Bicarbonate enhances the in vitro antibiotic activity of kanamycin in Escherichia coli.

UNLABELLED: Growth of enteropathogenic Escherichia coli E2348/69 was inhibited by bicarbonate in a dose-dependent manner, showing approximately 5% growth reduction at 5 mmol l(-1) while kanamycin at 3·12 µg ml(-1) inhibited growth by 15%, yet when kanamycin and bicarbonate were combined at these concentrations, inhibition increased to 80%. Unexpectedly, at bicarbonate concentrations >20 mmol l(-1) enhancement of the antibiotic activity virtually disappeared, i.e. there was a paradoxical Eagle-like effect. How bicarbonate acts is unclear, but neutral or alkaline pH also enhanced the activity of kanamycin. However, several differences indicated a separate effect of bicarbonate. First, bicarbonate inhibited growth more than the corresponding increments in pH. Second, at low concentration, the antibiotic enhancing effect of bicarbonate was stronger than the effect of pH alone. Third, 5 mmol l(-1) bicarbonate significantly enhanced the activity of kanamycin while the corresponding pH had no effect. Fourth, the Eagle-like effect was exclusive of bicarbonate because changes in pH did not induce an analogous behaviour. Notwithstanding the mechanism, the enhancing effect of bicarbonate was indubitable. Consequently, it seems worthwhile to explore further its potential to improve the efficacy of aminoglycosides and maybe even other antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Bicarbonate at a low concentration enhanced the in vitro antibiotic activity of kanamycin and gentamicin. Even though the action mechanism of bicarbonate is hitherto unknown, it seems worthwhile to explore further its capacity to improve the efficacy of aminoglycosides. Clearly, the well-known harmful side-effects of aminoglycosides are a concern. However, it has recently been shown in a fish model that bicarbonate may protect ciliary cells against the damage caused by aminoglycosides. So, it seems possible that bicarbonate could help reduce aminoglycoside dosage at the same time that it might help lessen the damage to auditory ciliary cells in humans.

Heterogeneity in the activity of Mexican Helicobacter pylori strains in gastric epithelial cells and its association with diversity in the cagA gene.

Helicobacter pylori CagA is translocated into gastric epithelial cells by a type IV secretion system and interacts with the Src homology 2 phosphatase, altering cell morphology. Multiple EPIYA motifs in CagA are associated with increased activity in cells and with gastric cancer. The aim of this work was to study the heterogeneity in activity in cells of multiple H. pylori single colonies isolated from a single patient and its association with polymorphism in cagA. The presence of cagA, cagE, cagT, and cag10 was studied with 318 H. pylori isolates from the antra and corpora of 18 patients. AGS gastric epithelial cells were infected with 75 isolates, and interleukin-8 (IL-8) secretion, cytoskeletal changes, CagA translocation, and tyrosine phosphorylation were measured. The cagA 3'-variable region was sequenced for 30 isolates to determine the number and types of EPIYA motifs. Isolates from an individual stomach were usually genetically related and had quantitatively similar phenotypic effects on cells (IL-8 induction and cytoskeletal changes). However, strains from different patients with similar CagA EPIYA motif patterns varied widely in these phenotypes. Among isolates with an EPIYA-ABC pattern, the phenotype was variable: IL-8 induction ranged from 200 to 1,200 pg/ml, and morphological changes occurred in 20 to 70% of cells. In several cases, cagA sequence diversity appeared to explain the lack of CagA activity, as isolates with an EPIYA-ACC pattern or a modified B motif had reduced cell activity. cag pathogenicity island-positive H. pylori isolates displayed a high level of heterogeneity in the capacity to induce IL-8 secretion and morphological changes; an absent or modified B motif was associated with low activity.

Phenotypic characterization of ipaH+ Escherichia coli strains associated with yolk sac infection.

