Contamination of Pig Carcasses at Two Abattoirs by Escherichia coli with Special Reference to O-serotypes and Antibiotic Resistance

Evidence of the source of carcass contamination of pigs at slaughter was obtained by determining presumptive coliform counts on faeces and on carcass surfaces, and comparing the O-serotypes and antibiotic sensitivity patterns of Escherichia coli from both sites. All of the 16 pig carcasses from the slaughter line of a commercial abattoir were contaminated with presumptive coliform bacilli on most sites examined; the carcasses of six out of eight pigs slaughtered at the Meat Research Institute (MRI) abattoir were also contaminated, but only small numbers of coliforms could be detected on a few of the sites. The proportion of O-serotypes of E. coli present in faeces which were also detected on carcass surfaces, indicating faecal contamination, varied between 0 and 8.6% in MRI slaughtered pigs but reached 66.6% in one group of commercially slaughtered pigs. O-serotypes found on carcass surfaces but not in the faeces of the pigs, were used as an indication of environmental contamination and this was very evident in the commercially slaughtered pigs. A high proportion of E. coli O-serotypes in the gut were resistant to antibiotics and these were also often found on the carcass surface and, since the range of O-serotypes in the pig is similar to that reported in man, the pig must be considered to be a potential reservoir of antibiotic resistant E. coli for man.     

An Investigation of Calf Carcass Contamination by Escherichia coli from the Gut Contents at Slaughter

The degree of carcass contamination at slaughter of three groups of calves (a total of 36 animals) was studied and the O-antigen types of Escherichia coli found on the carcass surface and in rectal contents were determined. In one-third of the animals E. coli strains found on the surface of a carcass belonged to the same O-serotype as those found in the rectal sample of the same calf. Nearly all the O-serotypes found on the carcasses were also found in the rectal contents of at least one calf, showing that cross-contamination of carcasses had occurred. The E. coli isolated from the rectal contents were all resistant to at least one antibacterial agent.     

Fluctuations in Escherichia coli O-serotypes in Pigs Throughout Life in the Presence and Absence of Antibiotic Treatment

A detailed study, throughout life, of the coliform and Escherichia coli gut flora of two pigs, is presented. One pig was given oral tetracycline for 3 d on two separate occasions; the other pig was not treated and housed separately. The fluctuations in numbers of coliforms, O-serotypes of E. coli , the occurrence and persistence of antibiotic resistance, and their interrelations, are analysed for both animals. Oral tetracycline profoundly affected the proportion of antibiotic sensitive and resistant E. coli , and, by selecting for R-factor bearing O-serotypes, limited the number of O-serotypes isolated from individual faceal specimens. Specific O-serotypes exhibited marked differences in their ability to persist in the gut of the control pig. The R-factor bearing O-serotypes, selected by tetracycline treatment, also showed differences in ability to persist after treatment indicating that properties other than the possession of R-factors, decide colonizing ability. The size of the colony sample for serotype analysis is discussed.     

The Effect of Tetracycline on the Coliform Gut Flora of Broiler Chickens with Special Reference to Antibiotic Resistance and O-Serotypes of Escherichia coli

The effect of terramycin, administered prophylactically in drinking water, on the gut flora of broiler birds was investigated. Exposure to the antibiotic for only 24 h profoundly affected the counts of tetracycline-resistant strains and selected O-serotypes carrying resistance determinants. Large numbers of Escherichia coli resistant to sulphonamides were found in treated and control birds and this is discussed in relation to the use of sulphaquinoxaline as a coccidiostat. Evidence of carcass contamination by antibiotic resistant E. coli found in the gut is presented.     

Cloning of the chromosomal determinants encoding hemolysin production and mannose-resistant hemagglutination in Escherichia coli.

We have cloned the chromosomal hemolysin determinants from Escherichia coli strains belonging to the four O-serotypes O4, O6, O18, and O75. The hemolysin-producing clones were isolated from gene banks of these strains which were constructed by inserting partial Sau3A fragments of chromosomal DNA into the cosmid pJC74. The hemolytic cosmid clones were relatively stable. The inserts were further subcloned either as SalI fragments in pACYC184 or as BamHI-SalI fragments in a recombinant plasmid (pANN202) containing cistron C (hlyC) of the plasmid-encoded hemolysin determinant. Detailed restriction maps of each of these determinants were constructed, and it was found that, despite sharing overall homology, the determinants exhibited minor specific differences in their structure. These appeared to be restricted to cistron A (hlyA), which is the structural gene for hemolysin. In the gene banks of two of these hemolytic strains, we could also identify clones which carried the genetic determinants for the mannose-resistant hemagglutination antigens Vb and VIc. Both of these fimbrial antigens were expressed in the E. coli K-12 clones to an extent similar to that observed in the wild-type strains. These recombinant cosmids were rather unstable, and, in the absence of selection, segregated at a high frequency.     

