Placental Transfer of IgG Antibodies Specific to Klebsiella and Pseudomonas LPS and to Group B Streptococcus in Twin Pregnancies

Group B Streptococcus (GBS), Klebsiella spp. and Pseudomonas spp. are important aetiological agents of neonatal infections in Brazil. There is a lack of data in the literature regarding the specific transport of immunoglobulin G (IgG) against these pathogens in multiple pregnancies. Maternal (n = 55) and umbilical cord (n = 110) blood samples were prospectively collected at birth from 55 twin pregnancies. The factors associated with cord levels and transfer ratios of IgG against GBS, Klebsiella and Pseudomonas were examined. The IgG umbilical cord serum levels specific to GBS, Klebsiella LPS and Pseudomonas LPS were significantly associated with maternal‐specific IgG concentrations and the presence of diabetes. The anti‐Klebsiella IgG cord serum concentrations were also related to birthweight and the presence of hypertension. The transfer ratios against GBS and Pseudomonas LPS were associated with maternal‐specific IgG concentrations. The transfer ratios for GBS and Pseudomonas LPS were associated with gestational age at delivery and the presence of diabetes, respectively. None of the examined parameters were related to Klebsiella LPS transfer ratios. We conclude that in twin pregnancies, specific maternal IgG serum concentrations and diabetes were the parameters associated with umbilical cord serum IgG concentrations reactive with the three pathogens investigated. All the other parameters investigated showed different associations with neonatal‐specific IgG levels according to the antigen studied. There was no uniformity of the investigated parameters regarding association with placental IgG transfer ratios against the GBS, Pseudomonas LPS and Klebsiella LPS.     

Systemic antibody response to diarrheagenic Escherichia coli and LPS O111, O157 and O55 in healthy Brazilian adults

Enterohaemorrhagic Escherichia coli (EHEC) can cause a variety of human illnesses ranging from uncomplicated diarrhoea to haemorrhagic colitis and haemolytic uremic syndrome. The serotype O157:H7 has been associated with numerous outbreaks worldwide, but in Brazil the infection is rare. Brazilian adults present antibodies reactive with the principal virulence factors of enteropathogenic E. coli (EPEC) that have many genetic and antigenic similarities with EHEC. Lipopolysaccharides (LPS) are components of outer membranes and important virulence factors of Gram‐negative bacteria. LPS O111 is present in EPEC and EHEC strains. LPS O157 is found only in EHEC strains, but it has some structural similarities with LPS O55 present in EPEC strains. This study investigates the levels of IgG and IgM seric antibodies reactive with EHEC O157:H7, EHEC O111:H–, EPEC O111:H– and the levels of anti‐LPS O111, LPS O157 and LPS O55 antibodies in healthy adults living in São Paulo, Brazil. The antibody levels were determined by an enzyme‐linked immunosorbent assay for 100 individual serum samples, and the presence of anti‐bacterial and anti‐LPS seric antibodies was confirmed. Positive correlations were found among the three kinds of antibodies. The concentrations of IgM anti‐LPS were significantly higher than those of IgG, and surprisingly, the concentrations of anti‐LPS O157 were high in view of the infrequent isolation of O157 bacteria in Brazil. Our results suggest that there is a cross‐reacting immunity to EHEC in the Brazilian population, which may be a result of the immunity to EPEC antigens. Alternatively, Brazilians may be exposed to EHEC more frequently than has previously been thought.     

