Yersinia Yop-specific IgA antibodies in Hungarian blood donors.

Sera of 112 healthy Hungarian blood donors were tested for the presence of Yersinia enterocolitica and Y. pseudotuberculosis-specific agglutinins by tube agglutination, and for that of yersinia outer membrane protein (Yop)-specific IgA antibodies by ELISA. The positive results of this latter assay were confirmed by immunoblot. Only one sample gave a positive agglutination reaction with Y. enterocolitica antigen (group 03) and four exhibited an equivocal reaction with Y. pseudotuberculosis antigens (groups II and IV). Contrary to the low incidence of agglutinins, 15.1% of the samples showed a positive Yop-specific IgA reaction, while further 5.3% samples fell into the equivocal range by ELISA (17 and 6 specimens, respectively). Eleven of these samples (9.8% of all specimens tested) were also positive by immunoblot for the presence of Yop-specific IgA antibodies. These data suggest a higher incidence of yersinia infections than the 1.0-1.4 per 10(5) population predicted on the basis of stool culture results.

Value of surveillance cultures in a bone marrow transplantation unit.

Because of the increased risk of infection with the associated diagnostic and therapeutic problems in bone marrow transplantation (BMT) patients, the usefulness of surveillance cultures (SC) at the BMT department of the National Institute of Haematology, Blood Transfusion, Transplantation and Immunology, Budapest, was reviewed. Between January 1992 and May 1995, 26 BMT operations were performed; 13 patients had 23 febrile espisodes. In 12 of these episodes infection was clinically documented; however, SC of these patients yielded bacteria identical with those in the blood culture in only two episodes (1 and 6 days before their blood cultures became positive, respectively). Out of a total of 1187 samples from these patients, potentially pathogenic bacteria were isolated from 145 SC and 43 blood cultures (drawn on 31 different days). Suppression of the gastrointestinal flora could be achieved by the department's decontamination regimen; however, overgrowth by gram-positive organisms (mainly coagulase-negative staphylococci) occurred in the intestine and at other body sites. On the basis of these results, SC are of limited value in predicting infection or identifying the causative organisms of fever. On the other hand, SC are useful in confirming the efficiency of suppression of the body flora by antimicrobial agents. Specific treatment was based on suitably sampled materials, and close contact between physicians, infectious disease specialists and microbiologists was essential.

Detection of VTEC using specific DNA probes and complex typing of Escherichia coli O157.

The incidence of E. coli causing hemorrhagic colitis (HC) or non-bloody enteritis in Hungary was studied using SLT-I and SLT-II gene-probes as well as Vero-cell toxicity and Verotox-F tests. Out of 41 E. coli O157 strains isolated in Hungary between 1987 and 1996 15 strains (O157:HNM 4, O157:H77 8, O157:HNT 3) derived from hemorrhagic colitis (HC). Hybridization was observed with SLT-I and/or SLT-II in 19 strains. Verocytotoxin production of E. coli of 23 other serotypes was proven by hybridization of DNA probes. SLT production were demonstrated in 24 strains. Complex typing (sero-, phage-, colicin-typing and plasmid profile analysis) was carried out in E. coli serogroup O157 strains isolated from different geographical areas. Using the Hungarian phages the E. coli O157:HNM, O157-H7 strains could be distributed into 6 phage groups each and these phage groups could be further divided according to colicin production and plasmid profile. The Hungarian phage typing method for E. coli strains used since 1978 was compared to the method elaborated in Canada in 1990. Out of the most frequent Canadian phage types (1, 4, 8, 31, 14) phage types 8, 31 and 14 were observed in Hungary.

Genotypic and phenotypic characters and nosocomial significance of bacteria endemic in neonatal intensive care units.

