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.     

Comparative characteristics of the adhesive properties of microorganisms isolated from the urine of children with chronic obstructive pyelonephritis

The adhesive properties of 215 cultures, including 215 Escherichia coli strains, 43 Klebsiella pneumoniae strains and 60 Pseudomonas aeruginosa strains isolated from the urine of 124 children with chronic obstructive pyelonephritis were studied in the direct hemagglutination test simultaneously with those of 30 E. coli strains and 20 K. pneumoniae strains isolated from the feces of 50 healthy children, as well as 60 P. aeruginosa strains isolated from children with parenteral infections of other localization. E. coli and K. pneumoniae strains isolated from the urine of children with chronic obstructive pyelonephritis were found to have D-mannose-resistant hemagglutinins (68% and 37.2%) and a combination of mannose-sensitive and mannose-resistant adhesins (44.6% and 13.3% respectively). P. aeruginosa strains isolated from the urine of urological patients in the postoperative period showed the presence of mannose-resistant hemagglutinins to a greater extent (76.6%) than those isolated from children with parenteral infections of other localization (45%).     

A comparative study of the biological properties of pyelonephritogenic Escherichia coli isolated at different times in the infectious process

The comparative study of the biological properties of E. coli cultures, isolated from the urine of 7 patients two times during the first 11 days from the beginning of clinical manifestations of the exacerbation of chronic pyelonephritis, was conducted. In most cases the strains obtained as the result of the inoculations of the first and second urine samples belonged to the same serological and enzymatic variants. Still bacteria isolated in the second investigation, in contrast to E. coli obtained by the earlier inoculation of urine samples, often had no hemagglutinins and showed low adhesive capacity with respect to uroepithelium. Only in one out of 4 patient E. coli with antigen K1+ could be detected not only after the first inoculation, but also after the second one. In 4 patients E. coli cultures obtained as the result of the second isolation of these bacteria had lower content of sialic acid. Besides, differences in the sensitivity of E. coli strains isolated from the same patients in the course of the infectious process to the action of nonspecific protection factors of the body were established. The results obtained in the course of this study give more precise understanding of the existing conception of the pathogenesis of pyelonephritis.     

Resistance to antibacterial drugs of Enterobacteriaceae isolated from healthy children and from those with Salmonella infections

A total of 2329 Enterobacteriaceae strains in bacterial associations isolated from healthy children and children with salmonellosis were tested for their resistance to antimicrobial drugs. It was shown that the aerobic microbial associations isolated from the healthy children contained higher numbers of strains sensitive or resistant to 1-3 antibiotics while the microbial associations from the children patients with salmonellosis treated with antibiotics contained higher numbers of strains resistant to 6-8 antibiotics. Resistance of the aerobic bacterial associations was mainly defined by resistance of E. coli and K. pneumoniae. The feces of the healthy children never treated with antimicrobial drugs contained strains resistant to them. The use of the antibiotics in the treatment led to increasing numbers of the resistant bacteria and changing species composition of the bacterial associations.     

Occurrence of type-1C fimbriae on Escherichia coli strains isolated from human extraintestinal infections

Two monoclonal antibodies specific for type-1C fimbriae of Escherichia coli were produced. In enzyme-linked immunosorbent assay and immunoblotting the antibodies, which were of the IgG1 isotype, reacted with type-1C, but not with P or type-1 fimbriae of E. coli strain KS71. Immunoblotting and immunoprecipitation of crude fimbrial extracts from 25 strains invariably gave an apparent molecular weight of 17 000 for the type-1C fimbrillin. A total of 313 E. coli strains, isolated from patients with extraintestinal infection or from faeces of healthy children, were screened for the presence of type-1C fimbriae using both the monoclonal and polyclonal antibodies. Of these, 45 (14%) strains had type-1C fimbriae, with the highest frequency (27%) on strains isolated from patients with pyelonephritis. No faecal strain had type-1C fimbriae, and the frequency on the other diagnostic groups ranged from 11 to 15%. Thus, no direct correlation between type-1C fimbriae and bacterial virulence in human extraintestinal infections was found. Type-1C fimbriae were detected on only a few E. coli serotypes, notably on all O6:K2:H1 and O22:K13:H1 strains tested.     

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.     