A Three-Layer Immunoradiometric Assay for Determination of IgG Subclass Antibodies in Human Sera ("IgG Subclass RAST")

We report the development of a three-layer immunoradiometric assay (TIRA) for measurement of IgG antibodies of all four subclasses in human sera. The first layer consists of diluted human serum, the second layer is monoclonal mouse antibodies to human IgG subclasses., and the third layer is ¹²⁵I-labelled rabbit anti-mouse IgG. Monoclonal anti-IgG1. anti-IgC3 and anti-IgG4 reacted only with their complementary IgG subclass, whereas the anti-lgG2 showed slight cross-reactivity to immunoglobins of other subclasses and classes and to light chain proteins. The observed cross-reactivity was found to be without importance, when the TIRA was applied to measurement of IgG subclass antibodies. Equipotency was established by use of appropriate dilutions of the monoclonal antibodies, and the assay was calibrated by use of human reference serum. The TIRA therefore permits reliable inter-individual and intra-individual comparisons of the IgG antibody response in all four subclasses. Von-sped fie binding obtained with pooled normal human serum was below 0.33'#. Inter-assay coefficient of variation was between 18 and 27%, The TIRA was applied to measurement of IgG subclass antibodies to timothy grass pollen in sera from grass pollen allergies undergoing immunotherapy.     

Sequencing of Escherichia coli O111 O-Antigen Gene Cluster and Identification of O111-Specific Genes

Shiga toxin (Stx)-producing Escherichia coli strains of serogroup O111 are the most frequently isolated non-O157 strains causing outbreaks of gastroenteritis with hemolytic-uremic syndrome. The O111 O-antigen gene cluster had been cloned and about half of it has been sequenced; we have now sequenced the remainder of the gene cluster, which is 12.5 kb in length and which comprises 11 genes. On the basis of sequence similarity, we have identified all the O-antigen genes expected, including five sugar biosynthetic pathway genes, three transferase genes, the O-unit flippase gene, and the O-antigen polymerase gene. By PCR testing with E. coli strains representing all 166 O-antigen forms, some randomly selected gram-negative bacteria, and Salmonella enterica serovar Adelaide, we showed that four O-antigen genes are highly specific to O111. This work provides the basis for a sensitive test for the rapid detection of E. coli O111. This is important both for decisions related to patient care, because early treatment may reduce the risk of life-threatening complications, and for the detection of sources of contamination.     

Outbreak of infantile enteritis caused by enterotoxigenic Escherichia coli O6.H16.

Ten out of 18 babies at risk developed enteritis in an outbreak in a special-care baby unit. Salmonella, Shigella, and Escherichia coli belonging to the traditional infantile enteropathogenic serogroups were not found in faeces from the babies and the staff, and no virus particles were found by electron microscopy. Detailed serotyping of E. coli showed that five of the 10 babies with diarrhoea and one of the eight without diarrhoea were excreting E. coli O6.H16. All six isolates of this serotype produced both heat-labile and heat-stable enterotoxin. Enterotoxigenic strains of E. coli O6.H16 have caused outbreaks of enteritis in adults in the USA and Japan.     

Subclass Distribution of IgA and IgG Antibodies Against Clq in Patients with Rheumatic Diseases

To obtain insight into the immunoregulatory mechanisms in patients with different rheumatic diseases, the occurrence and the subclass distribution of IgA and IgG antibodies against Clq (anti-ClqAb) was determined. In patients with systemic lupus erythaematosus (SLE) the highest frequency of increased serum levels of IgG anti-ClqAb were found, whereas IgA anti-ClqAb were predominantly present in patients with ankylosing spondylitis (AS) and patients with rheumatoid arthritis complicated by vasculitis (RV). In all the IgA anti-ClqAb positive AS and RV patients the antibody reactivity involved the IgAl subclass while the IgA2 subclass was found in 47% of the patients. Further characterization of the IgA anti-Clq binding activity in sera of AS patients revealed that both subclasses of IgA anti-ClqAb were predominantly polymeric; the binding of both IgA subclasses with solid phase CI q was inhibitable by aggregated fluid phase Clq; we found no delectable interference of rheumatoid factor in the test system for the measurement of IgA anti-ClqAb. In patients with SLE the IgG anti-ClqAb reactivity was mainly of the IgG2 and IgG3 subclass, whereas in the same patients the IgG anti-tetanus toxoid response was not restricted to these subclasses.
The predominance of IgG2 and IgG3 subclass of anti-ClqAb in sera of SLE patients, suggests a skewing of the anti-ClqAb response. The observation that the IgA anti-ClqAb of both subclasses is predominantly polymeric in nature and the notion that polymeric IgA is associated with activation of inflammation cascades, suggests that IgA anti-ClqAb may contribute to tissue damage.     

