as a source of iron. Hemolysin was detected, and the production of hemolysin was iron repressible. DNA sequences encoding hemolysin production and DNA sequences encoding the ability to use heme or hemoglobin as a sole source of iron were cloned from P. shigelloides and expressed in Escherichia coli. The abilities to use heme and hemoglobin as iron sources were closely linked, and the cloned sequences encoded the ability to transport the porphyrin, as well as iron, into the cells.

Cellular fatty acid composition of Plesiomonas shigelloides

The cellular fatty acid compositions of 29 strains of Plesiomonas shigelloides and 5 strains of Aeromonas hydrophila were studied. The cellular fatty acid compositions of all the Plesiomonas strains were identical and characterized by the presence of hexadecanoate (16:0) (33%), hexadecenoate (16:1) (28%), octadecenoate (18:1) (9%), and octadecanoate (18:0) (6%). The cellular fatty acid composition of A. hydrophila was similar to that of the Plesiomonas strains, except that the former contained an average of 25% 16:0, 29% 16:1, 12% 18:1, and 2% 18:0 acids compared with 33, 28, 9, and 6%, respectively, for the latter. The percentage ratios of 16:1 to 16:0 and 18:1 to 18:0 could be used to differentiate P. shigelloides from A. hydrophila. These ratios were 0.8 and 1.5 for the former and 1.2 and 6.0 for the latter.

PERITRICHOUS FLAGELLATION IN PLESIOMONAS SHIGELLOIDES STRAINS

In total, 131 strains of Plesiomonas shigelloides isolated from various sources were tested for peritrichous flagella by a flagella staining method. When incubated on a solid medium for 18 hr at 25 C, peritrichous flagella were demonstrated in 89 (68%) of them. With an electron microscope, the peritrichous flagella were clearly distinguished from the lophotrichous ones by their wavelength.

Laboratory investigations on the low pathogenic potential of Plesiomonas shigelloides

The pathogenic properties of 16 Plesiomonas shigelloides strains recovered from humans with extraintestinal and intestinal illnesses, infected animals, and environmental sources were investigated. Most strains possessed a high cell charge and low surface hydrophobicity analogous to those of Shigella spp.; additionally, serogroup O:17 strains reacted with Shigella group D antisera. However, unlike the shigellae, P. shigelloides strains did not universally bind Congo red, were noninvasive in HEp-2 cell assays, and did not produce a Shiga-like toxin on Vero cells. On HEp-2, Y1, and possibly Vero cells, a low-level cytolysin was consistently produced by all 16 P. shigelloides strains when grown in either Evan Casamino Acids-yeast extract or Penassay broth. The median 50% lethal dose for all 16 P. shigelloides strains in outbred Swiss Webster mice was 3.5 x 10(8) CFU (range, 3.2 x 10(7) to greater than 1 x 10(9) CFU). Animal pathogenicity did not correlate with cytolysin expression, possession of a greater than or equal to 120-MDa plasmid, protein profile, or resistance to complement-mediated lysis. No strain analyzed produced siderophores or a heat-stable enterotoxin. The results suggest that members of the genus Plesiomonas have an overall low pathogenic potential, irrespective of the site of isolation or phenotypic, serologic, or surface properties shared with other traditional enteropathogens.

Laboratory observations on Plesiomonas shigelloides strains isolated from children with diarrhea in Peru

Eleven strains of Plesiomonas shigelloides isolated from 10 Peruvian children with diarrhea were examined. All the strains were resistant to two or more antibiotics, most commonly ampicillin, gentamicin, erythromycin, kanamycin, and streptomycin. The strains were all negative in the Sereny and cell culture assays used to test for enteroinvasiveness. One strain showed cytotoxic activity on Vero cells. The strains showed no antigenic relationship with Shigella organisms. Both bioassays and enzyme-linked immunosorbent assays used for detection of Escherichia coli enterotoxins were negative. Nucleic acid probes for such toxins likewise gave negative results. The strains all possessed a large (approximately 200-megadalton) plasmid in addition to one or more other plasmids. Several different plasmid profiles were observed among these 11 P. shigelloides strains, indicating that the isolates were not acquired from a common source or from a single bacterial clone.