Differential effects of catecholamines on in vitro growth of pathogenic bacteria

Supplementation of minimal medium inoculated with bacterial cultures with norepinephrine, epinephrine, dopamine, or isoproterenol resulted in marked increases in growth compared to controls. Norepinephrine and dopamine had the greatest enhancing effects on growth of cultures of Pseudomonas aeruginosa and Klebsiella pneumoniae, while epinephrine and isoproterenol also enhanced growth to a lesser extent. The growth of Escherichia coli in the presence of norepinephrine was greater than growth in the presence of the three other neurochemicals used in the study. Growth of Staphylococcus aureus was also enhanced in the presence of norepinephrine, but not to the same degree as was the growth of gram negative bacteria. Addition of culture supernatants from E. coli cultures that had been grown in the presence of norepinephrine was able to enhance the growth of K. pneumoniae. Addition of the culture supernatant fluid culture from E. coli cultures that had been grown in the presence of norepinephrine did not enhance growth of P. aeruginosa or S. aureus. Culture supernatant fluids from bacteria other than E. coli grown in the presence of norepinephrine were not able to enhance the growth of any bacteria tested. The results suggest that catecholamines can enhance growth of pathogenic bacteria, which may contribute to development of pathogenesis; however, there is no uniform effect of catecholamines on bacterial growth.     

The effect of Lactobacillus spp. on the attachment of enterotoxigenic Escherichia coli to isolated porcine enterocytes

A total of 43 strains of lactobacilli were isolated from the gastrointestinal tract of piglets at the time of weaning. Isolates, grown on solid media, were allocated to strongly adherent or non/weakly adherent groups on the basis of numbers attaching to isolated porcine enterocytes. Strains of Lactobacillus fermentum were disproportionally represented amongst the strongly-adherent strains and Lact.acidophilus and Lact. salivarius amongst the non/weakly-adherent group. Lactobacilli showed significantly better attachment ability when grown on agar than when grown in broth culture. Strongly adherent strains were not found to effect the attachment of enterotoxigenic Escherichia coli to porcine enterocytes, tested under the conditions of exclusion (lactobacilli added to the enterocytes before E. coli ), competition (lactobacilli and E. coli added simultaneously) and displacement ( E. coli added before lactobacilli). Tests were made with [¹⁴C]-labelled E. coli. Suspensions of bacteria and enterocytes were passed through a filter selected to retain enterocytes but pass free bacterial cells. Counts (dpm) obtained from filters after solubilization were taken as a measure of E. coli attachment. Some strains of lactobacilli coaggregated with enterotoxigenic E. coli with K88 fimbriae, but not with a K88-negative mutant strain. These were excluded from the competitive exclusion experiments. In the apparent absence of a direct effect on the association of E. coli with host tissue, removal of potential gut pathogens by aggregation could contribute to the probiotic properties ascribed to lactic acid bacteria.     

Pathogenic enteric Escherichia coli in children with and without diarrhea in Maputo, Mozambique

A study was conducted on the circulation of potentially diarrheagenic Escherichia coli in two groups of children, both under the age of seven. The first group (548 children) suffered from mild diarrhea and attended the Xipamanine Health Center of Maputo, in Mozambique. The second group (380 children) included randomly chosen, asymptomatic, children from the same population. A total of 503 E. coli strains were isolated from the two groups of children (n=375 and 128, respectively). All E. coli strains were genotypically and phenotypically screened. The presence of virulence-associated genes was assessed by a set of multiplex PCR specific for st and lt genes of enterotoxic Escherichia coli (ETEC), eae and bfpA genes of enteropathogenic E. coli (EPEC), stx(1) and stx(2) of enterohemorrhagic E. coli (EHEC), ial of enteroinvasive E. coli (EIEC) and the species-specific gene uidA. Adhesion and citotoxicity of isolated E. coli were evaluated in vitro on different cell cultures. A total of 37 isolates harbored virulence-associated genes: 18 were classified as ETEC, (15 from symptomatic, and three from asymptomatic children), 16 as EPEC (respectively, 13 and 3) and three EIEC in the symptomatic group. No stx(1) or stx(2) genes, associated with enterohemorrhagic E. coli were found. On the basis of the adhesion pattern on HeLa cells, 167 E. coli were classified as diffusely adhering, (125 in patients and 42 in controls) and 67 as enteroaggregative, (50 and 17, respectively). To the best of our knowledge, this is the first report in the literature on the circulation of potentially diarrheagenic E. coli in Mozambique.     