Structural and functional differences between disease-associated genes of enterohaemorrhagic Escherichia coli O111

We analysed 72 clinical isolates of enterohaemorrhagic Escherichia coli (EHEC) O111 from patients with diarrhoea or haemolytic uraemic syndrome (HUS) isolated during the period from 1955 to 2005 and identified six motile strains (flagellar antigens 8, 10 and 11); the remaining 66 (92%) were nonmotile (NM) and could not be typed by conventional H serotyping. To improve subtyping methodologies, we determined genotypes of the flagellin-encoding fliC. Three fliC genotypes were found which were identical to those of motile EHEC O111 with H antigens 8, 10 and 11 and designated fliC(H8), fliC(H10) and fliC(H11). The IS629 insertion element was present, identically located, in six epidemiologically unrelated isolates with fliC(H8). The prevalence of the fliC genotypes in the 72 EHEC O111 strains were fliC(H8) (89%), fliC(H10) (7%) and fliC(H11) (4%). Within these fliC genotypes, a high degree of homogeneity for the presence of disease-associated genes was found. The adhesins-encoding genes eae and efa-1 were present in all strains with fliC(H8) and fliC(H11), but absent from strains with fliC(H10). The latter strains have not been reported previously. Strains with fliC(H10) and fliC(H11), but not those with fliC(H8), retained intact cadA and cadC loci and decarboxylated lysine. Three different stx genotypes including stx(1), stx(2) and stx(1)/stx(2) were determined among the 72 EHEC O111. We observed a significant increase over time in the frequency of strains harbouring both stx(1) and stx(2). The presence of stx(2) both alone and in combination with stx(1) was significantly (chi(2)=23.16, P<0.00001, CI(95) [2.29; 9.76]) associated with HUS. Therefore, the emergence of EHEC O111 should be monitored carefully. We conclude that EHEC O111 strains can be differentiated using specific loci required for motility, adherence, Stx production, and lysine decarboxylation. The divergence within EHEC O111 makes it possible to subtype these emerging pathogens in the laboratory thereby providing a basis for further investigations into their ecological niches and survival capabilities.     

Pathoadaptive mutation that mediates adherence of shiga toxin-producing Escherichia coli O111

The adherence of pathogenic Escherichia coli strains to intestinal epithelium is essential for initiation of infection. The cad operon encodes the lysine decarboxylase (LDC) system responsible for metabolizing lysine, and this operon has been proposed as an antivirulence mechanism in enteroinvasive E. coli and Shigella flexneri and as a factor mediating E. coli O157:H7 adherence. We sought to determine whether the LDC activity was present in a phylogenetically characterized collection of diarrheagenic E. coli (DEC) strains and to establish whether its expression was associated with their adherence to tissue culture cells. LDC activity was found in most of the pathogenic E. coli strains tested and was absent from Shiga toxin-producing E. coli (STEC) O111 strains (DEC pathotype 8). Analysis of the cad region in these O111 strains indicates that the operon has been rearranged and some of the genes are either missing or disrupted. A similar rearrangement was found in an E. coli O111:H8 strain recently isolated from an outbreak in Texas. Complementation of the LDC-negative strains with the cad operon in trans restored the LDC activity and resulted in a reduction in adherence to tissue culture cells. Initial analysis of the protein profiles on the surface of the O111 strains indicates that the LDC activity has an effect on the expression of the adhesin intimin. Cadaverine had a slight effect on LDC-negative strain adhesion but none on intimin expression. Our data suggest that this pathoadaptive mutation is an important mechanism to control functions potentially implicated in the pathogenesis of these organisms.     

