Rm Silva

The localized adherence pattern of an atypical enteropathogenic Escherichia coli is mediated by intimin omicron and unexpectedly promotes HeLa cell invasion.

Enteropathogenic Escherichia coli (EPEC) forms attaching and effacing lesions in the intestinal mucosa characterized by intimate attachment to the epithelium by means of intimin (an outer membrane adhesin encoded by eae). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC); only tEPEC carries the EAF (EPEC adherence factor) plasmid that encodes the bundle‐forming pilus (BFP). Characteristically, after 3 h of incubation, tEPEC produces localized adherence (LA) (with compact microcolonies) in HeLa/HEp‐2 cells by means of BFP, whereas most aEPEC form looser microcolonies. We have previously identified nine aEPEC strains displaying LA in extended (6 h) assays (LA6). In this study, we analysed the kinetics of LA6 pattern development and the role of intimin in the process. Transmission electron microscopy and confocal laser microscopy showed that the invasive process of strain 1551‐2 displays a LA phenotype. An eae‐defective mutant of strain 1551‐2 prevented the invasion although preserving intense diffused adherence. Sequencing of eae revealed that strain 1551‐2 expresses the omicron subtype of intimin. We propose that the LA phenotype of aEPEC strain 1551‐2 is mediated by intimin omicron and hypothesize that this strain expresses an additional novel adhesive structure. The present study is the first to report the association of compact microcolony formation and an intense invasive ability in aEPEC.

Prevalence and Characteristics of the O122 Pathogenicity Island in Typical and Atypical Enteropathogenic Escherichia coli Strains

ABSTRACT The presence of the pathogenicity island (PAI) O122 genes, efa1 (lifA), sen, pagC, nleB, and nleE, in typical and atypical enteropathogenic Escherichia coli (EPEC) strains was investigated. The simultaneous occurrence of all genes was statistically associated with diarrhea due to atypical EPEC. Detection of the complete PAI O122 could aid in the identification of potential pathogenic strains of atypical EPEC.

Adhesin-Encoding Genes from Shiga Toxin-Producing Escherichia coli Are More Prevalent in Atypical than in Typical Enteropathogenic E. coli

ABSTRACT Four of six adhesin-encoding genes (lpfA, paa, iha, and toxB) from Shiga toxin-producing Escherichia coli strains were detected in typical and atypical enteropathogenic E. coli (EPEC) strains of various serotypes. Although the most prevalent gene was lpfA in both groups, paa was the only potential diarrhea-associated gene in atypical EPEC.

Injuries to the mesenteric microcirculation due to bacterial translocation.

CFU/g or mL of tissue. The recovery was 60% positive inblood and 100% positive in tissues. Despite the absence ofclinical symptoms or mortality at all time periods studied, amicrocirculatory injury was detected between 2 hour and 28days. At 2 hours after BT the microcirculatory injuriesincluded obstruction of distal capillaries with low flow, anda lesser degree of obstruction in small and medium venules.In addition multiple focal hemorrhages in capillaries andsmall venules were distributed throughout the entire intes-tinal mesentery. However, the majority of medium andlarge venules and arterioles were not affected; they main-tained a normal blood flow pattern. Also, an augmentedvolume of lymphatic flow was observed in association withan increased contractile frequency of lymphatic vessels(control 6 and BT 12 contractions/min). When com-pared to the normal microcirculation, these alterationswere progressive reaching maximal severity on day 3. At day7, the injuries were persistent albeit improving. At 14 dayspost-BT, the microvascular circulatory pattern was mostlyrestored; resolved at 28 days. However, the vast majority ofpreviously damaged vessels remained without restoration ofblood flow, suggesting that the recuperation of the mesen-teric microvascular damage was most likely due to a neo-vascularization process. Neither BT nor microcirculatorydamage were evident in the control group receiving vehicle.

First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

Background In a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. The transfer mechanism of this insertion sequence to M. kansasii was investigated here. Methodology/Principal Findings A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. The linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau. Conclusions/Significance Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.

Assessing the diversity of the virulence potential of Escherichia coli isolated from bacteremia in São Paulo, Brazil.

Most of the knowledge of the virulence determinants of extraintestinal pathogenic Escherichia coli (ExPEC) comes from studies with human strains causing urinary tract infections and neonatal meningitis and animal strains causing avian colibacillosis. In this research, we analyzed the phylogenetic background, the presence of 20 ExPEC virulence factors, and the intrinsic virulence potential of 74 E. coli strains isolated in São Paulo, Brazil, from 74 hospitalized patients (43 males and 31 females) with unknown-source bacteremia. Unlike other places in the world, the bacteremic strains originated equally from phylogroups B2 (35%) and D (30%). A great variability in the profiles of virulence factors was noted in this survey. Nevertheless, 61% of the strains were classified as ExPEC, meaning that they possessed intrinsic virulent potential. Accordingly, these strains presented high virulence factor scores (average of 8.7), and were positively associated with 12 of 17 virulence factors detected. On the contrary, the non-ExPEC strains, isolated from 39% of the patients, presented a generally low virulence capacity (medium virulence factor score of 3.1), and were positively associated with only the colicin cvaC gene. These results show the importance of discriminating E. coli isolates that possess characteristics of true pathogens from those that may be merely opportunistic in order to better understand the virulence mechanisms involved in extraintestinal E. coli infections. Such knowledge is essential for epidemiological purposes as well as for development of control measures aimed to minimize the incidence of these life-threatening and costly infections.

