O-serogroups, virulence genes of pathogenic Escherichia coli and Pulsed-field gel electrophoresis (PFGE) patterns of O149 isolates from diarrhoeic piglets in Korea

A total of 116 Escherichia (E.) coli isolates isolated from neonatal diarrhoeic piglets were serogrouped and tested for the presence of virulence genes for fimbrial and non-fimbrial adhesins, intimin, and enterotoxins. Pulsed-field gel electrophoresis (PFGE) pulsotypes were also analyzed within O149 enterotoxigenic E. coli (ETEC) isolates. In total, Sixty eight (58.6%) isolates were serotyped. Among them, forty three (63.2%) belonged to 12 serogroups in the descending order: O149, O8, O157, O101, O60, O9, O117, O127, O138, O167, O27 and O97. The predominant pathotype was ETEC (68, 58.6%) which is closely associated with F4 (37, 31.9%) and LT:STb:EAST1 (23, 19.8%) out of the isolates harbouring at least one gene for toxin and/or fimbria. Among non-fimbrial adhesins, porcine attaching and effacing-associated factor (paa) was closely associated with F4-positive isolates (64.7%) rather than F18-positive isolates (5.9%). Adhesion involved in diffuse adherence (AIDA) was only detected in 3 isolates. No eae-positive isolates were detected. The PFGE pattern of 15 O149 isolates was grouped into 12 pulsotypes at 88% similarity level. The results show a wide variety of distinct restriction patterns though all belonged to the same serogroup O149. It is believed that a broad array of O serogroup and virulence genes are associated with neonatal diarrhoea in Korea.  

Apical targeting of the formin Diaphanous in Drosophila tubular epithelia

Apical secretion from epithelial tubes of the Drosophila embryo is mediated by apical F-actin cables generated by the formin-family protein Diaphanous (Dia). Apical localization and activity of Dia are at the core of restricting F-actin formation to the correct membrane domain. Here we identify the mechanisms that target Dia to the apical surface. PI(4,5)P2 levels at the apical membrane regulate Dia localization in both the MDCK cyst model and in Drosophila tubular epithelia. An N-terminal basic domain of Dia is crucial for apical localization, implying direct binding to PI(4,5)P2. Dia apical targeting also depends on binding to Rho1, which is critical for activation-induced conformational change, as well as physically anchoring Dia to the apical membrane. We demonstrate that binding to Rho1 facilitates interaction with PI(4,5)P2 at the plane of the membrane. Together these cues ensure efficient and distinct restriction of Dia to the apical membrane.

Prevalence and Genetic Characterization of Shiga Toxin-Producing Escherichia coli Isolates from Slaughtered Animals in Bangladesh

ABSTRACT To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx 1 and/or stx 2, respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx 2, eae, katP, etpD, and enterohemorrhagic E. coli hly (hly EHEC) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx 1. Only 7.0% (n = 5) of the isolates were positive for hly EHEC, and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpf O113, saa, lpfA O157/01-141, and lpfA O157/OI-154 genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.

Primate Lentivirus Capsid Sensitivity to TRIM5 Proteins

ABSTRACT Mammalian cells express several factors that inhibit lentiviral infection and that have been under strong selective pressure. One of these factors, TRIM5, targets the capsid protein of incoming retrovirus particles and inhibits subsequent steps of the replication cycle. By substituting human immunodeficiency virus type 1 capsid, we were able to show that a set of divergent primate lentivirus capsids was generally not susceptible to restriction by TRIM5 proteins from higher primates. TRIM5α proteins from other primates exhibited distinct restriction specificities for primate lentivirus capsids. Finally, we identified novel primate lentiviral capsids that are targeted by TRIMCyp proteins.

Genetic and phenotypic diversity of Bifidobacterium thermacidophilum fecal isolates from newborns

This study was undertaken to genetically identify and phenotypically characterize 14 bifidobacteria isolated from 20 breast-fed newborns. These isolates showed 98%–99% similarity to Bifidobacterium thermacidophilum subsp. suis based on 16S rDNA. Further analysis by pulsed-field gel electrophoresis of chromosomal DNA digested with XbaI revealed 4 distinct restriction patterns. The predominant pattern, shared by 8 (57%) isolates, produced a macro-restriction profile with about 13 large fragments ranging in size from >242.5 to 23.1 kb, whereas the other 6 displayed 3 distinct restriction profiles all characterized by more micro- than macro-restriction, with fragments ranging in size from 97 to 9.4 kb. Phenotypic characteristics, including carbohydrate fermentation profile, maximal growth temperature, and antibiotic susceptibility, varied widely even among strains showing the same restriction profile. The presence of B. thermacidophilum in stools of newborn infants may indicate the potential of these bacteria for aiding the development of the intestinal ecosystem.