The degree of colonization was determined by complex typing (sero-, phage, colicin-, pyocin typing, plasmid profile analysis) of 212 Escherichia coli, 232 Klebsiella, 117 Pseudomonas aeruginosa and 52 Staphylococcus aureus strains isolated from nose, throat, ear and other sources of 563 new-born infants in gynaecological and maternity wards of two neonatal intensive care units (NICU I and II) during a one year period. The presence of Klebsiella strains was more frequent in NICU I and E. coli and P. aeruginosa in NICU II, S. aureus occurred in a low level in both units. In NICU I 34 kinds, in NICU II 43 kinds of E. coli serotype were found. In NICU I the accumulation of serotypes O6:H-, O6:H1, O19:H-, in NICU II O4:H-, O6:H1 was observed. The Klebsiella strains belonged in NICU I into 21, in NICU II into 12 phage types. Klebsiella was more frequent in NICU I than in NICU II, though the strains belonged to the same phage type in NICU II in 50.7%, but in NICU I 4 frequent and 19 rare phage types occurred. Sero- and pyocin typing was effective for typing of P. aeruginosa. The most frequent sero- and pyocin types were in NICU I:O11a,11b; in NICU II: O2a,2d,2f; 12v. The rate of antibiotic resistance in E. coli, Klebsiella, P. aeruginosa and S. aureus was nearly the same in both units, multiple resistance was more frequent in NICU I (except P. aeruginosa, it was multiple resistant in 100% in both units). In NICU I 267, in NICU II 174 infants were treated with antibiotics. The administration of penicillin derivatives was nearly similar in the two care units and the resistance among E. coli and Klebsiella strains was nearly the same too. Though, cephalosporins were used more frequently in NICU II, resistance to cephalosporins among E. coli and Klebsiella was a bit higher in NICU I. Aminoglycosides were more often used in NICU I, resistance to aminoglycosides among E. coli and Klebsiella was higher in this unit. The rate of isolation of the examined bacteria was significantly lower in the group treated with antibiotics, than in the untreated group.

Typing of coagulase-negative staphylococci isolated from immunocompromised patients.

A total of 3121 coagulase-negative staphylococcal strains sourced from clinical samples were characterized during a 4-year period. Biotype, antibiotic resistance pattern, phage pattern and slime production was determined. Plasmid profile analysis was performed on related isolates. Thirty percent of strains originated from the Bone Marrow Transplantation Unit, The National Institute of Haematology, Blood Transfusion and Immunology (NIHBTI), Budapest. Staphylococcus epidermidis occurred most frequently (48.8% in total, 58.2% source from NIHBTI). Total bacteriophage typability was 75.9%, and 603 phage patterns were observed. NIHBTI isolates differed in the incidence of multiply resistance, slime positivity and average frequency of phage patterns from the total suggesting spread of a selected hospital population. Statistical analysis of data obtained by typing showed no predominance of any endemic clone: the strains colonizing the immunocompromised patients and isolated from staff and inanimated environment differed from each other in biotype, phage pattern, antibiotic susceptibility, slime production and/or plasmid profile.

Computerized complex typing of Escherichia coli strains from different clinical materials.

A multivariate analysis of 3334 Escherichia coli strains originating from different clinical materials revealed that 50.2% of isolates belonged to the most common 12 (O1, O2, O4, O6, O7, O8, O15, O18, O45, O75, O78, O83) out of 133 serogroups. Haemolysin (Hly) production, mannose resistant haemagglutinating activity for human erythrocytes (MRHA) and colicinogenicity (Col) were recorded in 30, 30 and 36%, respectively. Antigens K1 and K5 were present in 11% and 6.6%, respectively. Association were found among certain serotypes and virulence markers (O1, H-, H7, K1, MRHA, Col; O2, H-, Kl, Col; O4, H-, H5, MRHA, Hly; O6, H-, H1, MRHA, Hly; O6, K5, MRHA, Col; O7, H-, H4, K1, MRHA, Col; O18ac, H7, K1, Col; O18ac, H-, K5, MRHA, Hly; O78, H-, Col (V-type); O83, H-, K1, Col). There were associations among clinical specimens, age of patients, nosocomial group of diseases, serogroups and virulence markers, too (cerebrospinal fluid-CSF-O7, O18ac, O45, O83-K1-newborn meningitis; O78-ColV-meningitis, sepsis, inflammations diseases of premature babies; CFS-O6, MRHA, Hly-adult-meningitis, sepsis, urinary tract infection-UTI-, pneumonia, other inflammatory diseases; blood-O2, O4, O6, O18ac, ONT, K5, MRHA, Hly-sepsis, UTI, hepatic diseases; urine-O1, O2, O4, O6, O18ac, O75, virulence markers fall to differ among upper and lower UTI; faeces-O1, O4, O6, O18ac, O78, virulence markers rare). Associations were also found among animal pathogenicity tests, specimens, serogroups and virulence factors: highly virulent group strains (i.e. LD50 below 10(6)) belonged to serogroups O2, O6, O18ac, possessed antigen K1 (less frequently the presence of MRHA, Hly, K5) and originated mainly from CSF. With mouse lung toxicity test correlations of serogroups (O4, O6, O18ac), antigen K5, MRHA, Hly and specimens (blood) were also shown. However, association was found between the lack of virulence factors and phage insensitivity and also between K5 positivity and sensitivity to phages 16, 17, there were no correlations between serogroups and phage patterns. On the basis of the above-described associations one can find correlations among virulence markers, serotype, and nosological group of diseases. Animal pathogenicity tests give additional data in understanding the pathomechanism of diseases. Correlations between phage patterns and serogroups reveal certain epidemiological relatedness and also virulence of strains.