The Development of IgA-Specific Antibodies to Escherichia coli O Antigen in Children

One hundred and sixty-five infants were studied longitudinally from birth to 5 years of age. One hundred and twenty-three school-age children and 27 adults were examined cross-sectionally. Total salivary IgA levels and IgA antibodies against Escherichia coli O antigens were measured. Total IgA levels were low (less than 20 mg/l) from birth to 4 years of age. At 5 years of age there was a dramatic increase in the total IgA level (geometric mean = 100.7 mg/l), after which the levels fell to values similar to those observed in adults (adult geometric mean = 53.2 mg/l). Low levels of IgA-specific E. coli antibodies were observed for the first 4 years of life (less than 1.0 ELISA units). There was a gradual increase in specific antibodies between 5 and 9 years of age (geometric men at 9 years = 4.66 ELISA units) to levels similar to those observed in adults (adult geometric mean = 8.20 ELISA units). It is suggested that the patterns of development for these variables reflect a balance between antigenic exposure and immune control mechanisms.     

Direct Detection of Shiga Toxigenic Escherichia coli Strains Belonging to Serogroups O111, O157, and O113 by Multiplex PCR

Shiga toxigenic Escherichia coli (STEC) strains are a diverse group of organisms associated with severe gastrointestinal and systemic diseases in humans. Within the STEC family, eae-positive STEC strains, particularly those belonging to serogroups O157 and O111, appear to have greater virulence for humans. However, in spite of being eae negative, STEC strains belonging to serogroup O113 have frequently been associated with cases of severe STEC disease, including hemolytic-uremic syndrome (HUS). We have developed a modified multiplex PCR assay for detection of STEC strains belonging to these three serogroups in cultures of feces by using primers specific for portions of the genetic loci (rfb) encoding biosynthesis of the respective O antigen. These primers direct amplification of PCR products of 259, 406, and 593 bp for serogroups O157, O111, and O113, respectively. The assay was validated by testing 40 previously characterized STEC strains, with 100% agreement. It also detected STEC strains of the appropriate genotype in primary fecal cultures from 13 patients with HUS or bloody diarrhea. Thirty other primary fecal cultures from patients without evidence of STEC infection were negative.     

Subclass Distribution of Human Anti-Staphylococcus aureus Alpha Toxin Antibodies: Suggestion of an IgG1, IgA1, IgG4 Switch Pattern

The subclass distribution of antibodies against alpha toxin from Staphylococcus aureus in man was investigated. Antibodies were restricted to IgG1, IgA1, and IgG4 and appeared to develop sequentially. These data suggest a distinct pattern of class and subclass switches in response to protein antigens. However, studies in immunodeficient (class- or subclass- deficient) donors show that the proposed patterns is not obligatory.     

Neonatal Liver Disease Associated with Placental Transfer of Anti-mitochondrial Antibodies

Background: Anti-mitochondrial antibody is the diagnostic hallmark of primary biliary cirrhosis. Its role in the aetiology of primary biliary cirrhosis is controversial. Methods: Two cases of neonatal hepatitis seropositive for anti-mitochondrial antibody are described. Anti-mitochondrial antibody Ig isotype and epitopic specificity were investigated by immunofluorescence and enzyme immunoassays. Results: In both infants anti-mitochondrial antibody was of the G class, mainly G1 and G3 subclasses, and reacted with two synthetic peptides reproducing major M2 epitopic regions: inner lipoyl domain pyruvate dehydrogenase complex (PDC)-E2_162-176 and PDC-E3 binding protein (PDC-E3BP)_86-100. One infant also reacted with outer lipoyl domain PDC-E2_35-49, and 2-oxoglutarate dehydrogenase complex (OGDC)-E2_99-113. An identical pattern of reactivity was present in their mothers, indicating the maternal origin of the antibodies. Anti-mitochondrial antibody disappeared in the infants with the disappearance of the liver pathology. Conclusions: The simultaneous disappearance of hepatitis and anti-mitochondrial antibody in the infants suggests a possible causal link between the two.     