Typing of intimin (eae) genes from enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea in Montevideo, Uruguay: identification of two novel intimin variants (μB and ξR/β2B)

A total of 71 enteropathogenic Escherichia coli (EPEC) strains isolated from children with diarrhoea in Montevideo, Uruguay, were characterized in this study. PCR showed that 57 isolates carried eae and bfp genes (typical EPEC strains), and 14 possessed only the eae gene (atypical EPEC strains). These EPEC strains belonged to 21 O : H serotypes, including eight novel serotypes not previously reported among human EPEC in other studies. However, 72 % belonged to only four serotypes: O55 : H− (six strains), O111 : H2 (13 strains), O111 : H− (14 strains) and O119 : H6 (18 strains). Nine intimin types, namely, α1 (two O142 strains), β1 (29 strains, including 13 O111 : H2 and 14 O111 : H−), γ1 (three O55 : H− strains), θ (five strains, including three strains with H40 antigen), κ (two strains), ε1 (one strain), λ (one strain), μB (six strains of serotypes O55 : H51 and O55 : H−) and ξR/β2B (22 strains, including 18 O119 : H6) were detected among the 71 EPEC strains. The authors have identified two novel intimin genes (μB and ξR/β2B) in typical EPEC strains of serotypes O55 : H51/H− and O119 : H6/H−. The complete nucleotide sequences of the novel μB and ξR/β2 variant genes were determined. PFGE typing after XbaI DNA digestion was performed on 44 representative EPEC strains. Genomic DNA fingerprinting revealed 44 distinct restriction patterns and the strains were clustered in 12 groups. Only 15 strains clustered in six groups of closely related (similarity >85 %) PFGE patterns, suggesting the prevailing clonal diversity among EPEC strains isolated from children with diarrhoea in Montevideo.

Pantoea agglomerans Strain EH318 Produces Two Antibiotics That Inhibit Erwinia amylovoraIn Vitro

ABSTRACT Pantoea agglomerans (synonym: Erwinia herbicola) strain Eh318 produces through antibiosis a complex zone of inhibited growth in an overlay seeded with Erwinia amylovora, the causal agent of fire blight. This zone is caused by two antibiotics, named pantocin A and B. Using a genomic library of Eh318, two cosmids, pCPP702 and pCPP704, were identified that conferred on Escherichia coli the ability to inhibit growth ofE. amylovora. The two cosmids conferred different antibiotic activities on E. coli DH5α and had distinct restriction enzyme profiles. A smaller, antibiotic-conferring DNA segment from each cosmid was cloned. Each subclone was characterized and mutagenized with transposons to generate clones that were deficient in conferring pantocin A and B production, respectively. Mutated subclones were introduced into Eh318 to create three antibiotic-defective marker exchange mutants: strain Eh421 (pantocin A deficient); strain Eh439 (pantocin B deficient), and Eh440 (deficient in both pantocins). Cross-hybridization results, restriction maps, and spectrum-of-activity data using the subclones and marker exchange mutants, supported the presence of two distinct antibiotics, pantocin A and pantocin B, whose biosynthetic genes were present in pCPP702 and pCPP704, respectively. The structure of pantocin A is unknown, whereas that of pantocin B has been determined as (R)-N-[((S)-2-amino-propanoylamino)-methyl]-2-methanesulfonyl-succinamic acid. The two pantocins mainly affect other enteric bacteria, based on limited testing.

Structure of the human CR1 gene. Molecular basis of the structural and quantitative polymorphisms and identification of a new CR1-like allele.

Structural and quantitative polymorphisms have been described in human CR1. In the former, the S allotype is larger than the F allotype by 40-50 kD, the size of a long homologous repeat (LHR). In the latter, homozygotes for a 7.4-kb Hind III fragment express fourfold more CR1 per erythrocyte than do homozygotes for the allelic 6.9-kb restriction fragment. The basis for these genomic polymorphisms has been determined by restriction mapping the entire S allele and part of the F allele. The S allele is 158 kb and contains 5 LHRs of 20-30 kb, designated -A, -B/A, -B, -C, and -D, respectively, 5' to 3'. Extensive homology was found among the LHRs in their restriction maps, exon organization, and the coding and noncoding sequences. The presence of LHR-B/A in the S allele but not in the F allele accounts for the longer transcripts and polypeptide associated with the former allotype. At least 42 exons are present in the S allele, with distinct exons for the leader sequence, the transmembrane and cytoplasmic regions and most of the SCRs comprising the extracellular portion of CR1. Consistent with the mapping of the ligand binding site to the first two SCRs in each LHR, the second SCRs in LHR-A, -B/A, -B, and -C are encoded by two exons, reflecting a specialized function for this unit. The allelic 7.4/6.9-kb Hind III fragments extend from the 3' region of LHR-C to LHR-D. The 6.9-kb restriction fragment is the result of a new Hind III site generated by a single base change in the intron between the exons encoding the second SCR of LHR-D. A second cluster of genomic clones has been identified by hybridization to CR1 probes. Although they contain regions of hybridization to the cDNA and genomic probes derived from CR1, these cannot be overlapped with the structural gene owing to their distinct restriction maps. Three genomic polymorphisms previously identified by CR1 cDNA probes map to this region. These additional clones may represent part of a duplicated allele located nearby within the CR1 locus.