High colonisation by probiotic Escherichia coli A0 34/86 strain is associated with a less diverse microbiome related to children's age.

Probiotic supplementation in childhood serves as an additional source of bacterial colonisers and represents an opportunity to beneficially manipulate the intestinal microbiome. Differences in the ability of probiotic strains to colonise the gut may be related to the variously diversified gut microbiome. We report the results of the association between composition of the gut microbiome and the colonisation capacity of the probiotic strain Escherichia coli A0 34/86 (CNB - Colinfant New Born supplement) in the cases of three healthy children in different development stages (infant, toddler, and pre-school), as a preliminary insight to possible future prospective studies of this subject. Microbiome composition was estimated by 16S rRNA gene sequencing of 55 stool samples collected during approximately 3.5-13 months long periods. Detailed characterisation of the E. coli population was performed using colony PCR to detect 33 E. coli genetic determinants. In all children, genetic determinants typical for the probiotic E. coli A0 34/86 strain were detected immediately after administration of the probiotics. Analysis of the initial sample composition (the last sample taken before the probiotic administration) showed that the gut microbiome of infant and toddler with lower bacterial diversity was more successfully colonised by the probiotic strain. In our case report of three children, we showed for the first that supplementation with CNB probiotics in early infancy and toddlerhood was associated with high E. coli A0 34/86 colonisation and a significant change in the composition of the gut microbiome. Our results indicate that administration of CNB for its recommended duration might be efficient only in very early childhood.

Bejel in Cuba: molecular identification of Treponema pallidum subsp. endemicum in patients diagnosed with venereal syphilis.

OBJECTIVES: Bejel, caused by Treponema pallidum subsp. endemicum (TEN), was until now considered as a non-venereal disease endemic in areas with hot and dry climates. This study has identified TEN in clinical samples from Cuban patients previously diagnosed with syphilis. METHODS: We performed sequencing-based molecular typing on 92 samples from Cuban individuals diagnosed with syphilis. Moreover, to differentiate T. pallidum subspecies, multi-locus sequence analysis (MLSA) was designed and was applied to suspicious samples. RESULTS: Nine samples, from six patients, had a nucleotide sequence similarity (at all typing loci) to the Bosnia A genome, which is the infectious agent of bejel. Additionally, MLSA clearly supported a TEN classification for the treponemal samples. Clinical and epidemiological data from the six patients also suggested sexual transmission of bejel as well as the endemicity of this rare treponematosis in Cuba. CONCLUSIONS: Molecular identification of Treponema pallidum subsp. endemicum, the agent of bejel, in Cuban patients diagnosed with syphilis indicates the clear limitations of a diagnosis based exclusively on serology, geographical occurrence, clinical symptoms and anamnestic data. This finding has important implications for Global Public Health Systems, including paradigm changes regarding the location of endemic outbreaks, clinical aspects and transmission of this neglected disease.

[Macrolide resistance in Treponema pallidum subsp. pallidum in the Czech Republic and in other countries]. / Rezistence puvodce syfilis, Treponema pallidum subsp. pallidum, k makrolidovým antibiotikum v Ceské republice a ve svete.

Treponema pallidum subsp. pallidum (TPA) is the causative agent of the sexually transmitted disease syphilis. In the Czech Republic, several hundred cases of syphilis are reported annually; e.g. in 2012, 696 syphilis cases were documented. In the last decades, an increasing prevalence of macrolide resistant TPA strains harboring A2058G or A2059G mutations in the 23S rRNA gene has been reported. Macrolides were used (and rarely are still being used) in the Czech Republic for the treatment of syphilis in patients allergic to penicillin. While 37% of TPA strains were resistant to macrolides between 2004 and 2010, this rate increased to 67% between 2011-2013. High prevalence of A2058G or A2059G mutations and increasing rates of macrolide resistant TPA strains have also been documented in other developed countries. Therefore, macrolides should not be used in the treatment of syphilis.

Incidence of lysogenic, colicinogenic and siderophore-producing strains among human non-pathogenic Escherichia coli.