Surfactant protein D binds selectively to Klebsiella pneumoniae lipopolysaccharides containing mannose-rich O-antigens

Surfactant protein D (SP-D) plays important roles in the regulation of innate immune responses in the lung. We have previously shown that SP-D can agglutinate and enhance the macrophage-dependent killing of specific unencapsulated phase variants of Klebsiella pneumoniae. In the present studies, we used 16 clinical isolates of Klebsiella representing four O-serotypes and examined the interaction of SP-D with their isolated LPSs. Although SP-D bound to the core oligosaccharide of rough LPS from all isolates, it selectively bound to smooth forms of LPS expressed by O-serotypes with mannose-rich repeating units in their O-polysaccharides. SP-D was more potent in agglutinating unencapsulated phase variants of O-serotypes expressing these SP-D "reactive" O-polysaccharides, and more effectively inhibited the adhesion of these serotypes to lung epithelial cells. This novel anti-adhesion activity required the multimerization of trimeric SP-D subunits (dodecamers). Klebsiella serotypes expressing "nonreactive" LPS O-Ags were isolated at a significantly higher frequency from patients with K. pneumoniae. Our findings suggest that SP-D plays important roles in the clearance of opportunistic Gram-negative bacteria and contributes to known serotypic differences in the pathogenicity of Klebsiella through specific interactions with O-polysaccharides.     

Antigenic polysaccharides of bacteria. 21. The structure of O-specific polysaccharide chains and serological specificity of lipopolysaccharides from 7 Pseudomonas aeruginosa immunotypes

On mild acid degradation on lipopolysaccharides of seven Pseudomonas aeruginosa immunotypes, O-specific polysaccharides were obtained and their structures established. A peculiar feature of the polysaccharides is the presence of various, mostly acidic, mono- and diaminosugars, many of which have not previously been found in nature. The absence of serological cross-reactions (inhibition of passive haemagglutination) between lipopolysaccharides of seven immunotypes correlates with the absence of any common oligosaccharide fragments in their O-specific chains. The data obtained revealed structural and serological interrelations between O-antigens of seven immunotypes and P. aeruginosa O-serotypes, and showed that immunotypes 1 and 7 should be included into the serological classification scheme as individual O-serotypes.     

Immunological detection of heterogeneous O-antigen containing lipopolysaccharides in Escherichia coli

The components of the cell envelopes of Escherichia coli O1:K1, O7:K1, O18:K1 and O83:K1 strains were separated on SDS-polyacrylamide gels. Longitudinal slices (50 microns thick) of the gel were incubated with typing sera for E. coli O1, O7, O18 and O83, followed by detection of the bound antibodies with 125I-labelled protein A and autoradiography. The antisera reacted with many cell envelope components of strains both with the homologous O-serotype and heterologous O-serotypes. With O-typing sera cross-reactions with heterologous cells and cells boiled for 2 h were found. Up to 40 serotype-specific bands at regular positions with molecular weights between 12000 and 100000 were demonstrated. Since these bands were also observed when purified lipopolysaccharide and unabsorbed homologous O-typing sera were used, it was concluded that these bands represented lipopolysaccharide molecules with increasing molecular weight, all of which contained O-antigen specific immunodeterminants. The band patterns were not influenced by the growth conditions of the cells or the various isolation procedures for the cell envelopes. Comparison of various strains serotyped as O18 revealed strain differences with respect to their lipopolysaccharide band patterns. In the case of O21- and O83-serotyped strains lipopolysaccharide cross-reactions, which were detected by agglutination, were analysed in detail using the gel immunoradioassay method. These cross-reactions appeared to be caused by the presence of common determinants on their lipopolysaccharides and polysaccharide-like material. The cross-reacting antibodies could be removed by cross-absorption. It is concluded that the immunological detection of lipopolysaccharides and other components of E. coli in gels is an important tool in (1) the control of the specificity of typing antisera, (2) the study of the nature of cross-reacting antigens and (3) the study of the nature and uniformity of the various O- and K-serotypes.     