[Significance of phenotypical properties in extra-intestinal E. coli infections]. / Az Escherichia coli fenotípusos tulajdonságainak jelentösége az extraintestinalis fertözésekben.

Virulence factors (serogroup, haemolysis, haemagglutination, antigens K1, K5, colicinogenicity) and their association with diseases of 3334 Escherichia coli strains isolated from different clinical specimens between 1979-1990 were analysed. Strains, that were isolated from cerebrospinal fluid of newborns under one month were characterized by certain serogroups (O7, O18, O45, O78, O83), possession of antigen K1 and production of colicin. On the basis of their LD50 they belonged to the pathogenic group (i.e. < 10 10(6)) significantly more frequently than those isolated from other materials. Strains originating from blood cultures belonged frequently to serogroup O4, O6, O18 and were haemolytic. Their pathogenicity was proved by mouse lung toxicity test. Properties of strains isolated from wound and throat swabs, urinary samples resembled to that of strains originating from blood cultures in many respects, expressing the fact that bacteria settle in different organs before sepsis is developing. Frequent occurrence of strains with antigen K1, haemolysin and haemagglutination positivity in vaginal swabs expose newborns to danger. Knowledge of virulence markers and prevention of infections are associated.

Comparison of rapid methods for detection of heat-labile (LT) and heat-stable (ST) enterotoxin in Escherichia coli.

Oxoid VET-RPLA, ST-EIA and Pharmacia Phadebact ETEC-LT enterotoxin tests were compared to find a simple but reliable method for detecting enterotoxigenic Escherichia coli (ETEC) in Hungary. In the Oxoid tests, all six reference LT- or ST-producing strains, except one ST-producer, gave positive results. Of 11 reference porcine enterotoxigenic strains, all four LT-producers gave positive reactions for LT but three of 10 ST-producers gave negative reactions for ST. Thirteen of 50 strains from culture collections of H. Steinrück (Germany) were LT+ and nine of 33 were ST+. When 31 isolates were tested simultaneously with the Oxoid and the Pharmacia LT tests, 12 strains were LT+ by the Oxoid LT test but by the Phadebact LT test only seven of these strains were LT+ and, of the remainder, three gave uncertain results and two gave negative results. Of 69 porcine strains, seven were LT+ and three ST+. Of 901 human strains isolated in Hungary, 10 were LT+ and one of 24 tested was ST+. In two cases, ETEC strains were isolated from contacts of travellers returning from Mongolia and Bangladesh. Results of comparative studies with reference strains corresponded well to those of the classical toxin detection tests. The Oxoid test was rapid, sensitive, specific and easy to perform and is recommended for use in screening ETEC isolates.

Specific binding of lactoferrin to Escherichia coli isolated from human intestinal infections.