Incidence of enteritis of Enterobacteriaceae isolates possessing human colonization factor antigen. New human colonization factors

Of 462 Enterobacteriaceae strains including 435 Escherichia coli isolated from 250 patients, 298 haemagglutinating (HA) cultures were classified into 36 different HA groups. Sixteen of them belonged to Evan's I or II groups, although none possessed CF I or CF II antigen detectable by slide agglutination. Seventy-seven strains showed 4+ mannose resistant (MR) HA with human (53), bovine (2), chicken (6), guinea pig (7) or human and guinea pig (9) erythrocytes. These strains were significantly more frequent in patients under one year of age. Eighty-eight percent of the typable strains belonged to E. coli serogroups O1, O2, O4, O6, O18. HA positivity and fimbrial structures were correlated in 2 isolates (15/1, O18a, c:-K77: H-; 12/2/1 O1: K1: H .). Fimbriae of the two strains exhibited adhesive properties. Their fimbrial antigens differed serologically from each other and from those of the reference strains H 10407 and PB 176. Forty-nine of 4+ human MRHA strains showed variable reactions in the two sera for the new fimbrial antigens.     

The adhesive properties of a nonenteropathogenic strain of Escherichia coli in systems in vitro on isolated rat enterocytes and in vivo

The adhesive properties and colonizing capacity of E. coli strain O83, isolated from feces of healthy humans and marked according to its resistance to rifampicin and nalidixic acid, were studied. In vivo experiments on germ-free rats revealed that these bacteria were capable of colonizing intestinal mucosa; colonization increased from the small to large intestine and E. coli cells were mainly concentrated in the intestinal lumen and in mucin. In vitro studies showed that this nonenteropathogenic E. coli strain possessed pronounced adhesive properties with respect to the colonic cells of germ-free rats; these properties were considerably less pronounced with respect to the enteric cells of the small intestine. The electron microscopic study of E. coli cells revealed the presence of fimbriae and fibrillae on their surface.     

The production of F41 fimbriae by piglet strains of enterotoxigenic Escherichia coli that lack K88, K99 and 987P fimbriae

The enterotoxigenic Escherichia coli strains 1676, 1706, 1751 and KEC96a, which do not produce fimbrial adhesive antigens of the K88, K99 or 987P antigen type reacted both in vitro and in vivo with antiserum to F41 fimbriae in an indirect immunofluorescent antibody technique. Antiserum used to demonstrate material B, an adhesive antigen thought to mediate the adhesive and mannose-resistant (MR) haemagglutinating properties of E. coli strains 1676, 1706 and 1751, reacted in vitro with an F41+ strain. The antiserum also inhibited the MR haemagglutinating activity of F41 antigen and gave an anionic precipitation line in immunoelectrophoresis experiments with an extract containing F41 antigen. The MR haemagglutinating properties of an antigen extract containing material B from E. coli strain 1706 was neutralized by antiserum to F41 fimbriae and by OK antisera to E. coli strains that produce both F41 and K99 fimbriae. These sera also gave an anionic precipitation line with the MR haemagglutinin from E. coli strain 1706 and the MR haemagglutinin gave a line of identity with F41 in gel diffusion experiments with antiserum to F41 fimbriae. OK antisera to K99+ F41- bacteria and OK antisera to K88+ bacteria and 987P+ bacteria did not react with this haemagglutinin. Transmission electron microscopy on the ileum of newborn gnotobiotic piglets infected with E. coli strain 1706 showed irregular, poorly defined filamentous material surrounding some,though not all, bacteria but regular fimbrial structures were not visible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pathogenicity islands in opportunistic Enterobacteria

The presence of fragments of genomes hlyA, hlyB, papAH, papC, sfaG, sfaA and kps MT, associated with the pathogenicity islands of Escherichia coli, in clinical strains of other genera of the family Enterobacteriaceae, has been experimentally evaluated with the use of PCR. The presence of DNA fragments specific to the known genes of the pathogenicity clusters of E. coli in representatives of the genera Citrobacter, Enterobacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Serratia and Yersinia of rarely occurring groups has been established. In Enterobacteriaceae cultures isolated from the intestine amplicons homologous to hlyB were detected significantly less frequently than among strains of nonintestinal origin. In Enterobacteriaceae strains isolated in respiratory pathology amplicons of the pili gene (sfaG) were detected significantly more frequently than in collection cultures. The total evaluation of the detection rate of the genes of pathogenicity islands among Enterobacteriaceae clinical strains under study in comparison with E. coli showed that they occurred significantly less frequently. Klebsiella spp. were found to differ most essentially from E. coli as regards the occurrence of fragments of the genes of pathogenicity islands. The conclusion was made on the high probability of genetic exchange in DNA fragments between different species of bacteria with corresponding changes in their pathogenicity.     