Seventy-six Escherichia coli serotypes possessing the ipaH gene typical of enteroinvasive E. coli (EIEC) strains were characterized. Biochemical identification of our strains shows positive reactions for lactose fermentation (100% of strains), lysine decarboxylase (98.7% of strains) and motility (67.1% of strains), properties that do not correspond with those described to the EIEC group. The serotypes agree with an initial classification. In this, some common O antigens identified among ipaH+ strains were O2 (n=20), OR (n=11) and non-determined O? (n=10). The O2:NM serotype was the most common. Sixty-six percent (n=50) of the ipaH+ E. coli strains were colicin producers, of them, 26 (34%) produced Col V and other colicins, 13 (17%) produced colicins other than Col V, and 11 (14.5%) produced Col V only. Trimethoprim/Sulfa (72%), ampicillin (64.5%), enrofloxacin (55.3%), and ciprofloxacin (47.4%) were the major antimicrobial resistance frequencies observed. Twenty-five different multiresistance patterns were observed, where sixty-six strains (86.8%) were included. A MIC test showed that most of the strains were sensitive to low gentamicin and kanamycin concentrations, whereas most of the strains were resistant to tetracycline. An invasiveness assay showed that the predominant alterations caused to HEp-2 cells were changes in shape and staining, and in most of the specimens, a partial monolayer detachment was also seen. Fifteen strains invaded more than 30% of the monolayer cells, causing the formation of intercellular bridges or filipoidal-like protrusions. The results suggest the existence of specific clone complexes derived from EIEC strains adapted to the avian host. To our knowledge, this is the first study that demonstrates the presence of extraintestinal invasive E. coli (ExIEC) strains.

Estudio clínico de un grupo familiar español con síndrome de Hermansky-Pudlak / Clinical study of a Spanish family with Hermansky-Pudlak syndrome

El síndrome de Hermansky-Pudlak (SHP) es una enfermedad autosómica recesiva rara, en la que están afectadas organelas intracitoplasmáticas como melanosomas, cuerpos densos plaquetarios y lisosomas, y son la causa de albinismo oculocutáneo, diátesis hemorrágica y, en algunos pacientes, depósito de material ceroide en tejidos. Se han descrito 7 genes implicados en el SHP, muchos de ellos codifican proteínas de función desconocida. El SHP es una enfermedad rara; sin embargo, es la enfermedad monogénica más frecuente en el noroeste de Puerto Rico, debido a una mutación con efecto fundador. El objetivo de este artículo es presentar una familia con 15 pacientes afectados por esta enfermedad (AU)

Enterohaemorrhagic and enteropathogenic Escherichia coli use a different Tir-based mechanism for pedestal formation.

Enterohaemorrhagic Escherichia coli (EHEC) adheres to the host intestinal epithelium, resulting in the formation of actin pedestals beneath adhering bacteria. EHEC and a related pathogen, enteropathogenic E. coli (EPEC), insert a bacterial receptor, Tir, into the host plasma membrane, which is required for pedestal formation. An important difference between EPEC and EHEC Tir is that EPEC but not EHEC Tir is tyrosine phosphorylated once delivered into the host. In this study, we assessed the role of Tir tyrosine phosphorylation in pedestal formation by EPEC and EHEC. In EPEC, pedestal formation is absolutely dependent on Tir tyrosine phosphorylation and is not complemented by EHEC Tir. The protein sequence surrounding EPEC Tir tyrosine 474 is critical for Tir tyrosine phosphorylation and pedestal formation by EPEC. In contrast, Tir tyrosine phosphorylation is not required for pedestal formation by EHEC. EHEC forms pedestals with both wild-type EPEC Tir and the non-tyrosine-phosphorylatable EPEC Tir Y474F. Pedestal formation by EHEC requires the type III delivery of additional EHEC factors into the host cell. These findings highlight differences in the mechanisms of pedestal formation by these closely related pathogens and indicate that EPEC and EHEC modulate different signalling pathways to affect the host actin cytoskeleton.

Transcriptional regulation of the orf19 gene and the tir-cesT-eae operon of enteropathogenic Escherichia coli.