Isolation and characterization of bacteriophages for avian pathogenic E. coli strains

Aims:  To isolate and characterize bacteriophages, and to evaluate its lytic performance against avian pathogenic Escherichia coli (APEC) strains with high patterns of antibiotic resistance, in order to select phages for a therapeutic product to treat colibacillosis in chickens.
Methods and Results:  Bacteriophages were isolated from poultry sewage and tested against 148 O-serotyped APEC strains. The morphological characterization of the bacteriophages was made by transmission electronic microscopy (TEM) observations and the genetic comparison between bacteriophages DNA was performed by restriction fragment length polymorphism (RFLP) patterns. Results showed that 70·5% of the tested E. coli strains were sensitive to a combination of three of the five isolated phages, that seemed to be virulent and taxonomically belong to the Caudovirales order. Two of them look like 16–19, T4-like phages ( Myoviridae ) and the third is a T1-like phage and belongs to Syphoviridae family. All of them are genetically different.
Conclusions:  It was possible to obtain a combination of three different lytic bacteriophages with broad lytic spectra against the most prevalent O-serotypes of APEC.
Significance and Impact of the Study:  Data reported in this study, presents an in vitro well studied phage product to be used as antimicrobial agent to treat colibacillosis in poultry industry.     

O-Specific Polysaccharides (O-Antigens) of a New Species of Enteric Bacteria Escherichia albertii Closely Related to Escherichia coli

Russian Journal of Bioorganic Chemistry - The data on the structure of O-specific polysaccharides (O-antigens) of all nine known molecular types (potential O-serotypes) of a new type of...     

Membrane receptors on rat hepatocytes for the inner core region of bacterial lipopolysaccharides.

Bacterial lipopolysaccharides (LPS) are potent endotoxins that are thought to be involved in the pathogenesis of Gram-negative septicemia. The liver is known to be the primary organ responsible for the clearance of LPS from the systemic circulation in mammals. In this work, 125I-labeled LPS have been used in a filtration assay for the specific binding of LPS to intact rat hepatocytes. Eight S-form (smooth) LPS with complete O-specific polysaccharide chains isolated from different O-serotypes of Salmonella and Escherichia coli as well as nine R-form (rough) LPS isolated from Salmonella mutants deficient in synthesis of their core oligosaccharides were used in this study. All 125I-labeled S-form LPS and R-form LPS, except Re, show specific binding to isolated hepatocytes. The binding is saturable, is inhibited with excess unlabeled homologous or heterologous LPS but not lipid A, and is trypsin sensitive. L-Glycero-D-mannoheptose (heptose), a constituent of the inner core region of almost all LPS, is a potent inhibitor of the specific binding of 125I-labeled Rb2 LPS, whereas other monosaccharides, including 3-deoxy-D-manno-2-octulosonic acid (KDO), have weak or negligible inhibitor activity. These results strongly suggest the presence of a lectin-like receptor for the LPS inner core region (heptose-KDO region) on the plasma membrane of rat hepatocytes.     

Protective cross activity of the extracellular mucus of Pseudomonas aeruginosa

Extracellular slime was isolated from 15 P. aeruginosa typing strains of different O-serotypes (immunotypes). The isolated slime, partially purified by ethanol precipitation, was later referred to as crude slime. Glycolipoprotein was obtained from crude slime and lipopolysaccharide (LPS) was obtained from acetone-dried microbial cells by the method of aqueous-phenol extraction. All these antigenic preparations were studied in the active mouse cross-protection tests: immunized mice were challenged with 7 strains of different immunotypes, strain No. 170 019 or toxigenic strain PA-103. In experiments on mice the slime of different P. aeruginosa serotypes (immunotypes) was found to stimulate immunity to intraperitoneal infection with P. aeruginosa, both homologous or heterologous in respect to their immunotype, including toxigenic strains. Slime glycoprotein also stimulated active cross-immunity in mice, but the level of this immunity was higher than that of immunity stimulated by crude slime. LPS showed mostly weak protective activity in experiments on mice.     

Experimental immunological effectiveness and safety of pyoimmunogen vaccine against Pseudomonas aeruginosa infection

Pyoimmunogen, a polycomponent vaccine against P. aeruginosa infection, has been obtained in laboratory and semi-industrial conditions. The microbial biomass obtained from the strains belonging to O-serotypes (immunotypes) most frequently occurring in clinical practice has been used for producing protective antigens. The preparations have been found to contain proteins (peptides) and carbohydrates in the ratio 6 : 1 to 8 : 1, as well as traces of 2-keto-3-desoxyoctanate, which is indicative of the low content of endotoxin. The immunogenicity of the preparations has been studied experimentally by the active immunization of mice. In these experiments the animals vaccinated in a single injection were found to be protected from challenge with both homologous and heterologous P. aeruginosa strains. The high level of protection from infection caused by toxigenic strain PA-103 was registered. The preparations have low toxicity: LD50 for mice exceeds 2 mg (in protein content): after the multiple administration (7-10 times) of the preparation to mice and rats the weight of the experimental animals was not significantly different from the weight of the control animals.     