Microevolution of epidemiological highly relevant non-O157 enterohemorrhagic Escherichia coli of serogroups O26 and O111

Enterohemorrhagic Escherichia coli (EHEC) are a cause of bloody diarrhea, hemorrhagic colitis (HC) and the potentially fatal hemolytic uremic syndrome (HUS). While O157:H7 is the dominant EHEC serotype, non-O157 EHEC have emerged as serious causes of disease. In Germany, the most important non-O157 O-serogroups causing one third of EHEC infections, including diarrhea as well as HUS, are O26, O103, O111 and O145. Interestingly, we identified EHEC O-serogroups O26 and O111 in one single sequence type complex, STC29, that also harbours atypical enteropathogenic E. coli (aEPEC). aEPEC differ from typical EHEC merely in the absence of stx-genes. These findings inspired us to unravel a putative microevolutionary scenario of these non-O157 EHEC by whole genome analyses.Analysis of single nucleotide polymorphisms (SNPs) of the maximum common genome (MCG) of 20 aEPEC (11 human/ 9 bovine) and 79 EHEC (42 human/ 36 bovine/ 1 food source) of STC29 identified three distinct clusters: Cluster 1 harboured strains of O-serogroup O111, the central Cluster 2 harboured only O26 aEPEC strains, while the more heterogeneous Cluster 3 contained both EHEC and aEPEC strains of O-serogroup O26. Further combined analyses of accessory virulence associated genes (VAGs) and insertion sites for mobile genetic elements suggested a parallel evolution of the MCG and the acquisition of virulence genes. The resulting microevolutionary model suggests the development of two distinct EHEC lineages from one common aEPEC ancestor of ST29 by lysogenic conversion with stx-converting bacteriophages, independent of the host species the strains had been isolated from.In conclusion, our cumulative data indicate that EHEC of O-serogroups O26 and O111 of STC29 originate from a common aEPEC ancestor and are bona fide zoonotic agents. The role of aEPEC in the emergence of O26 and O111 EHEC should be considered for infection control measures to prevent possible lysogenic conversion with stx-converting bacteriophages as major vehicle driving the emergence of EHEC lineages with direct Public Health consequences.     

Characterization of Enterohemorrhagic Escherichia coli O111 and O157 Strains Isolated from Outbreak Patients in Japan

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx₂ and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx₂ isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx₁, stx₂, and stx₁ stx₂), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx₂ isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx₂ to an stx-negative strain may have occurred during infection.     

Human IgG but not IgM antibodies can protect mice from the challenge with live O6 Escherichia coli

We evaluated the ability of human anti-lipopolysaccharide (LPS) O6 immunoglobulin G (IgG) and IgM antibodies to protect mice challenged with Escherichia coli serotype O6:K2ac. Purified whole IgG, commercial gammaglobulin, whole IgM-effluent, pool of normal human serum (NHS), agammaglobulinaemic serum (test groups) or phosphate-buffered saline (control group) was injected into adult male 18 h before a challenge with viable O6 E. coli. The mortality rate was assessed over a period of 72 h. To determine the opsonic and phagocytic activity of the antibody isotypes, we incubated peritoneal macrophages from the control and test groups collected at different times after challenge with the live bacteria with acridine orange for fluorescent analysis. Tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 were quantified in serum of both the test and control groups. All mice that received commercial gammaglobulin or NHS survived. Purified whole IgG (containing 1.1 mg/l of anti-LPS O6 IgG antibodies) protected 87.5% of the animals tested in this experiment, while whole IgM-enriched effluent with 1.5 mg/l of anti-LPS O6 IgM antibodies protected only 12.5%. The agamma serum showed no protective capacity compared with PBS (serving as control). The minimal concentration of anti-LPS O6 IgG antibodies able to protect 50% of animals was 0.137 mg/l of purified whole IgG. Whole IgM-enriched effluent showed no protective capacity independently of the concentration tested (0.048-17.0 mg/l of anti-LPS O6 IgM antibodies). Fluorescent analysis of peritoneal macrophages from animals pretreated with purified whole IgG showed no bacteria at 8 h after the challenge. By contrast, whole IgM effluent showed an increasing number of live bacteria at the same time. Mice that had received whole IgM effluent (1.5 mg/l of anti-LPS O6 IgM antibodies) before the challenge with LPS O6 presented 20.5 microg/l of IL-6 and 1.5 microg/l of TNF-alpha. Serum from animals pretreated with purified IgG did not present any detectable pro-inflammatory cytokine. Our findings suggest that IgG but not IgM antibodies protect animals from a challenge with E. coli O6 serotype.     