To establish an intimate interaction with the host epithelial cell surface, enteropathogenic Escherichia coli (EPEC) produces Tir, a bacterial protein that upon translocation and insertion into the epithelial cell membrane constitutes the receptor for intimin. The tir gene is encoded by the locus for enterocyte effacement (LEE), where it is flanked upstream by orf19 and downstream by the cesT and eae genes. With the use of a series of cat transcriptional fusions and primer extension analysis, we confirmed that tir, cesT, and eae form the LEE5 operon, which is under the control of a promoter located upstream from tir, and found that the orf19 gene is transcribed as a monocistronic unit. We also demonstrated that the LEE-encoded regulator Ler was required for efficient activation of both the tir and the orf19 promoters and that a sequence motif located between positions -204 and -157 was needed for the Ler-dependent activation of the tir operon. Sequence elements located between positions -204 and -97 were determined to be required for the differential negative modulatory effects exerted by unknown regulatory factors under specific growth conditions. Upon deletion of the upstream sequences, the tir promoter was fully active even in the absence of Ler, indicating that tir expression is subject to a repression mechanism that is counteracted by this regulatory protein. However, its full activation was still repressed by growth in rich medium or at 25 degrees C, suggesting that negative regulation also occurs at or downstream of the promoter. Expression of orf19, but not of the tir operon, became Ler independent in an hns mutant strain, suggesting that Ler overcomes the repression exerted by H-NS (histone-like nucleoid structuring protein) on this gene.

Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression.

Secretion of effector proteins in enteropathogenic Escherichia coli (EPEC) is mediated by a specialized type III secretion system whose components are encoded in the LEE1, LEE2 and LEE3 operons. Using cat transcriptional fusions and primer extension analysis, we determined that the LEE2 and LEE3 operons are expressed from two overlapping divergent promoters, whose expression is negatively regulated by flanking common upstream and downstream silencing regulatory sequences (SRS1 and SRS2). In the absence of either SRS1 or SRS2, expression of the LEE2 and LEE3 operons became independent of Ler, a positive regulatory protein encoded by the first gene of the LEE1 operon. Similarly, in the absence of the histone-like protein H-NS, expression from both promoters became Ler independent even if both SRSs were present. In addition, the efficient expression of both the LEE2 and the LEE3 promoters required PerC (BfpW), a protein coded by the third gene of the per (bfpTVW) locus, but only in the presence of the EAF plasmid. Our deletion analysis also showed that the negative regulation observed in the presence of ammonium or at temperatures above 37 degrees C (e.g. 40 degrees C) required the SRSs or elements located therein. In contrast, the negative regulation observed in LB or at temperatures below 37 degrees C (e.g. 25 degrees C) was still observed even in the absence of both SRSs and seems to act only on the promoters. Together, these results suggest that Ler acts as an antirepressor protein that overcomes the H-NS-mediated silencing on the LEE2/LEE3 divergent promoter region, which is probably caused by the formation of a repressing H-NS-nucleoprotein complex.

Macrocarpins A-D, new cytotoxic nor-triterpenes from Maytenus macrocarpa.

Macrocarpins A (1), B (2), C (3) and D (4), four new nor-triterpenes, have been isolated from the roots of Maytenus macrocarpa. The structures were established by spectroscopic examinations. Natural compounds 1, 2, 4 and the acetyl derivative 1a are cytotoxic against four tumoral cell lines with IC50 values ranging between 0.4 and 5.2 microM.

Tumour prothymosin alpha content, a potential prognostic marker for primary breast cancer.

In a previous report we suggested that the estimation of prothymosin alpha (PTA) levels in primary breast tumours might be used to identify breast cancer patients at high risk for distant metastasis (Dominguez F et al (1993) Eur J Cancer 29A: 893-897). Here the role of tumour PTA levels as predictor was investigated with respect to both disease-free survival (DFS) and survival. Tumours were obtained from a series of 210 consecutive female patients with ductal carcinoma who underwent surgery at the Hospital Xeral de Galicia (Santiago de Compostela, Spain). Characteristics including PTA tumour levels, number of positive axillary nodes, patient's age at surgery and tumour histological grade were significantly associated with DFS and survival, as determined by univariate analysis. Patients with tumours with low or moderate PTA levels demonstrated a statistically decreased rate of tumour recurrence and a statistically significant increased overall survival in comparison with those whose tumours had high PTA levels. Patient's relative risk of dying was 2.1 times greater for tumours with high PTA levels than for those tumours with low or moderate PTA levels. In conclusion, these data support the hypothesis that tumour high PTA levels is associated with a worse outcome.