The degrees of human lactoferrin (HLf) and bovine lactoferrin (BLf) binding in 169 Escherichia coli strains isolated from human intestinal infections, and in an additional 68 strains isolated from healthy individuals, were examined in a 125I-labelled protein binding assay. The binding was expressed as a percentage calculated from the total labelled ligand added to bacteria. The HLf and BLf binding to E. coli was in the range 3.7 to 73.4% and 4.8 to 61.6%, respectively. Enterotoxigenic strains demonstrated a significantly higher HLf binding (median = 19%) than enteropathogenic, enteroinvasive, enterohaemorrhagic strains or normal intestinal E. coli isolates (medians 6 to 9). Enteropathogenic strains belonging to serotypes O44 and O127 demonstrated significantly higher HLf binding compared to O26, O55, O111, O119 and O126. No significant differences in the degree of HLf or BLf binding were found between aerobactin-producing and non-producing strains. The interaction was further characterized in a high Lf-binding EPEC strain, E34663 (serotype O127). The binding was stable in the pH range 4.0 to 7.5, did not dissociate in the presence of 2M NaCl or 2M urea, and reached saturation within two h. Unlabelled HLf and BLf displaced the 125I-HLf binding to E34663 in a dose-dependent manner. Apo- and iron-saturated forms of Lf demonstrated similar binding to E34663. Among various unlabelled subepithelial matrix proteins and carbohydrates tested (in 10(4)-fold excess) only fibronectin and fibrinogen caused a moderate inhibition of 125I-HLf binding. According to Scatchard plot analysis, 5,400 HLf-binding sites/cell, with an affinity constant (Ka) of 1.4 x 10(-7) M, were estimated in strain E34663. These data establish the presence of a specific Lf-binding mechanism in E. coli.

Correlation between human lactoferrin binding and colicin susceptibility in Escherichia coli.

Escherichia coli H10407 demonstrated low 125I-human lactoferrin (HLf) binding (7%) and was insusceptible to group A (A, E1, E2, E3, E6, and K) and group B (B, D, Ia, Ib, and V) colicins. Conversely, a spontaneous HLf high-binding (44%) variant, H10407(Lf), demonstrated an increase susceptibility to both colicin groups. Colicin-insusceptible E. coli wild-type strains 75ColT, 84ColT, and 981ColT showed a low degree of HLf binding, i.e., 4, 8, and 10%, respectively. The HLf binding capacity was high in the corresponding colicin-susceptible mutants 75ColS (43%), 84ColS (32%), and 981ColS (43%). Furthermore, HLf low- (less than 5%) and high- (greater than 35%) binding E. coli clinical isolates (10 in each category) were tested for susceptibility against 11 colicins. Colicin V susceptibility did not correlate with HLf binding in either categories. However, with the remaining colicins, three distinct HLf-binding, colicin susceptibility patterns were observed; (i) 10 of 10 HLf low-binding strains were colicin insusceptible, (ii) 6 of 10 HLf high-binding strains were also colicin insusceptible, and (iii) the remaining HLf high binders were highly colicin susceptible. Certain proteins in the cell envelope and outer membrane of wild-type H10407 (HLf low binder, colicin insusceptible) showed a lower mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared to the corresponding proteins of mutant H10407(Lf) (HLf high binder, colicin susceptible). These mobility differences were also associated with HLf-binding proteins in Western blot (ligand blot) analysis. The wild type showed a smooth form of lipopolysaccharide (LPS) with a distinct ladder of O-chains, compared to the rough LPS of the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

[Determining the pattern of outer membrane proteins of gram-negative bacteria as a contribution to complex typing]. / Die Bestimmung des Musters der Proteine der äusseren Membran gramnegativer Bakterien als Beitrag zur komplexen Typisierung.

The aim of these investigations was to study relations between the serotype of E. coli strains and the pattern of their outer membrane proteins ("OMP") in sodium dodecylsulfate polyacrylamide gel electrophoresis. Three groups of strains being well characterized at least serologically (01, 02, 018ac containing different K, H, and in part F antigens) were submitted to this analysis. In all cases a nearly complete paralellity between OMP pattern and O:K:H(F:) type was observed, provided that the strains were epidemiologically related. The possibility is discussed that the OMP type could be used as a guide marker for the complex typing of E. coli strains.

Relative surface hydrophobicity, antigen K1 and haemagglutinating activity are associated in Escherichia coli.

Hydrophobic property of 136 Escherichia coli strains was examined by salt aggregation test (SAT). Out of the tested strains 61 were SAT positive. The correlations among the surface properties characterized by SAT and other phenotypical properties, e.g. mannose resistant haemagglutinating activity (MRHA), mannose sensitive haemagglutinating activity (MSHA), presence of antigen K1 and adsorption to Al(OH)3 gel were examined. The results showed that (i) Possession of antigen K1 provides the bacterial cell a hydrophilic character and covers its relative surface hydrophobicity; (ii) Correlation exists between the relative hydrophobicity of the bacteria determined by SAT and their haemagglutinating activity. SAT values are also influenced by non haemagglutinating fimbriae and also by other non fimbrial structures; (iii) The hydrophilic surface characters are mainly expressed by the results of adsorption to Al(OH)3 gel and the hydrophobic characters rather by the SAT values.