Structural characterization of the carbohydrate backbone of the lipopolysaccharide of Vibrio parahaemolyticus O-untypeable strain KX-V212 isolated from a patient

Vibrio parahaemolyticus strain KX-V212 of a novel serotype, which does not belong to any of the known 13 O-serotypes of this vibrio, was isolated from a patient. Its O-antigen harbors a unique strain-specific O-antigenic factor(s), in addition to that shared by the O-antigen of V. parahaemolyticus serotype O2. A carbohydrate backbone nonasaccharide was isolated from the lipopolysaccharide (LPS) of strain KX-V212 by dephosphorylation, reduction and deacylation and found to consist of one residue each of D-glucose, D-galactose, D-GlcN, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and 5-acetamido-7-(N-acetyl-D-alanyl)amino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (Non5Ac7Ala), and two residues each of D-GlcA and L-glycero-D-manno-heptose (LD-Hep). Analysis of the isolated and deacylated lipid A showed that this oligosaccharide was an artifact resulting from a loss of one GlcN residue from the lipid A backbone. Therefore, the carbohydrate backbone of the LPS is a decasaccharide having the structure shown below. The initial LPS contains also D-GalA and phosphoethanolamine at unknown positions. Both similarity and differences are observed between the LPS of V. parahaemolyticus serotype O2 and strain KX-V212. [carbohydrate structure: see text]     

Chemical and serological properties of lipopolysaccharides from Vibrio parahaemolyticus O-untypeable strains isolated from patients

Chemical and serological studies have been carried out on the O-antigenic lipopolysaccharides (LPS) of six strains, U-6443, W-90144, X-3972, AD-7999, 90A-6611 and KX-V212, of Vibrio parahaemolyticus isolated from patients. The O-serotypes of these strains have not been identified because they were not agglutinated by any diagnostic antisera against known O-serotype strains. A compositional sugar analysis of their LPS revealed that out of the six O-untypeable (OUT) strains, U-6443, W-90144 and AD-7999 strains belonged to chemotype II (chemotype of O2), 90A-6611 and KX-V212 strains to chemotype III (chemotype of O3, O5, O11 and O13) and X-3972 strain to chemotype IV (chemotype of O4). A structural analysis of LPS isolated from KX-V212 revealed that the inner core region of the LPS consisted of only one mole of 2-keto-3-deoxy-D-manno-octonic acid, which carried a phosphate group at position C4 and the outer core at position C5. In passive hemolysis tests performed by using LPS as the antigen to sensitize sheep red blood cells (SRBC), and diagnostic antisera (O1 to O11) or anti-whole-cell rabbit antisera raised against O12, O13 and the six OUT strains, strong cross-reactivity was observed among LPS derived from the strains belonging to chemotype II (U-6443, W-90144, AD-7999 and O2). Strong cross-reactivity was also observed between X-3972 (chemotype IV) and O4 LPS. In contrast, LPS from two of the strains belonging to chemotype III (90A-6611 and KX-V212) did not react with any of the antisera raised against known O-serotypes. Cross-absorption tests showed that the O-antigens of U-6443, W-90144 and AD-7999 were identical to that of O2, and the O-antigen of X-3972 to that of O4. On the other hand, after the absorption of antisera raised against 90A-6611 and KX-V212 with O2 cells, the hemolytic activities against SRBC sensitized with homologous LPS were still retained at a high titer, whereas the hemolytic activities against SRBC sensitized with LPS from other O-serotype strains were completely eliminated. A cross-absorption test revealed that the O-antigens of these two strains were identical to each other. Thus, it was demonstrated that the O-serotype of OUT strains 90A-6611 and KX-V212 was not involved in the known O-serotypes; rather it represented a novel serotype which has not hitherto been reported.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa II (Sandvik) and V (IM-1, Verder-Evans)