Enteropathogenic Escherichia coli translocated intimin receptor, Tir, requires a specific chaperone for stable secretion.

Enteropathogenic Escherichia coli (EPEC) secretes several Esps (E. coli-secreted proteins) that are required for full virulence. Insertion of the bacterial protein Tir into the host epithelial cell membrane is facilitated by a type III secretion apparatus, and at least EspA and EspB are required for Tir translocation. An EPEC outer membrane protein, intimin, interacts with Tir on the host membrane to establish intimate attachment and formation of a pedestal-like structure. In this study, we identified a Tir chaperone, CesT, whose gene is located between tir and eae (which encodes intimin). A mutation in cesT abolished Tir secretion into culture supernatants and significantly decreased the amount of Tir in the bacterial cytoplasm. In contrast, this mutation did not affect the secretion of the Esp proteins. The level of tir mRNA was not affected by the cesT mutation, indicating that CesT acts at the post-transcriptional level. The cesT mutant could not induce host cytoskeletal rearrangements, and displayed the same phenotype as the tir mutant. Gel overlay and GST pulldown assays demonstrated that CesT specifically interacts with Tir, but not with other Esp proteins. Furthermore, by using a series of Tir deletion derivatives, we determined that the CesT binding domain is located within the first 100 amino-terminal residues of Tir, and that the pool of Tir in the bacterial cytoplasm was greatly reduced when this domain was disrupted. Interestingly, this domain was not sufficient for Tir secretion, and at least the first 200 residues of Tir were required for efficient secretion. Gel filtration studies showed that Tir-CesT forms a large multimeric complex. Collectively, these results indicate that CesT is a Tir chaperone that may act as an anti-degradation factor by specifically binding to its amino-terminus, forming a multimeric stabilized complex.

Autoactivation and environmental regulation of bfpT expression, the gene coding for the transcriptional activator of bfpA in enteropathogenic Escherichia coli.

Expression of bfpA, the gene coding for the structural subunit of the bundle-forming pili (BFP) in enteropathogenic Escherichia coli (EPEC), requires the product of bfpT (also called perA), a member of the AraC family of transcriptional regulators. Here, we show that bfpT-cat fusions were not expressed in a bfpT - or in a non-EPEC strain, unless a functional bfpT was present, indicating that an autoregulatory mechanism is involved in expression. Further experiments with bfpT-cat fusions and primer extension analysis showed that bfpT is transcribed from a conventional sigma-70 promoter and that it is expressed throughout the growth curve. It is regulated in response to the ammonium concentration, temperature and growth media, in the same proportions as those described previously for bfpA. In addition, bfpT and bfpA expression was also modulated by osmolarity, but was not affected by pH, iron excess or limitation. Deletion analysis of the bfpT upstream region revealed that a DNA segment of 81 bp, extending upstream from the transcriptional start site, contained all the sequence elements required for maximal expression of bfpT. Furthermore, it shares significant homology with a bfpA upstream AT-rich region, which has been shown to be involved in the BfpT-dependent regulation of bfpA. Interestingly, ammonium repression was observed only when bfpT-cat or bfpA-cat expression was complemented in an EPEC background, whereas low-temperature regulation was observed in both EPEC and non-EPEC strains. This suggests that specific regulatory elements are present in EPEC, while others are shared with non-pathogenic E. coli.

Negative and positive regulation of the non-osmoregulated ompS1 porin gene in Salmonella typhi: a novel regulatory mechanism that involves OmpR.