Natural Occurrence of Horizontal Transfer of Mycobacterium avium- Specific Insertion Sequence IS1245 to Mycobacterium kansasii

ABSTRACT Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. The insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome.

Primary fecal culture used as template for PCR detection of diarrheagenic E. coli virulence factors

The PCR technique applied to primary fecal cultures (PFC-PCR) was compared to the usual method employing isolated colonies (IC-PCR) in order to assess its sensitivity in the detection of virulence markers of diarrheagenic Escherichia coli in fecal samples obtained from children with diarrhea. Among the 149 samples analysed, PFC-PCR detected 81(54.4%) samples presenting one or two virulence markers, while IC-PCR detected only 59 (39.6%) positive samples. The markers detected in order of frequency were: pAA, LT-I, eaeA, ST-I, daaE, and ipaH. The PFC-PCR method of detection of diarrheagenic E. coli virulence markers proved to be reliable and more sensitive (p<0.05) than the usual method employing isolated colonies. It has also the advantage of being faster and less expensive than the detection methods in current use.

Uropathogenic Escherichia coli pathogenicity islands and other ExPEC virulence genes may contribute to the genome variability of enteroinvasive E. coli

BackgroundEnteroinvasive Escherichia coli (EIEC) may be the causative agent of part of those million cases of diarrhea illness reported worldwide every year and attributable to Shigella. That is because both enteropathogens have many common characteristics that difficult their identification either by traditional microbiological methods or by molecular tools used in the clinical laboratory settings. While Shigella has been extensively studied, EIEC remains barely characterized at the molecular level. Recent EIEC important outbreaks, apparently generating more life-threatening cases, have prompted us to screen EIEC for virulence traits usually related to extraintestinal pathogenic E. coli (ExPEC). That could explain the appearance of EIEC strains presenting higher virulence potential.ResultsEIEC strains were distributed mainly in three phylogroups in a serogroup-dependent manner. Serogroups O124, O136, O144, and O152 were exclusively classified in phylogroup A; O143 in group E; and O28ac and O29 in group B1. Only two serogroups showed diverse phylogenetic origin as follows: O164 was assigned to groups A, B1, C, and B2 (one strain each), and O167 in groups E (five strains), and A (one strain) (Table 1). Eleven of 20 virulence genes (VGs) searched were detected, and the majority of the 19 different VGs combinations found were serogroup-specific. Uropathogenic E. coli (UPEC) PAI genetic markers were detected in all EIEC strains. PAIs IJ96 and IICFT073 were the most frequent (92.1 and 80.4%, respectively). PAI IV536 was restricted to some serogroups from phylogroups A, B1 and E. PAI ICFT073 was uniquely detected in phylogroups B2 and E. A total of 45 (88%) strains presented multiple PAI markers (two to four). PAIs IJ96 and IICFT073 were found together in 80% of strains.ConclusionsEIEC is a DEC pathovar that presents VGs and pathogenicity island genetic markers typically associated with ExPEC, especially UPEC. These features are distributed in a phylogenetic and serogroup-dependent manner suggesting the existence of stable EIEC subclones. The presence of phylogroups B2 and E strains allied to the presence of UPEC virulence-associated genes may underscore the ongoing evolution of EIEC towards a hypervirulent pathotype.

Substantial changes in the intestine-derived lymph during bacterial translocation.

NFECTION CONTINUES to be an important concern in clinical small bowel transplantation (SBT), and increasing evidence has credited bacterial translocation (BT) as being a possible source for the pathogenesis of postsurgical infection. 1– 4 In addition, the so-called gut hypothesis of the pathogenesis of sepsis progressing to multiple organ failure has been attributed to enhanced BT in septic states by increasing vascular permeability. 5,6 In previous works, we have shown two distinctive routes of BT, lymphatic and hematological. 7 However, the lymphatic via only showed positive BT up to the mesenteric lymph node (MLN) through the intestinal afferent ducts, and, surprisingly, negative BT by efferent mesenteric lymph duct (MLD). 7 Thus, we raised the hypothesis that the lymphatic via of BT was possibly linked to host immune activation and the hematological via would be the true route for intestinal bacteria to reach the systemic circulation. In this study, we investigated the role of the lymphatic via of BT on host immune response by examining changes in efferent MLD contents using a previously established BT model in rats. 2