Characterisation of verotoxin-producing Escherichia coli strains isolated from human patients in Hungary over a 7-year period.

The purpose of this study was to characterise verotoxigenic Escherichia coli (VTEC) strains isolated in Hungary from 2000 to 2006. Altogether, 33 human VTEC strains were investigated to define the O:H antigens, verotoxin 1, 2 (vtx1 and 2), intimin (eae), enteroaggregative heat-stable toxin (ast1), autoagglutinating adhesin (saa) and enterohaemolysin (ehlyA) genes and sensitivity to 11 antimicrobial agents. The strains belonged to 14 different O:H serotypes, among which O157:NM (non-motile) was the most prevalent (45%, 15/33). Patients infected with O157 more often presented bloody diarrhoea or haemorrhagic colitis (63%, 12/19) than those infected with non-O157 (46%, 6/14). Haemolytic uraemic syndrome evolved in two patients infected with O26:H11. The vtx1vtx2c toxin gene combination was found in 58% (11/19) and vtx2c alone in 31% (6/19) of the O157 strains. All of the O157 strains possessed gamma1, while two O26 strains had the beta1 intimin gene. Twenty strains (75%, 25/33) carried the ehlyA gene and five non-O157 strains had ast1. The majority of the strains (76%) were resistant to at least one antimicrobial agent, but none of them showed the extended-spectrum beta-lactamase (ESBL) phenotype.

CTX-M-15-producing multidrug-resistant enteroaggregative Escherichia coli in the United Arab Emirates.

Extended-spectrum beta-lactamase (ESBL) production was demonstrated in five independent, multidrug-resistant isolates of enteroaggregative Escherichia coli (EAEC) from the United Arab Emirates, representing 11.3% of the EAEC isolates recovered during 1 year. All five isolates carried the bla(CTX-M-15) and the bla(TEM-1) genes, the former positioned 48 bp downstream of an ISecp1 element. In two isolates, the bla(CTX-M-15 )and bla(TEM-1) genes were located on a 95-kb plasmid. This is the first detailed description and characterisation of ESBL production in enteroaggregative E. coli and also the first report of CTX-M-producing organisms encountered on the Arabian Peninsula.

Phenotypic and genetic characterization of enteropathogenic Escherichia coli (EPEC) and entero-aggregative E. coli (EAEC) from diarrhoeal and non-diarrhoeal children in Libya.

A total of 50 Escherichia coli strains isolated in a Libyan hospital (20 from children with diarrhoea and 30 from healthy children) were investigated for their pathotypes and virulence traits. Altogether nine eae-positive (enteropathogenic E. coli, EPEC) and nine aggR-positive (entero-aggregative E. coli, EAEC) strains were identified. Significantly (P=0.001) more EPEC strains were identified from diarrhoeal patients (n=8) than from healthy controls (n=1), while six EAEC strains were identified from diarrhoeal and three from healthy children. Typical (eae(+), EAF(+), bfp(+)) EPEC strains (n=6) belonged to classical EPEC serogroups O55, O114, O127 and showed localized adherence on Hela cells. EAEC strains revealed genetic heterogeneity but uniformly adhered to HeLa cultures in an entero-aggregative adherence pattern. Antibiotic resistance frequently, characterized the strains. Sixty-eight percentage of the strains were resistant against at least one antibiotic and 30% harbored a class 1 integron independently of their clinical background. This is the first report from North Africa demonstrating the significance of EPEC and EAEC.

Identification of enteroinvasive Escherichia coli and Shigella strains in pediatric patients by an IpaC-specific enzyme-linked immunosorbent assay.

A new method, a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) recognizing a secreted, invasion plasmid-coded protein antigen (IpaC), was used to identify enteroinvasive Escherichia coli and Shigella strains among colonies from 859 cultures of fecal samples from children in Kuwait. A total of 33.8% of the samples were diarrheal. By the immunoassay, enteroinvasive E. coli strains were identified from two diarrheal samples but from none of the samples from children without diarrhea. These strains were fully virulent and belonged to serogroup O28ac. In addition, 26 Shigella strains were also recognized by the ELISA, while only 23 were isolated by routine biotyping and serotyping. For two diarrheal patients, Shigella was identified by culture only. The study showed that the IpaC-specific immunoassay is a simple and useful tool for identifying enteroinvasive strains. Furthermore, by reporting the first enteroinvasive E. coli isolates from Kuwait, the study indicates the presence of this group of pathogens as a potential source of diarrhea in the region.

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.

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.

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.

What is the diagnostic value of beta-D-glucuronidase (BDG) activity of bacteria using Fluorocult ECD agar for their cultivation?

A total of 1510 strains from 15 genera of Gram-negative and Gram-positive bacteria were studied. More than 94% of 327 Escherichia coli strains showed beta-D-glucuronidase (BDG) activity. Seventeen serotypes from 170 E. coli O serogroup representatives were negative. Relationship between the existence of BDG positive and negative E. coli strains in the same serogroup or serotype has not been observed. The rate of BDG positivity was 42% among Salmonella arizonae strains and 42.2% among Shigella strains. Only one Citrobacter strain out of the 971 strains belonging to Citrobacter, Edwardsiella, Enterobacter, Hafnia, Klebsiella, Proteus, Serratia, Yersinia, Pseudomonas, Aeromonas, Vibrio and Listeria was BDG positive. A screening method based on only BDG activity is not sufficient for the primary diagnosis of E. coli.

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.

beta-D-glucuronidase (BDG) activity of gram-negative bacteria.

BDG is an inducible enzyme that is encoded by the uidA gene in Escherichia coli. Genetic sequences of this gene are present in most if not all E. coli strains regardless of the BDG phenotype. Expression of BDG activity can be influenced by lactose-induced catabolite repression or genetic mutations. Salmonella, Shigella and Yersinia strains frequently exhibit positive BDG reaction. BDG activity of strains belonging to genus Edwardsiella, Serratia, Yersinia, Vibrio, Erwinia, Alcaligenes, Acinetobacter, Moraxella, Plesiomonas, Achromobacter, Flavobacterium, Chromobacterium and Pasteurella awaits examination.

The influence of growth conditions of Pasteurella multocida on its ability to colonise the nasal mucosa of SPF piglets.

Colonisation of type D Pasteurella multocida was studied in five groups of seven SPF piglets each. Piglets of Group 1 were kept together with seven 5-week-old piglets obtained from a large herd infected with toxigenic P. multocida for 16 weeks (contact infection). These piglets were made free from toxigenic Bordetella bronchiseptica by local immunisation. Piglets of Group 2 were inoculated with 5 x 10(7) colony-forming units (cfu) of P. multocida washed from the nasal mucosa of piglets free from toxigenic B. bronchiseptica with fetal calf serum. Piglets of Group 3 were inoculated intranasally with 5 x 10(7) cfu of P. multocida washed from yeast-extract proteose-peptone cystine (YPC)-blood agar with fetal calf serum. Piglets of Group 4 were inoculated with 5 x 10(7) cfu of P. multocida grown in a YPC-based broth without blood. Piglets of Group 5 served as controls. The piglets of Group 1 did not contract P. multocida infection from infected contact piglets. After a single inoculation one of four, while after three inoculations two of three piglets of Group 2 became infected by P. multocida. After a single inoculation none of four, while after three inoculations one of three piglets of Group 3 were colonised by P. multocida. Both single and repeated inoculation failed in piglets of Group 4.

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.

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.

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.