The Salmonella typhi ompS1 gene codes for an outer membrane protein of the OmpC/OmpF porin family. It is expressed at very low levels, relative to the major porins. However, deletion analysis of the 5' regulatory region showed that the gradual removal of nucleotides -310 to -88, upstream from the P1 major transcriptional start-point, resulted in a stepwise increase in expression, reaching levels 10-fold above those for the ompC major porin gene. Hence, this 222 bp segment contains cis-acting regulatory elements involved in negative control. Primer extension analysis revealed the presence of three promoters: P1 activity was OmpR dependent; P2 was expressed at a lower level in the absence of OmpR; and P3 had a minor constitutive activity. OmpR bound preferentially to box II, an 18 bp F1/C1 canonical site, the removal (-88 to -66) of which resulted in a decrease in expression thus supporting its role in positive control. Expression of ompS1 was not induced by a set of stress conditions, including a shift in osmolarity, nor was the IHF regulator involved in negative control. An ompS1 homologue was found in E. coli K-12, which contains a nonsense codon and a shift in the reading frame, whereas Salmonella typhimurium contains an open reading frame in this region. Thus, S. typhi ompS1 provides novel features in OmpR regulation.

The ompB operon partially determines differential expression of OmpC in Salmonella typhi and Escherichia coli.

Expression of the Escherichia coli OmpC and OmpF outer membrane proteins is regulated by the osmolarity of the culture media. In contrast, expression of OmpC in Salmonella typhi is not influenced by osmolarity, while OmpF is regulated as in E. coli. To better understand the lack of osmoregulation of OmpC expression in S. typhi, we compared the expression of the ompC gene in S. typhi and E. coli, using ompC-lacZ fusions and outer membrane protein (OMP) electrophoretic profiles. S. typhi ompC expression levels in S. typhi were similar at low and high osmolarity along the growth curve, whereas osmoregulation of E. coli ompC in E. coli was observed during the exponential phase. Both genes were highly expressed at high and low osmolarity when present in S. typhi, while expression of both was regulated by osmolarity in E. coli. Complementation experiments with either the S. typhi or E. coli ompB operon in an S. typhi DeltaompB strain carrying the ompC-lacZ fusions showed that both S. typhi and E. coli ompC were not regulated by osmolarity when they were under the control of S. typhi ompB. Interestingly, in the same strain, both genes were osmoregulated under E. coli ompB. Surprisingly, in E. coli DeltaompB, they were both osmoregulated under S. typhi or E. coli ompB. Thus, the lack of osmoregulation of OmpC expression in S. typhi is determined in part by the ompB operon, as well as by other unknown trans-acting elements present in S. typhi.

Fimbrial expression in enteric bacteria: a critical step in intestinal pathogenesis.

The ability of species of enteric bacteria to recognize and colonize unique niches along the intestine is mainly based on receptor distribution and interpretation of a combination of environmental signals leading to the expression of specific adherence factors. Such elaborate orchestration of events is critical during the initial steps of pathogenesis.

Analysis of cis-acting elements required for bfpA expression in enteropathogenic Escherichia coli.

bfpA expression in enteropathogenic Escherichia coli is regulated by growth medium, temperature, and ammonium concentration and requires the BfpT protein (also called PerA), a member of the AraC family of transcriptional activators. Site-directed and PCR random mutagenesis, as well as deletion analysis of the bfpA upstream regulatory region, supported assignment of the promoter elements and demonstrated that the cis-acting elements that mediate BfpT-dependent regulation of bfpA are located between positions -85 and -46. Interestingly, this region shares 73% identity with a 40-bp-long AT-rich tract located upstream of the bfpT gene, which is essential for bfpT autoregulation.

Distinctive IS200 insertion between gyrA and rcsC genes in Salmonella typhi.

While probing the vicinity of ompC, a copy of the IS200 insertion element was found between the gyrA and rcsC genes of Salmonella typhi, the causal agent of typhoid fever. This distinctive feature was conserved throughout 63 S. typhi isolates of different geographical origins and was absent from 46 other Salmonella serotypes, including those most associated with human infections, as well as from 19 other enteric bacteria. Furthermore, the location of this IS200 copy corresponds to a constant band, present throughout the 14 PstI S. typhi IS200 fingerprints, encompassing several Vi phage types. Interestingly, an apparently unrelated serotype not frequently associated with human disease, Salmonella weltevreden, contained an IS200 copy at the same position, although it was accompanied by an additional segment of cryptic DNA. On the basis of these findings, PCR assays were designed for molecular typing of S. typhi, and these are potentially useful in studying the epidemiology of typhoid fever.