Clonal distribution of K1 and K5 antigen possessing Escherichia coli isolates.

A significant difference was observed in the occurrence of the examined markers (Col+, ColV+, Hly+, Aer+, AbR) and in the plasmid carrier state between strains with and without K1 and K5 antigens. Plasmids of the same size were harboured by serotypes possessing K1 and K5 antigens, e.g. among O1: K1: H- strains plasmids of 60-79 Md, among O1: K1: H7, O18ac: K1: H7, O45: K1: H7 and O83: K1: H- strains plasmids of 80-95 Md were frequent. The average plasmid number was higher in K1 strains than in K5 strains. In serogroup O1 the frequency of the plasmid carrier state was associated with the O serogroup and not with the K antigen. The plasmid number in K5 of serogroups O6 and O18 was lower than in K5- strains. Plasmids of 80-95 Md were predominant among the strains derived from blood and cerebrospinal fluid, whereas these plasmids were rare among the K1 and K5 strains isolated from other sources. Plasmids of 60-79 Md were frequent among strains derived from different sources. The 30-40 Md plasmids were relatively frequent among strains isolated from urine. In contrast with literary data, O1: K1: H-, O1: K1: H7 and other frequent serotypes consisted of different clones. Different clones were found within a single serotype, too.

Cloacin tolerance and aspecific colicin insensitivity of human Escherichia coli strains.

Of 182 wild-type human, aerobactin producer Escherichia coli strains 86.3% were insensitive to cloacin. All randomly chosen 51 strains were relatively cloacin tolerant. Cloacin tolerant strains were not considerably more sensitive to hydrophobic drugs than the cloacin sensitive descendant strains. Pathogenicity of the cloacin sensitive strains was significantly lower (p less than 0.05) in intraperitoneal mice infection than that of the cloacin tolerant ones. Suggesting a new aspect of the uptake mechanism of colicins, cloacin tolerance was very frequently associated with an aspecific insensitivity to a broad spectrum of colicins.

Association of virulence markers with animal pathogenicity of Escherichia coli in different models.

Employing chicken and several strains of mice, different routes (intraperitoneal, subcutaneous) of infections and isogenic pairs of strains, association of virulence markers with animal pathogenicity was studied in Escherichia coli. Mouse virulence of avian strains was less significant than the lethality for chicks of human strains. LD50 in various animals did not differ significantly. Strains with antigen K1 were more virulent for mice than their K1- derivatives. Loss of haemolysin (Hly), mannose resistant haemagglutinating capacity or antigen K5 less markedly decreased the virulence. As opposed to other virulence factors, increased virulence of K1+ strains could also be demonstrated in mouse sepsis assay based on bacterial counts in the liver. Loss of Hly alone did not influence the persistence in the liver, however, these strains killed less mice. Aerobactin acts together with other factors, it is not per se a virulence factor. In organotropic experiments 19 strains out of 36 belonging to serotypes O7:K1:H-, O18:K1:H-, O78:H- and spontaneously agglutinable K1+ cultures, caused ophthalmitis with purulent discharge, and 4 out of 22 strains that belonged to serotype O78:H- induced uncoordinated movement of mice. Because of its special organotropic affinity to the brain and as it caused two epidemics of meningitis among newborns in Hungary, serotype O78:H- has a special pathogenic property and differs from other O78 strains that were isolated in other countries.

In vitro and in vivo (LD50) effects of human lactoferrin on bacteria.