Characterization of Escherichia coli serogroups causing meningitis, sepsis and enteritis. I. Serological properties and animal pathogenicity of O18, O78 and O83 isolates.

Escherichia coli O78: K80 strains isolated from an outbreak among premature and newborn infants with meningitis, sepsis and enteritis, from sporadic cases of enteritis and from healthy carriers were compared with one another and with different E. coli serogroups. The O78: K80 cultures uniformly failed to give the rabbit intestinal loop test and the guinea pig eye reaction and none of them contained L1 antigen. After intraperitoneal injection into mice, the organisms multiplied in the peritoneal cavity and caused bacteriaemia lasting at least 2 weeks. E. coli strains originating from septicaemia (O78: K80, O18a,c: K?, O83: K?) showed significantly lower LD50 values for mice (9 x 10(3)--7 x 10(5)) than did E. coli serogroups associated with infantile enteritis only (3 x 10(8)--7 x 10(8)). It is assumed that the isolates differ in pathogenicity not only from E. coli strains associated with "cholera-like" disease and with "dysenteriform" infection, but also from L1 antigen-containing cultures described in neonatal meningitis, and constitute a separate group characterized by an ability to cause meningitis, sepsis and enteritis within the same outbreak.     

Virulence characteristics of Escherichia coli isolates from children with urinary tract infections

The urinary tract is among the most common sites of bacterial infection and E. coli is by far the most common infecting agent in children and adults of both sexes. In an attempt to evaluate the intrinsic virulence of E. coli uroisolates from children, 54 strains were assessed by using PCR for the presence of five representative genetic determinants coding for adherence systems (pap, sfa/foc, afa), and toxins (hly and cnf). The prevalence of pap, sfa/foc and afa genes was 55%, 54%, and 44%, respectively. Hemolysin-encoding gene hly was detected in 55% strains, while cnf was exhibited by 35% of the screened E. coli isolates. Among the 39 PCR positive strains isolated from children's urine cultures the co-occurrence of the various targeted virulence genes was detected in 30 strains, the virulence profiles identified suggesting the presence of their localization on chromosomal regions known as pathogencity-associated islands. The rapid and reliable detection of the intrinsic virulence potential by this molecular approach could be very useful when evaluating the importance of microorganism pathogenicity versus host's susceptibility for developing an overt symptomatology of infection.     

Escherichia coli bacteriocins: antimicrobial efficacy and prevalence among isolates from patients with bacteraemia

Bacteriocins are antimicrobial peptides generally active against bacteria closely related to the producer. Escherichia coli produces two types of bacteriocins, colicins and microcins. The in vitro efficacy of isolated colicins E1, E6, E7, K and M, was assessed against Escherichia coli strains from patients with bacteraemia of urinary tract origin. Colicin E7 was most effective, as only 13% of the tested strains were resistant. On the other hand, 32%, 33%, 43% and 53% of the tested strains exhibited resistance to colicins E6, K, M and E1. Moreover, the inhibitory activity of individual colicins E1, E6, E7, K and M and combinations of colicins K, M, E7 and E1, E6, E7, K, M were followed in liquid broth for 24 hours. Resistance against individual colicins developed after 9 hours of treatment. On the contrary, resistance development against the combined action of 5 colicins was not observed. One hundred and five E. coli strains from patients with bacteraemia were screened by PCR for the presence of 5 colicins and 7 microcins. Sixty-six percent of the strains encoded at least one bacteriocin, 43% one or more colicins, and 54% one or more microcins. Microcins were found to co-occur with toxins, siderophores, adhesins and with the Toll/Interleukin-1 receptor domain-containing protein involved in suppression of innate immunity, and were significantly more prevalent among strains from non-immunocompromised patients. In addition, microcins were highly prevalent among non-multidrug-resistant strains compared to multidrug-resistant strains. Our results indicate that microcins contribute to virulence of E. coli instigating bacteraemia of urinary tract origin.     

