Frequency of detecting bacteria of the genera Citrobacter and Hafnia in different contingents of examined adults]

A study of the incidence of bacteria of the Citrobacter and Hafnia genus in adults permitted to establish a greater occurrence of the mentioned microbes in the patients with various acute intestinal diseases in comparison with the healthy ones. Bacteria of the Citrobacter and Hafnia genus were revealed in the patients with acute intestinal diseases of obscure etiology, which often were diagnosed as gastroenteritis, enteritis, etc. Bacteria of the Citrobacter and Hafnia genus were revealed in the dysentery patients. Results of studies carried out among various healthy groups of the population indicated no significant differences in respect to the carrier state of the Citrobacter and Hafnia bacteria both within each of the groups of the persons examined and between them. Further studies directed to the investigation of the etiological role of bacteria of the Citrobacter and Hafnia genus in the pathology of enteric disturbances are necessary.     

Biological properties of opportunistic microorganisms isolated from patients with acute intestinal diseases

The comparative study of the incidence of the pathogenicity markers (DNAase, RNAase, phosphatase and hemolytic activity) in shigellae and salmonellae, acknowledged as the causative agents of intestinal infections, and in opportunistic bacteria isolated from the feces of patients with acute intestinal diseases and healthy persons has been made. The study has revealed that DNAase and RNAase activity occurs most frequently in Shigella flexneri, in salmonellae and in opportunistic enterobacteria isolated from the intestinal contents of patients with acute intestinal diseases. In this respect they essentially differ from the same species of opportunistic enterobacteria isolated from healthy persons.     

Pathogenic characteristics of Escherichia coli strains in cases of asymptomatic bacteriuria and symptomatic urinary tract infections

The aim of this study was to examine if E. coli isolated from asymptomatic bacteriuria differed in pathogenic features from strains isolated from symptomatic infections of urinary tract. In this study 130 strains of E. coli isolated from women having asymptomatic bacteriuria and 112 strains isolated from patients with symptoms of urinary tract infection were examined. It was shown that E. coli isolated from patients with symptomatic urinary tract infection showed the more frequently ability to cause mannose-resistant haemagglutination of human erythrocytes, resistance to bactericidal activity of serum and haemolytic properties than those isolated from asymptomatic bacteriuria. These strains showed also the higher ability to adhere to Vero cells in tissue culture. Among E. coli strains isolated from persons with asymptomatic bacteriuria the pathogenic features were most frequently found in strains from healthy women and the most rarely in isolated from diabetic women.     

Study of the role of conditionally pathogenic enterobacteria in acute intestinal diseases]

Conditionally pathogenic enterobacteria were isolated in 34.2% of the patients with acute intestinal diseases (1559 cases examined in all). Such representatives of enterobacteria as Klebsiella, Hafnia, Citrobacter of 4, 8, and 22 serological groups. Proteus mirabilis of 6, 10, 13, 26, and 28 serological groups, and Proteus morganii mostly played the etiological role. In some of the patients conditionally pathogenic microbes only aggravated the main disease, taking part in the development of dysbacteriosis. In healthy individuals these microbes were much more rare and were encountered inconstantly.     

Investigation of the cytotoxic capacity of some adherent opportunistic enterobacterial strains by the MTT assay and transmission electron microscopy

The purpose of this study was to determine the cytotoxic effect on CaCo-2 intestinal cells of dialysates obtained from bacterial cultures of some enterobacterial opportunistic strains with different sources of isolation (food, stool culture, acute diarrhoea, urine culture), previously tested and selected for their intensive adherence and invasion capacity to the cellular substratum and also for their cytotoxic effect on cell monolayers. In this study the level of cytotoxicity was measured quantitatively by means of the MTT assay and qualitatively by transmission electron microscopy (TEM). The MTT method uses a tetrazolium salt for the quantitative spectrophotometric assay of CaCo-2 cells survival and proliferation rates in the presence of bacterial dialysates. This test detects the viable cells, which are able to reduce the tetrazolium salt and offers the advantages of a very simple, rapid and precise method. For TEM examination the ultrathin sections were prepared following the standard protocols. The most cytotoxic strains proved to be Citrobacter freundii 93 strain isolated from stool culture, and Enterobacter cloacae 43, isolated from food followed by E. coli 115 strain isolated from acute diarrhoea. These results correlate well with TEM results pointing out the cytotoxic effect of Enterobacter cloacae 43 strain and also its ability to induce attachment and to destroy the cell surface (A/E) of HEp-2 cells. Besides their great adherence and invasion capacity, the production and release of cytotoxic factors into the extracellular medium represent virulence factors in these strains. This could be responsible for the increase of the pathogenic potential of opportunistic bacteria and explain their implication in the etiology of severe infections and food-borne diseases. This study proved that the virulence of opportunistic pathogens is not correlated with the strain's origin, the most evident virulence features being exhibited by an Enterobacter cloacae strain isolated from food.     