The current incidence of Escherichia coli strains in healthy humans capable of producing the inhibitory exoproducts, such as temperate bacteriophages, corpuscular or HMW (high-molar mass) and proteinaceous or LMW (low-molar mass) colicins and siderophores was determined. Fifty-three E. coli strains were collected from the colons of 53 healthy human volunteers in Brno (Czechia) and tested for spontaneous and induced production of inhibitory exoproducts in a cross-test against each other. Of the strains tested, 37.7% produced bacteriophages, 41.5% produced from one to several LMW colicins, 11.3% formed HMW colicins and 15.1% (eight strains) produced exocellular siderophores different from enterochelin. Of these, seven strains formed aerobactin and one strain formed an untyped siderophore. E. coli strains differ greatly in the incidence of colicinogeny and lysogeny from its closest systemic relatives in the genus Escherichia and therefore should not be regarded as a model bacterium in this respect.

[Molecular detection and subtyping of Treponema pallidum subsp. pallidum in clinical specimens]. / Molekulární detekce a typizace Treponema pallidum subsp. pallidum v klinickém materiálu.

An in-house two-step nested PCR amplification targeting the tmpC gene (TP0319, encoding putative membrane lipoprotein) was used for detection of chromosomal DNA of Treponema pallidum subsp. pallidum in clinical specimens. We tested 138 blood serum samples from 111 adult patients with suspected, primary, secondary, early or late latent syphilis. T. p. pallidum DNA was not detected in any of the analyzed specimens. Out of 11 mucocutaneous swabs (7 genital and 4 pharyngeal), 6 collected from 3 patients with primary or secondary syphilis tested positive. One skin swab from a patient with early congenital syphilis was also positive as were his serum and cerebrospinal fluid samples. DNA sequencing of the genes TP0136 and TP0548 from the positive samples revealed two strains with DNA sequences identical to that of T. p. pallidum strain SS14 and two unique previously undescribed T. p. pallidum strains. The advances in molecular typing of T. p. pallidum in clinical specimens will be of relevance to the epidemiology of syphilis and will allow for clinical discrimination between reinfection and syphilitic reactivation.

Exoproducts of the Escherichia coli strain H22 inhibiting some enteric pathogens both in vitro and in vivo.

AIMS: The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo. METHODS AND RESULTS: In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae. CONCLUSIONS: Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria.

The Escherichia fergusonii iucABCD iutA genes are located within a larger chromosomal region similar to pathogenicity Islands.

Three strains of Escherichia fergusonii (EF873, EF1496, EF939) of 50 strains tested produced the hydroxamate siderophore aerobactin. Screening of a cosmid library of the strain EF873 chromosomal DNA (in aerobactin nonproducing Escherichia coli VCS257) for aerobactin production identified iucABCD and iutA gene orthologues. The predicted IucABCD and IutA proteins showed 59-65% identity to the corresponding proteins of Shigella flexneri and E. coli. Aerobactin molecules synthesized by E. fergusonii and E. coli strains stimulated growth of aerobactin indicator strains harboring either E. coli or E. fergusonii iutA genes. In the 12 kb upstream and 17 kb downstream regions of the iuc and iut genes, 20 additional ORFs were identified. Their gene products showed homology to proteins from E. coli, S. flexneri, Klebsiella aerogenes, Pseudomonas aeruginosa and Vibrio cholerae. Probes recognizing DNA sequences from a region of more than 25 kb, which included the iucABCD and iutA genes, hybridized with chromosomal DNA of two aerobactin-producing strains (EF873 and EF939), but not with other nonproducing E. fergusonii strains tested. These data, together with the genetic organization of this region, suggest that E. fergusonii iucABCD iutA genes are a portion of a larger segment of DNA similar to pathogenicity islands of other bacteria.

Genetic organization of plasmid ColJs, encoding colicin Js activity, immunity, and release genes.

The 5.2-kb ColJs plasmid of a colicinogenic strain of Shigella sonnei (colicin type 7) was isolated and sequenced. pColJs was partly homologous to pColE1 and to pesticin-encoding plasmid pPCP1, mainly in the rep, mob, and cer regions. A 1.2-kb unique region of pColJs showed significantly different G+C content (34%) compared to the rest of pColJs (53%). Within the unique region, seven open reading frames (ORFs) were identified. ORF94 was shown to code for colicin Js activity (cja), a 94-amino-acid polypeptide (molecular mass, 10.4 kDa); ORF129 (cji) was shown to code for the 129-amino-acid colicin Js immunity protein (molecular mass, 14.3 kDa); and ORF65 was shown to be involved in colicin Js release by producer bacteria (cjl) coding for a 65-amino-acid polypeptide (molecular mass, 7.5 kDa). In contrast to the gene order in other colicin operons, the cjl gene was found upstream from cja. Moreover, the promoter upstream from cjl was similar to promoters described upstream from several colicin activity genes. The cji gene was found to be located downstream from cja with a transcription polarity opposite to that of the cjl and cja genes. The cja, cji, and cjl genes were not similar to other known colicin genes. Colicin Js was purified as an inactive fusion protein with an N-terminal histidine tag. Activity of the purified fusion form of colicin Js was restored after cleavage of the amino acids fused to the colicin Js N terminus.