Infectivity and resistance to antibiotics of bacterial strains isolated from patients hospitalised in intensive care units

The aim of this study was to evaluate the frequency of isolation and antimicrobial resistance testing of bacterial strains isolated from clinical specimens from patients hospitalized in three Intensive Care Units in Wroc?aw. The susceptibility of bacteria (107 strains) to selected antibiotics was determined. The results clearly show that non-fermentative rods were identified as the main agents causing pneumonia (58% of isolates). The second commonest pathogens were Gram-positive cocci (29%). The P. aeruginosa and E. cloacae strains were resistant to ampicillin, amoxicillin/clavulanate, cefuroxime and cefotaxime. All isolates of A. baumanii were susceptible only to imipenem. The rods of K. pneumoniae and E. coli were resistant to ampicillin, about 55% strains of both bacteria were sensitive to other antibiotics, except piperacillin/tazobactam, imipenem and ciprofloxacin. About 90% of methicillin resistant S. epidermidis strains were resistant to all antibiotics, except vancomycin (100% isolates were sensitive). ESBL were detected among E. cloace, K. pneumoniae and E. coli. We found P. aeruginosa rods producing MBL.     

Genetic determinants of Escherichia coli pathogenicity isolated from urine and feces of children with different clinical variants of urinary system infection

In the process of examination of 156 children of different age groups 176 E. coli cultures were isolated; of these, 98 cultures were isolated from acute cystitis and pyelonephritis patients, 28--from urine in cases of aysmptomatic bacteriuria, 30--from feces in cases of asymtomatc bacteriuria and intestinal dysbacteriosis, while 20 cultures--from feces of healthy children. In these bacteria the presence of genes associated with pathogenicity islets (PI) hlyA, hlyB, cnf-1, papC, sfaG and gene irp-2 (iron-regulated protein) was established with PCR. The detection rate of PI determinants in uropathogenic E. coli (UPEC) was shown to depend on the variants of the clinical manifestation of urinary tract infection. The total detection rate of PI gene fragments in UPEC cultures of different origin was indicative of their definitely less frequent occurrence in asymptomatic bacteriuria, observed simultaneously with intestinal dysbacteriosis, in comparison with acute urological infection. Practically the same detection rate of PI determinants in E. coli, isolated in asymptomatic bacteriuria in children, reflected high probability of genetic exchange in the above-mentioned fragments and made it possible to presume the existence of DNA sites, characteristic mainly of pathogenic clones. The established heterogeneity of the detection rate of PI determinants in E. coli clinical isolates requires further study.     

Spent media from cultures of environmental isolates of Escherichia coli can suppress the deficiency of biofilm formation under anoxic conditions of laboratory E. coli strains

The prevailing lifestyle of bacteria is sessile and they attach to surfaces in structures known as biofilms. In Escherichia coli, as in many other bacteria, biofilms are formed at the air-liquid interface, suggesting that oxygen has a critical role in the biofilm formation process. It has been reported that anaerobically growing E. coli laboratory strains are unable to form biofilms even after 96 h of incubation on Luria Bertani (LB) medium. After analyzing 22,000 transposon-induced and 26,000 chemically-induced mutants we failed to isolate an E. coli laboratory strain with the ability to form biofilm under anaerobic growth conditions. Notably, seven strains from a collection of E. coli isolated from different hosts and the environment had the ability to form biofilm in the absence of oxygen. Interestingly, spent medium from cultures of one strain, Souza298, can promote biofilm formation of E. coli laboratory strains growing under anaerobic conditions. Our results led us to propose that laboratory E. coli strains do not release (or synthesize) a molecule needed for biofilm formation under anoxic conditions but that they bear all the required machinery needed for this process.     

Combinations of pathogenicity factors in Escherichia coli isolated from children and domestic animals

The study has shown that in E. coli strains isolated from children aged up to 1 year with acute intestinal diseases of unknown etiology different combinations of pathogenicity factors (Ent. K88, K99, Vir, CFAI, CFAII, HIy, ColV, ColI) can be detected in 47.1 +/- 3.8% of cases, while in E. coli strains isolated from practically healthy children of the same age combined carriership of these factors does not exceed 5.8 +/- 2.5%. In E. coli strains isolated from swine and cattle with diarrhea the combined carriership of pathogenicity factors is 68.5 +/- 2.8% and 58.4 +/- 4.9% respectively.