Analysis of P fimbriae on Escherichia coli O2, O4, and O6 strains by immunoprecipitation.

P fimbriae on Escherichia coli O2, O4, and O6 strains were analyzed by immunoprecipitation. Fimbrial extracts were prepared from a total of 35 strains and tested for precipitation with four anti-P-fimbria sera. The overall fimbrial composition of the strains was related to the O:K:H serotype, and two to three P fimbrial variants per strain were found on most of the O4 and some of the O6 strains. The O2 strains, in contrast, showed only one antigenic variant of P fimbriae per strain, which was serologically unrelated to those of the O4 and O6 strains. The results stress the multiplicity and serological complexity of E. coli P fimbriae.     

Characterization of antimicrobial resistance among Escherichia coli O111 isolates of animal and human origin

Fifty isolates of Escherichia coli serogroup O111 recovered from humans and various animal species over a 24-year period (1976-1999) were examined for typical virulence-associated factors and susceptibilities to antimicrobials of human and veterinary significance. Nine H (flagellar) types were identified including nonmotile (n = 24), 32 (n = 12), negative (n = 5), and 56 (n = 3). Thirty-five (70%) isolates possessed at least one Shiga-toxin-producing E. coli (STEC)-associated virulence determinants (eae, stxl, stx2, hlyA) via PCR analysis. Of these 35 isolates, 20 possessed eae, stxl, and hlyA genes, whereas three isolates possessed eae, stxl, stx2, and hylA genes. Multiple antibiotic resistance was observed in 70% of the 50 E. coli O111 isolates. The majority of isolates displayed resistance to streptomycin, sulfamethoxazole, tetracycline, and kanamycin. Bacterial resistance to ampicillin, gentamicin, chloramphenicol, trimethoprim and apramycin was also observed. Integrons were identified in 23 (46%) of the E. coli isolates assayed, with a 1-kb amplicon being most frequently observed. DNA sequencing of these integrons revealed the presence of the aadA gene, encoding resistance to streptomycin. Two integrons of 1.5 and 2 kb contained the aadA2 and either dfrI or dfrXII genes, encoding resistance to streptomycin and trimethoprim, respectively. Integrons were also identified from isolates dating back to 1982. Isolates were further genetically characterized via ribotyping, which identified 15 distinct ribogroups, with 62% of isolates clustering into four major ribogroups. Certain riboprint patterns from different animal species, including humans, were observed in isolates spanning the 24-year collection period, suggesting the dissemination of specialized pathogenic O111 clones.     

Molecular characterisation of verocytotoxin-producing Escherichia coli of serogroup O111 from different countries.

A collection of epidemiologically unrelated verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O111 isolated from human patients and cattle with diarrhoeal disease in five different countries were characterised by determination of their VT genotypes, the presence of other virulence factors such as the intimin-coding eae gene and the enterohaemorrhagic E. coli (EHEC) plasmid, and their antibiotic susceptibility patterns. The genetic relatedness among isolates was evaluated by genomic DNA fingerprinting techniques such as restriction fragment length polymorphism analysis of ribosomal RNA genes (ribotyping) and pulsed-field gel electrophoresis. The results indicated that the VTEC O111 examined belong to two distinct clonal lineages. The first group was constituted mainly of non-motile, eae-positive, EHEC plasmid-positive isolates from both man and cattle. The second lineage was represented by an O111:H2 epidemic strain, isolated during an outbreak of haemolytic uraemic syndrome in France and exhibiting an unusual combination of virulence factors: VT production and aggregative adhesion to HEp-2 cells associated with an enteroaggregative E. coli (EAEC) plasmid.