The in vitro and in vivo effects of human lactoferrin (LF), apoLF, iron saturated LF and of different iron containing compounds (ferric chloride, ferric sodium citrate) were studied on Escherichia coli, Salmonella typhi-murium and Pseudomonas aeruginosa reference and wild-type strains with well-defined virulence markers (i.e. enterochelin, aerobactin production). LF exert in vitro antibacterial effect, and iron-free Vogel-Bonner medium proved to be suitable for its determination. The effect of intraperitoneally administered LF could not be evaluated because of its aspecificity, as any treatment (e.g. saline, Ringer solution) before bacterial challenge activated macrophages. In contrast to the in vitro results, intramuscular challenge failed to inhibit bacterial growth in vivo, as siderophores produced by bacteria were able to acquire lactoferrin-bound iron. LF treatment, like iron addition, enhanced the virulence of bacteria in mice, whereas apoLF - using iron present in the body fluids - turned to LF being unable to acquire siderophore-bound iron from bacteria. These findings do not support the literary view that LF would be useful as an antimicrobial drug.

Bacteriological proficiency testing in Hungarian clinical microbiology laboratories.

In course of a proficiency testing programme carried out in 1989, a total of 47 clinical microbiology laboratories of public health stations and of hospitals received freeze-dried cultures for isolation, identification and antimicrobial susceptibility testing. The specimens contained bacteria that occur in everyday work, including those that require improved methods of cultivation and identification. Nine public health laboratories and one hospital laboratory achieved excellent results. Good results were attained by 11 public health and 6 hospital laboratories. Four public health and 10 hospital laboratories were on the medium level and 4 hospital laboratories did not reach even this degree. The main failures were due to an insufficient anaerobic cultivation, unreliable identification and negligation of controls for drug susceptibility tests.

The frequency of aerobactin production and its effect on the pathogenicity of human Escherichia coli strains.

A total of 981 human Escherichia coli strains (including 632 strains isolated between 1979 and 1983 and 349 strains isolated in 1987) was examined for aerobactin production by biological qualitative test. Aerobactin positivity was found in 55.1% and 47.3%, respectively, in the two groups of strains, while enterochelin was produced nearly by 100% of the strains. Aerobactin production was significantly more frequent than the average among blood and CSF strains samples and serogroup O2 and O6 strains. Aerobactin was more frequent among isolates with K1 or K5 antigens and producing haemolysin and mannose-resistant haemagglutination than among the ones lacking these virulence factors. A strict correlation was found between the pathogenicity in mouse following intraperitoneal infection and the frequency of aerobactin production. The distribution of the LD50 values of the aerobactin positive strains was shifted towards the lower values comparing to the aerobactin negative ones, proving statistically the effect of aerobactin in the increase of pathogenicity.

Adhesion properties of E. coli cells in the presence of promethazine.

Some E. coli strains were tested for adsorption to HEp-2 cells and on aluminium hydroxide gel. The adhesiveness of E. coli to HEp-2 cells was inhibited by promethazine. MRHA (mannose-resistant haemagglutinating activity) positive plasmid-carrying E. coli strains were found to be adsorbed to tissue culture cells more effectively than the MRHA-negative strains. Fifty percent of the clinical isolates contained antibiotic resistance plasmids, but only 40% of these strains were able to transfer the antibiotic resistance properties to E. coli as recipient. It is presumed that the hydrophobic adsorption of bacteria depends on the fimbriae, while aluminium hydroxide gel adsorption correlates with surface properties other than the fimbriae.

Adsorption to Al(OH), gel of Escherichia coli is correlated with O and K antigens and with type of extraintestinal infection.

Living suspensions of 89 Escherichia coli strains were tested for adsorption to Al(OH)3 gel in the presence of phosphate ions. On the basis of AC50 (phosphate molarity inhibiting 50% adsorption of the strain examined), E. coli strains could be classified into two main groups. Forty-three strains belonged to group 1 (AC50, 0.01-0.04), and 42 of them fell into serogroups O1, O2, O5, O7, O18ac, O83 or were spontaneously agglutinable. One strain in group 1 was exceptional as it had antigen O4. Of these 43 strains 33 had K antigen K1. Serogroup distribution of 46 group 2 strains (AC50, 0.001-0.009) was O2, O4, O6, O18ac, O75 and O78; 20 out of 46 possessed antigen K5. No correlation existed between H antigens or haemagglutinating capacity and AC50 of the strains. A close correlation was shown between AC50 pattern and the two main pathogenecity groups (i.e. "newborns' meningitis" and "sepsis and organotropic diseases") on one hand and between AC50 pattern and O, K serotypes on the other. The findings indicate that these E. coli strains with identical markers had a clonal connection.