Development of ELISA to estimate thymosin alpha1, the N terminus of prothymosin alpha, in human tumors.

We reported that tumor content of prothymosin alpha (ProT alpha) is a proliferation index of human breast tumors that might be used to identify patients at high risk for distant metastasis (Dominguez et al., Eur J Cancer 1993; 29A:893-7). In that study ProT alpha concentrations were measured by a RIA; here we present an alternative nonisotopic assay that could be used in a standard clinical laboratory. Main features of the ELISA are: (a) A recombinant fusion protein glutathione S-transferase (GST)-human ProT alpha was used to coat the microtiter plates; (b) we used a polyclonal antiserum raised in rabbits that detects thymosin alpha1, the NH2-terminal fragment of ProT alpha; (c) it is as sensitive as the RIA; (d) it is faster than the RIA. ProT alpha concentrations in various human tumors (skin, esophagus, colorectal, and breast) as assessed by ELISA were comparable with, although twofold greater than, the values previously estimated by RIA.

Cloning and characterization of bfpTVW, genes required for the transcriptional activation of bfpA in enteropathogenic Escherichia coli.

Expression of the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is regulated at the transcriptional level by growth phase, temperature, calcium and ammonium. Genes required for the transcriptional activation of bfpA were localized to a 1.8 kb fragment of the enteroadherent factor (EAF) plasmid of EPEC that is separated from the bfp operon by 6 kb. Within this fragment three identically oriented and closely spaced open reading frames (ORFs) were identified and designated bfpT, bfpV and bfpW. bfpT is predicted to encode a 31.8 kDa protein that shares homology with the AraC family of transcriptional regulators, including the presence of a conserved C-terminal DNA-binding helix-turn-helix motif. Insertional inactivation of bfpT led to the loss of bfpA transcription, BfpA protein production and the localized adherence (LA) phenotype; this mutant phenotype could be complemented by introduction of bfpTVW and, on separate plasmids, bfpT + bfpW. However, introduction of bfpT + bfpV, bfpV alone, bfpW alone, or bfpV + bfpW did not enable recovery of the wild-type phenotype. Maximal efficiency of bfpA transcription required all three genes, but bfpV and bfpW each enhanced transcription providing bfpT was also present. A series of deletions of the bfpA upstream promoter region was prepared; with respect to the bfpA transcription start site, sequence between nucleotides -94 and -55 was found to bind bfpT. BfpT also bound a DNA fragment containing the eaeA promoter region on the EPEC chromosome. From these results we conclude that bfpTV W causes transcriptional activation of bfpA, and possibly eaeA, by a trans-acting mechanism that may co-ordinately regulate the expression of EPEC virulence determinants.

Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.

Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion. Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase. Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype.

The bundle-forming pili of enteropathogenic Escherichia coli: transcriptional regulation by environmental signals.

The bundle-forming pili (BFP) of enteropathogenic Escherichia coil (EPEC) are required for the development of circumscribed colonies of bacteria attached to the surfaces of cultured epithelial cells, a process termed the localized adherence (LA) phenotype. Similar lesions are evident in jejunal biopsies from EPEC-infected children. BFP production is not constitutive, but instead occurs upon transfer of bacteria from nutrient broth to tissue culture media, indicating that the expression of BFP may be environmentally regulated. To learn more about how BFP protein expression is induced during epithelial-cell adherence, bfpA-cat transcriptional fusions and northern blot analyses were employed to monitor bfpA expression as a function of environmental signals and growth kinetics. bfpA expression was found to be regulated at the transcriptional level, and to require a separate locus on the EPEC adherence factor (EAF) plasmid. Expression occurred selectively during exponential-growth phase and was greatest between 35 and 37 degrees C, and in the presence of calcium. Ammonium (20 mM) significantly reduced bfpA mRNA and protein expression and the development of the LA phenotype. Analysis of the bfpA upstream sequence and identification of the transcription initiation site revealed a conventional sigma (70)-dependent promoter and an AT-rich tract that might affect promoter activity. Taken together, these findings further support the pathogenic role of BFP by explaining how BFP production would commence in the small intestine and terminate in the colon and in external habitats.