Incidence of virulence-encoding genes among enteric Escherichia coli strains isolated from healthy subjects

Escherichia coli, heterogeneous species consisting of commensal and pathogenic strains, is causing a broad spectrum of intestinal and extra intestinal diseases, ranging from asymptomatic infections to septicaemia, according to its capacity to produce different virulence factors. The incidence of different virulence-associated genes among the strains isolated from healthy subjects, taking into account that the human gastrointestinal tract is considered an important source for spreading E. coli strains, was evaluated. A total of 241 E. coli strains isolated from 41 healthy subjects, working in the food chain and coming to the laboratory for periodical medical control, were investigated for harbouring patogenicity factors--encoding genes. Extra intestinal virulence-associated genes, pap, sfa/foc, afa, hly, cnf and intestinal ones eaea, bfp, agg, It, st, vtx1 (stx1), vtx2 (stx2) and ipaH, were targeted by PCR using cellular lysate for total DNA. Genes encoding for adherence were the most prevalent. A number of 67 strains (27.80%) were positive for pap genes and 34 strains (14.11%) presented PCR positive results when afa genes were targeted, but sfa/foc genes were identified in only 10 strains (4.15%). Genes encoding for toxigenesis were less prevalent. A total of 9 strains amplified hly genes, 2.49% were positive for cnf genes and only 2 strains presented vtx1(stx1) gene. The results are in concordance with those which demonstrate that healthy subjects carrying strains possessing virulence-encoding genes could represent a reservoir for environmental circulation of such strains, considered life-threatening when a receptive host is encountered.     

Hemagglutinating and adhesive capacities of Klebsiella and Enterobacter strains

The authors analyze the data of studies on the hemagglutinating and adhesive capacity of 290 cultures, including 118 K. pneumoniae strains and 64 E. cloacae strains isolated from sick children, as well as 59 K. pneumoniae strains and 49 E. cloacae strains isolated from healthy children. The hemagglutinating properties of the strains were determined in the hemagglutination test with fresh, formalin- and tannin-treated red blood cells, the adhesive properties were studied by light microscopy. Among K. pneumoniae and E. cloacae strains isolated in acute intestinal infections, mannose-sensitive hemagglutination and pronounced adhesive activity were prevalent in most cases. Poorly adhesive and nonadhesive strains were characteristic of K. pneumoniae and E. cloacae cultures isolated from healthy children. The strains isolated from sick and healthy children differed only by the prevalence of adhesive cultures.     

traT gene sequences, serum resistance and pathogenicity-related factors in clinical isolates of Escherichia coli and other gram-negative bacteria

The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gram-negative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (50-70%) than in that of strains isolated from normal faeces (20-40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.     

Characteristics of bacteria in the genus Proteus isolated from patients with sporadic and group intestinal diseases

The biochemical and biological properties of 148 Proteus strains isolated from patients both in sporadic intestinal infections and in a case of group infection in children's hospital was studied. The study revealed that the etiological factor of the group infection was P. mirabilis belonging to rare serovar 48:2. Proteus organisms isolated in sporadic infections belonged to a great number of serovars. No relationship between the isolated serovar and the nosological form of the intestinal disease was established. Among the Proteus strains under study, 82 strains showed atypical biochemical properties in 1 test or more. No correlation between the clinical diagnosis and the occurrence of atypical strains was established.     

Interrelation of the enterotoxigenic properties and the antigenic structure of Escherichia

The enzymatic signs and serological characteristics of Escherichia enterotoxigenic strains isolated from patients with acute intestinal diseases and from healthy persons were studied. The cultures were subdivided into 24 enzymatic variants and classified with 48 serogroups and 61 serovars. The enterotoxigenic properties of the strains were compared with their serological characteristics and enzymatic signs. The strains, isolated from different persons and classified with the same serovar, belonged to the same variant with respect to the type of enterotoxin they produced (only thermostable enterotoxin, only thermolabile enterotoxin, or both), were similar in the degree of their toxigenicity and belonged, as a rule, to the same enzymatic variant. The data on the presence of manifest interrelation between the enteropathogenicity of Escherichia and their structure, as well as on the stability of the enterotoxigenic properties of these organisms, indicate that in acute intestinal diseases the determination of Escherichia enterotoxigenic strains can be carried out by common bacteriological techniques with the use of specific agglutinating sera.     

Persistense of mono- and associated cultures of conditionally pathogenic enterobacteria

The comparative study of adhesive, hemolytic, DNA-ase, lecithinase, antilysozymic, anticomplementary activities of mono- and associated cultures of 57 Enterobacter spp., 61 Citrobacter spp. and 55 Serratia spp. strains, isolated from patients with pyoinflammatory, intestinal and urological diseases is carried out. Different variations of cocultivated bacteria including Enterobacter and Citrobacter, Enterobacter and Serratia, Citrobacter and Serratia are used. It was shown, that cocultivated Enterobacter, Citrobacter and Serratia bacteria increased the persistent properties of mixt cultures.     