The iron- and temperature-regulated cjrBC genes of Shigella and enteroinvasive Escherichia coli strains code for colicin Js uptake.

A cosmid library of DNA from colicin Js-sensitive enteroinvasive Escherichia coli (EIEC) strain O164 was made in colicin Js-resistant strain E. coli VCS257, and colicin Js-sensitive clones were identified. Sensitivity to colicin Js was associated with the carriage of a three-gene operon upstream of and partially overlapping senB. The open reading frames were designated cjrABC (for colicin Js receptor), coding for proteins of 291, 258, and 753 amino acids, respectively. Tn7 insertions in any of them led to complete resistance to colicin Js. A near-consensus Fur box was found upstream of cjrA, suggesting regulation of the cjr operon by iron levels. CjrA protein was homologous to iron-regulated Pseudomonas aeruginosa protein PhuW, whose function is unknown; CjrB was homologous to the TonB protein from Pseudomonas putida; and CjrC was homologous to a putative outer membrane siderophore receptor from Campylobacter jejuni. Cloning experiments showed that the cjrB and cjrC genes are sufficient for colicin Js sensitivity. Uptake of colicin Js into sensitive bacteria was dependent on the ExbB protein but not on the E. coli K-12 TonB and TolA, -B, and -Q proteins. Sensitivity to colicin Js is positively regulated by temperature via the VirB protein and negatively controlled by the iron source through the Fur protein. Among EIEC strains, two types of colicin Js-sensitive phenotypes were identified that differed in sensitivity to colicin Js by 1 order of magnitude. The difference in sensitivity to colicin Js is not due to differences between the sequences of the CjrB and CjrC proteins.

From microbial genome sequence to applications.

Whole genome sequences of microbial pathogens present new opportunities for clinical applications. Chief among these are development of antimicrobials, diagnostics, and vaccines. While antimicrobial development is a more difficult, long-term prospect, new diagnostics and vaccines are likely to be the first products of microbial genomics. To take advantage of whole genome sequences, methods for production of gene products in surrogate hosts (heterologous expression) are required that will work for large-scale, high-throughput gene expression. This will allow genomic information from even the most experimentally difficult pathogens to be mined for applications. In addition, screening methods to test gene products for their potential as vaccine candidates are needed for large-scale screening. These areas for technological development should be stimulated by the potential for converting genomic sequence information into applications.

Characterization of colicin S4 and its receptor, OmpW, a minor protein of the Escherichia coli outer membrane.

Analysis of the nucleotide sequence of an Escherichia coli colicin S4 determinant revealed 76% identity to the pore-forming domain of the colicin A protein, 77% identity to the colicin A immunity protein, and 82% identity to the colicin A lysis protein. The N-terminal region, which is responsible for the Tol-dependent uptake of colicin S4, has 94% identity to the N-terminal region of colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other colicins. Mutants that lacked the OmpW protein were resistant to colicin S4.

The tip of the hydrophobic hairpin of colicin U is dispensable for colicin U activity but is important for interaction with the immunity protein.

The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of colicin U of Shigella boydii was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was no longer inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicin U hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575-582ColB), which was inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U. The colicin B immunity protein inactivated colicin U(575-582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.

Colicins--exocellular lethal proteins of Escherichia coli.

Colicins are toxic exoproteins produced by bacteria of colicinogenic strains of Escherichia coli and some related species of Enterobacteriaceae, during the growth of their cultures. They inhibit sensitive bacteria of the same family. About 35% E. coli strains appearing in human intestinal tract are colicinogenic. Synthesis of colicins is coded by genes located on Col plasmids. Until now more than 34 types of colicins have been described, 21 of them in greater detail, viz. colicins A, B, D, E1-E9, Ia, Ib, JS, K, M, N, U, 5, 10. In general, their interaction with sensitive bacteria includes three steps: (1) binding of the colicin molecule to a specific receptor in the bacterial outer membrane; (2) its translocation through the cell envelope; and (3) its lethal interaction with the specific molecular target in the cell. The classification of colicins is based on differences in the molecular events of these three steps.

Colicin U, a novel colicin produced by Shigella boydii.

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.