The lipopolysaccharides from two serologically related strains of Pseudomonas aeruginosa II (Sandvik classification) and V (IM-1, Verder-Evans classification) were isolated by the Westphal method and cleaved with 1% acetic acid. The O-specific polysaccharide from Sandvik II lipopolysaccharide involved L-rhamnose, N-acetyl-D-quinovosamine, and N-acetyl-D-galactosaminuronic acid in the ratio 2:1:1, as well as O-acetyl groups. The Verder-Evans V O-specific polysaccharide was found to contain just the same sugars and, in addition, D-glucose, the monosaccharide ratio being 2:1:1:1. On the basis of methylation analysis, Smith degradation, solvolysis with anhydrous hydrogen fluoride and 1H and 13C nuclear magnetic resonance data, it was concluded that the polysaccharides have identical main chains, and there were established the following structures of their repeating units: -4)-alpha-D-GalNAcA-(1-3)-beta-D-QuiNAc-(1-2)-alpha-L-Rha-(1 -3)-alpha-L-Rha-(1- increases 3 60% OAc Sandvik II -4)-alpha-D-GalNAcA-(1-3)-beta-D-QuiNAc-(1-2)-alpha-L-Rha-(1 -3)-alpha-L-Rha-(1- increases 3 alpha-D-Glc Verder-Evans V The data obtained allow us to substantiate the combining of the strains studied into one O-serogroup as two individual O-serotypes.     

Transposition of the phage D3112 genome in Escherichia coli cells

It has been demonstrated that the genome of phage D3112 of Preudomonas aeruginosa can be transposed into Escherichia coli chromosome as a component of the hybrid plasmid RP4 TcrKms::D3112. Also, transposition of D3112 from E. coli (D3112) chromosome into RP4 plasmid occurs. The phage stimulates the chromosome mobilizing activity of RP4 plasmid, similar to other transposons. E. coli (RP4::D3112) cells were previously shown to form no colonies at 30 degrees C. Auxotrophic mutants and mutants incapable of utilizing different carbohydrates were found among E. coli clones survived after a long incubation at 30 degrees C (at frequencies approximately 10(-3) - 10(-4). These mutants inherited stably the capability to produce D3112 phage. E. coli auxotrophic mutants have arisen indeed as a consequence of phage integration into the E. coli chromosome, since prototrophic transductants derived from these mutants after their treatment with generalized transducing P1 phage have lost the ability to produce D3112 phage. Clones with mutations in Km or Tc genes of RP4 plasmid, occurring at high frequencies (about 3%) were found after introduction of RP4 into E. coli (D3112). These mutant RP4 plasmids carry insertions of D3112 genomes. Clones of E. coli which lost mutant plasmids still produce D3112 and retain their initial auxotrophic mutations.     

Transformation of Escherichia coli in foodstuffs.

The plasmid transfer by transformation of Escherichia coli in 12 foods was investigated under conditions commonly found in processing and storage of food. Transformation occurred in all foods with frequencies of at least 10(-8) when a simplified standard transformation protocol with non-growing cells was applied. Higher rates (ca. 10(-7)) were found in milk, soy drink, tomato and orange juice. Furthermore, E. coli became transformed at temperatures below 5 degrees C, i.e. under conditions highly relevant in storage of perishable foods. In soy drink this condition resulted in frequencies which were even higher than those determined after application of a temperature shift to 37 degrees C. The transformation of cells growing in milk and carrot juice at a constantly kept temperature of 37 degrees C provides evidence for the potential of E. coli to become transformed naturally. With purified DNA frequencies were determined in these substrates of ca. 2.5 x 10(-7) and 2.5 x 10(-8), respectively. Similar frequencies were also obtained in milk containing the crude nucleic acids of homogenised cell suspensions of E. coli (pUC18). Moreover, the release of plasmid DNA from E. coli during food processing and the subsequent uptake of this DNA by growing E. coli cells was shown to take place after homogenisation in milk indicating a horizontal plasmid transfer by transformation of E. coli.     

The role of lipopolysaccharide as a receptor for some bacteriophages of Pseudomonas aeruginosa

Typing phages of the Colindale typing set for Pseudomonas aeruginosa have been tested for the use of lipopolysaccharide (LPS) as a receptor. Studies using the reference strains of the International Antigenic Typing Scheme for O-serotypes of P. aeruginosa supported earlier indications that none of the phages were O-specific. Studies of the adsorption of phages to LPS showed that typing phages 16, 44, F8, 68, 109, 352, and 1214 (as well as other phages 2 and H22) were LPS-specific, but were not consistently adsorbed by isolated LPS from all sensitive strains. Water-soluble fractions from LPS did not adsorb phages and did not inhibit their neutralization by whole LPS. No endoglycosidase activity against LPS was detected for any phage. The significance of these results for the roles of LPS in the adsorption process and phage sensitivity are discussed.