Characteristics of staphylococci isolated from newborn infants

The microflora of the skin and the nasal mucosa was studied in 14 healthy newborns, 3 newborns with purulent infections and a nurse working in a neonatal ward. To make this study, washings were obtained with the use of 0.1% Triton X-100 solution. The determination of the number of microorganisms, the percentage of staphylococci, the percentage of lecithinase-positive staphylococci and the number of colonies with antagonistic properties revealed that these characteristics differed according to the state of health of the newborns and the time elapsed after their birth, and that changes in the number of antagonistically active bacteria occurred in parallel with changes in the total number of bacteria. Among S. aureus strains isolated from the subjects covered by the survey strains belonging to phagovar 80, as well as untyped strains, and among S. epidermidis strains those belonging to biovars I and II occurred most frequently. Strains belonging to the same biovar or phagovar differed in their plasmid markers.     

Adhesion of four Bifidobacterium strains to human intestinal mucus from subjects in different age groups

The number of bifidobacteria in faeces and intestinal contents has been shown to be reduced with increasing age of the subject. The adhesion of four Bifidobacterium strains was tested to mucus isolated from subjects of different age. All strains bound significantly less to mucus isolated from elderly subjects, compared to mucus from the other age groups. Two of the tested strains also showed decreased adhesion to mucus isolated from 6-month-old and adult subjects compared to the adhesion to mucus from 2-month-old subjects. The results suggest that reduced adhesion may be a factor involved in the decreasing colonisation of elderly subjects by bifidobacteria.     

Various biological properties of enterobacteria, isolated from patients with diarrhea

381 enterobacterial strains isolated from patients with acute enteric diseases were studied. Of these, 279 strains, as well as 20 strains isolated from 50 healthy children, were studied for the presence of adhesins, hemolysins and catalase. The comparison of the hemagglutinating activity of enterobacteria isolated from sick and healthy children revealed no essential differences between them. 15.8% of enterobacterial strains isolated from sick children possessed hemolytic activity, while strains isolated from healthy children did not induce the hemolysis of erythrocytes. All enterobacterial strains isolated from patients with acute enteric diseases were multi-resistant to antibiotics. A multitude of different antibiograms was obtained, most of them occurring only once. In 1985 the number of multiresistant (i. e. resistant to 11 and more antibiotics) strains dropped from 61% to 26..9% in comparison with 1981.     

Taxonomy of Citrobacter rods found in human specimens

The aim of this study was the identification of 181 Citrobacter strains on the basis of the recently proposed taxonomic changes of Brenner. All strains were isolated from diarrhoeic patients; 124 strains were originally sent for identification to Laboratory of Enterobacteriaceae DB NIH, 57 strains was isolated in Czech Republic. Citrobacter isolates were initially identified as C. koseri (3 strains), C. amalonaticus (1 strain) or as members of the C. freundii complex (177 strains). Additionally some biochemical tests were performed. The ability to grow in medium containing KCN, lysine decarboxylase production, lactose fermentation and PYR test were examined. Strains belonging to the C. freundii complex were identified to the species level by biochemical methods on the basis of the results of Brenner, who found some tests to be useful in separating Citrobacter species. These test included citrate and acetate utilization, arginine dihydrolase and ornithine decarboxylase activities, motility, urease production, esculin hydrolysis, and acid production from sucrose, dulcitol, melibiose, raffinose and salicin. On the basis of the criteria described above, 96.6% of the strains tested could be assigned to one of the recently named species of C. freundii complex. Using biochemical tests suggested by Brenner we were able to identify Citrobacter strains members of newly recognised species. A five-test system is proposed to identify the most frequently encountered species currently residing in the C. freundii complex.     

Isolation and Characterization of Corynebacteria from Burned Children

A total of 221 strains of corynebacteria were isolated and characterized by methods which included tests encompassing five schemes proposed for grouping cutaneous diphtheroids. Seventy-one strains (group I) were isolated from the hospital air in patient areas and from the normal skins of children admitted for reconstructive surgery of old healed burns and from the normal skins of nursing personnel. One hundred and fifty strains (group II) were isolated from various clinical specimens and from normal skins of a population of acutely burned children. The majority of the strains in group I were lipophilic and contained the largest number of fluorescent strains. Among the group II strains, there was a subgroup which was nonsusceptible to oxacillin, lincomycin, erythromycin, and kanamycin and also had in common the fermentation of glucose and galactose, reduction of both nitrate and nitrite, and growth on 40% bile agar. These strains were the most commonly recognized types isolated from acutely burned patients and possibly originated from the patient's intestinal tract. Data indicated that the air was not a means of transmission for these corynebacteria among acute patients. Corynebacteria were isolated from 11% of the burn wound cultures by using a selective medium but were found in 66% of the acute patients. Over 90% of the strains in groups I and II did not conform sufficiently with described characteristics of common human indigenous corynebacteria to be accurately speciated.     