Production of a heat stable enterotoxin by Plesiomonas shigelloides

Eight strains of Plesiomonas shigelloides were assayed for enterotoxin production in the rabbit ileal loop assay. Seven strains required serial in vivo passage in the rabbit's intestine before enterotoxin activity was detected in the cells' filtrate. Enterotoxin production was readily lost with subculture of these toxinogenic cells. Heat treatment of the cells' filtrate from three strains that had never been passed in vivo led to detectable enterotoxin activity in three of six separate assays. Using LT, CT, STIa, STIb and STII as probes, no homology to the whole cell DNA of the eight strains was detected on Southern hybridization under low stringency conditions. The enterotoxin of P. shigelloides appears to be novel since production is induced by in vivo passage, activity is seen with heat treatment of cells' filtrate and there is no DNA homology to the cloned enterotoxin genes of Escherichia coli and Vibrio choleraea.

Identification of Enterobacteriaceae, Aeromonas spp. and Plesiomonas shigelloides with the ATB 32GN system

We have evaluated a new identification system based on 32 assimilation tests with 209 strains of Enterobacteriaceae, Aeromonas spp. and Plesiomonas shigelloides. Results of single tests are independent of the culture medium used (98.1% concordant results between blood agar and MacConkey agar). After incubation for 24 h (48 h) 82.3% (87.1%) of strains were correctly indentified to the species levek and 4.8% (5.3%) to the genus level. Misidentification occurred in only 2.4% of the strains. The excellent performance of the system with a difficult collection of strains will allow its use in routine identification of gram-negative rods in the clinical laboratory.

Evaluation of a small “Conventional” identification system for fermentative gramnegative rods using a computerized data base

A small “conventional” identification system consisting of six tubed media totalling eleven reactions was used to identify 467 strains of Enterobacteriaceae, 15 strains of Aeromonas sp., and 3 strains of Plesiomonas shigelloides. The system identified 371 strains (76.5%) correctly to maximum identification (species or genus) level and an additional 13 strains (2.7%) to genus level where species level identification would have been possible. Ninety strains (18.6%) were not identified. Only 11 strains(2.3%) were misidentified. The system is inexpensive compared with commercial systems and can be used for primary identification of fermentative gramnegative bacteria in a clinical microbiology laboratory. It comes with a computerized data base in the form of an identification manual.

Effect of acidity and antimicrobial agent-like compounds on viability of Plesiomonas shigelloides.

Nineteen Plesiomonas shigelloides strains were evaluated for their stability at acidic and slightly alkaline pHs and for their susceptibility to antimicrobial agent-like compounds produced by enteric flora. Most P. shigelloides isolates were rapidly inactivated under high-acid (pH 4 or less) conditions. Screening of enteric bacteria for elaboration of factors active against P. shigelloides revealed two organisms (Pseudomonas aeruginosa and Streptococcus [Enterococcus] faecium) capable of secreting such inhibitory substances. The results of this study suggest some factors potentially important in regulating gastrointestinal colonization by P. shigelloides from environmental sources.

Serotyping of plesiomonas shigelloides strains with our own antigenic scheme. An attempted epidemiological study

Thirty-four O and 19 H sera were used to test 169 strains of Plesiomonas shigelloides from several countries of three continents. The most frequent O serovar was 017 (O antigen identical with Shigella sonnei phase I) which occurred in the OH serovar combinations: 017:c, 017:d and 017:f. Some OH serovars were represented by a single strain; others, despite the small number of strains in hand, were evidently ubiquitous, having been isolated in mutually distant countries or at long time intervals. The source of our strains was most often human stools, diarrhoeal or collected at preventive examinations during epidemiological and ecological investigations, the droppings of animals (dogs, cats, pigs, sewer rats, water birds) and surface, sewer or aquarium water. The usefulness of serotyping in epidemiological investigation in the patients' environment was confirmed by a finding of two different serovars in one of our patients and her cat.

Tolerance of Plesiomonas shigelloides to pH, Sodium Chloride and Temperature

Forty strains of Plesiomonas shigelloides representing environmental and clinical isolates were characterized as to their tolerance to sodium chloride, acid and alkaline conditions and growth temperatures. In Tryptone broth all isolates grew in 3% but not 4% sodium chloride; whereas, in Trypticase Soy broth all isolates grew in 4% and 65% of them grew in 5% sodium chloride. All isolates initiated growth at pH 4.5 and 8.5 and 58% of the isolates grew at pH 4.0. Only 22% of the isolates grew at 8°C and 25% of them grew at 45°C. Heating at 60°C for 30 min destroyed all isolates tested.