Production of enterotoxin,verotoxin, hemolysin and cytotoxic necrotizing factor byEscherichia coli of intestinal and extraintestinal origin

Seventy-sixEscherichia coli strains were examined for heat-labile enterotoxin (LT), verotoxin (VT), hemolysin (HLy) and cytotoxic necrotizing factor (CNF). Thirty-six strains were isolated from patients suffering from diarrhea and forty from different extraintestinal infections. The number of LT-producing strains was low (2.6%) (one of intestinal and one of extraintestinal origin). Verotoxin was produced only by one extraintestinal strain. Four intestinal strains were hemolytic (11.2%) and also positive for CNF. From 24 hemolytic strains of extraintestinal origin (60%), 17 produced also CNF. Most of the hemolytic (30%) as well as CNF-producing strains (22.5%) were isolated from urine. Our results are similar to those of other studies confirming the close association between hemolysin and CNF production as well as a possible role of these toxic factors in pathogenesis of extraintestinal, infections caused byE. coli.     

Evaluation of the indirect haemagglutination test in the diagnosis of amoebiasis

The IHA test was evaluated in the diagnosis of amoebiasis. Axenically-grown E. histolytica was used as an antigen. A total of 427 sera from symptomatic (intestinal and extra-intestinal) amoebiasis patients. asymptomatic carriers, patients with parasitic intestinal infections other than E. histolytica, and healthy controls were tested. From 288 symptomatic cases of amoebiasis, 232 (80.6 per cent) gave positive reactions. In 93 asymptomatic cases of amoebiasis, 55 (59.1 percent) were seropositive. In testing of sera from 16 subjects with parasitic intestinal infections other than E. histolytica, a low-level positive IHA titres occurred in 2 (12.5 per cent). The test was also positive with a low titre in 3 (10.0 per cent) of the 30 subjects of the healthy control group. The results indicate that the IHA test is of value in the confirmation of intestinal and extra-intestinal amoebic infections especially in cases where the parasite is difficult to demonstrate.     

Enterotoxigenic Bacteroides fragilis in Hungary

Bacteroides fragilis, which constitutes about 1% of the colonic microflora in humans, is the most frequent anaerobic species involved in abscesses, soft-tissue infections and bacteraemias. Additionally, enterotoxigenic strains of B. fragilis have been demonstrated to be associated with diarrhoea in domestic animals and humans. Enterotoxigenic strains of B. fragilis derived from stool specimens and from infectious processes produce a toxin which induces a cytotoxic response in HT-29 colon carcinoma cells. These findings prompted us to investigate the prevalence of enterotoxigenic strains of B. fragilis isolated from various clinical specimens in Hungary. A total of 134 strains were collected from different clinical settings: 74 from infectious processes, 20 from stools of healthy subjects and 40 from the faeces of patients with diarrhoea where no other enteric pathogen could be isolated. Cell culture assays with HT-29 cells were performed on the filtered culture supernatants of the isolated strains. Of the 134 strains, 34 (25.3%) proved toxin-positive. The presence of free toxin was also observed in 20 of 50 (40%) of the faeces of adults with diarrhoea.     

Enterotoxigenic Escherichia in patients with acute intestinal infection

Out of 4,082 Escherichia strains isolated from 989 hospitalized patients with acute enteric infections 2,979 strains belonged to 139 O-groups and the remaining strains could not by typed with O-sera (O1-O164) or were in the serological R-form. 19.3% of the strains isolated from 354 patients showed a positive reaction for thermolabile enterotoxin (TLET) determined in the radioimmunoassay. Escherichia with pronounced reaction for TLET were isolated from 5.5% of the patients. The serological picture of Escherichia with a positive reaction for TLET comprised 78 O-groups, showed practically no variations in children of different age and adult patients and was related to the intensity of reaction for TLET. The number of enterotoxigenic Escherichia greatly varied within individual O-groups: from 3.1 +/- 3.63% (O11) to 100 +/- 9.81% (O114) of the total number of known strains in these O-groups. In most O-groups the number of enterotoxigenic strains varied from 10% to 50%. The most widely spread serovars of etiologically important enterotoxigenic Escherichia belonged to 24 O-groups and were often isolated as monocultures. The capacity for producing TLET (and, probably, some other antigenically related substances) in widely spread among Escherichia; still, the determination of enterotoxicity is a necessary, but not sufficient criterion of their etiological importance in acute enteric infections.