Aeromonas and plesiomonas as possible causes of diarrhoea.

The number of reports on the isolation of Aeromonas from patients with diarrhoeal disease is now large and suggests an etiological role of the bacterium. It is well established that strains of Aeromonas produce an enterotoxin. This enterotoxin is cytotonic, i.e. it does not damage the membrane, and it does not cross-react immunologically with cholera toxin and Escherichia coli heat-labile (LT) toxin. Most enterotoxigenic strains also produce a cytotoxic protein (hemolysin), the role of which is probably limited in diarrhoea but potentially toxic in humans in other kinds of infections. Strains of Plesiomonas shigelloides also seem to be able to cause diarrhoea in some cases. The pathogenesis of Plesiomonas-induced diarrhoea remains to be elucidated, but a heat-stable enterotoxin may be involved.

Invasive Ability of Plesiomonas shigelloides

Five of 16 strains of Plesiomonas shigelloides freshly isolated from children with acute diarrhoea, exhibited invasiveness for HeLa cells comparable to that of Shigella sonnei. None of the five, however, gave a positive reaction in the Sereny test. Although three of the invasive strains shared antigens with Shigella, no correlation was observed between invasiveness and the possession of such antigens. Only one of the invasive strains produced a toxin detectable as causing rounding of HeLa cells, although others released a product which inhibited protein synthesis in HeLa cells but which failed to provoke any dramatic morphological change.

Differentiation of Vibrionaceae Species by Their Cellular Fatty Acid Composition

The cellular fatty acid compositions of 10 Vibrio species, two Aeromoas species, three Photobacterium species, Plesiomonas shigelloides, and Escherichia coli Were determined by using capillary gas-liquid chromatography (GLC). The major fatty acids in all Vibrionaceae species and E. coli were hexadecenoic, hexadecanoic, and octadecenoic acids. Qualitative and quantitative differences in hydroxy, branched, and cyclopropane fatty acids and in isomers of unsaturated 16- and 18-carbon acids were used to divide the 29 strains belonging to 17 species tested into 13 GLC groups. Of the 13 groups, 10 contained one species, 2 contained two species, and 1 contained three species. All of the Vibrionaceae cultures were differentiated from E. coli (GLC group I) because the concentration of hexadecenoic acid was greater than the concentration of hexadecanoic acid; in E. coli, this ratio was reversed. Aeromonas hydrophila (GLC group II) and Aeromonas salmonicida (GLC group III) were differentiated from the Vibrio and Photobacterium species and from Plesiomonas shigelloides because the Aeromonas cultures did not contain 3-hydroxylauric acid. Seven of 10 Vibrio species, including Vibrio fluvialis (GLC group IV), Vibrio parahaemolyticus (GLC group V), Vibrio alginolyticus (GLC group V), Vibrio harveyi (GLC group V), Vibrio splendidus (GLC group VI), Vibrio vulnificus (GLC group VII), and Vibrio cholerae (GLC group VIII), contained both cis-9-hexadecenoic and cis-11-hexadecenoic acids. These seven species could be differentiated from Vibrio gazogenes (GLC group IX), Vibrio metschnikovii (GLC group XII), Vibrio anguillarum (GLC group XIII), Photobacterium leiognathi (GLC group XIII), Photobacterium phosphoreum (GLC group XI), Photobacterium angustum (GLC group XI), and Plesiomonas shigelloides (GLC group X) because these latter seven species did not contain cis-11-hexadecnoic acid. The only Vibrionaceae cultures which contained cyclopropane acids were Photobacterium phosphoreum, Photobacterium angustum, and one of the two strains of Plesiomonas shigelloides examined. Branched-chain acids were found in all species tested, and their concentrations ranged from less than 1 to 22%. Although the 16 Vibrionaceae species tested had many similarities in their cellular fatty acid compositions, there were differences which could be used for differentiation of members of this family at the genus and species levels.

On the serology of Plesiomonas shigelloides.

The serology of 87 strains of Plesiomonas shigelloides was studied. Thirty O antigenic groups and 11 H antigens were defined within the 87 strains, and an antigenic schema consisting of 40 serovars was established. Some O antigens of P. shigelloides were identical or closely related to those of some Shigella serovars.