Marshall R. Monteville

Secretion of Virulence Proteins from Campylobacter jejuni Is Dependent on a Functional Flagellar Export Apparatus

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.

Fibronectin-Facilitated Invasion of T84 Eukaryotic Cells by Campylobacter jejuni Occurs Preferentially at the Basolateral Cell Surface

ABSTRACT Previous studies have indicated that the ability to bind to fibronectin is a key feature in successful cell invasion by Campylobacter jejuni. Given the spatial distribution of fibronectin and the architecture of the epithelium, this suggests the possibility that C. jejuni cell invasion might preferentially occur at the basolateral cell surface. To test this hypothesis, we examined the interaction of C. jejuni with T84 human colonic cells. When grown under the appropriate conditions, T84 cells form a polarized cell monolayer. C. jejuni translocation of a T84 cell monolayer appeared to occur via a paracellular (extracellular) route as opposed to a transcellular (intracellular) route based on the finding that a C. jejuni noninvasive mutant translocated as efficiently as its isogenic parent. Additional studies revealed that two distinct C. jejuni wild-type isolates could compete with one another for host cell receptors, whereas a C. jejuni fibronectin-binding-deficient mutant could not compete with a wild-type isolate for host cell receptors. Further, C. jejuni adherence and internalization were significantly inhibited by antifibronectin antibodies but only when cells were first treated with EGTA to expose basolateral cell surfaces. Together, these results support the theory that C. jejuni invasion occurs preferentially at the basolateral surface of eukaryotic cells.

Identification of a fibronectin-binding domain within the Campylobacter jejuni CadF protein

The binding of Campylobacter jejuni to fibronectin (Fn), a component of the extracellular matrix, is mediated by a 37 kDa outer membrane protein termed CadF for Campylobacter adhesion to Fn. Previous studies have indicated that C. jejuni binds to Fn on the basolateral surface of T84 human colonic cells. To further characterize the interaction of the CadF protein with Fn, enzyme‐linked immunosorbent assays were performed to identify the Fn‐binding domain (Fn‐BD). Using overlapping 30‐mer and 16‐mer peptides derived from translated cadF nucleotide sequence, maximal Fn‐binding activity was localized to four amino acids (AA 134–137) consisting of the residues phenylalanine‐arginine‐leucine‐serine (FRLS). A mouse α‐CadF peptide polyclonal antibody (M α‐CadF peptide pAb) was generated using FRLS containing peptides and found to react with viable C. jejuni as judged by indirect fluorescent microscopy, suggesting that the FRLS residues are surface‐exposed. Binding of CadF to purified Fn and INT 407 human epithelial cells was significantly inhibited with peptides containing the Fn‐BD. Moreover, a CadF recombinant variant protein, in which the Phe‐Arg‐Leu residues (CadF AA 134–136) were altered to Ala‐Ala‐Gly, exhibited a 91% decrease in Fn‐binding activity as compared with the wild‐type CadF protein. Collectively, these data indicate that the FRLS residues (CadF AA 134–137) of the C. jejuni CadF protein possess Fn‐binding activity.

Azithromycin and loperamide are comparable to levofloxacin and loperamide for the treatment of traveler's diarrhea in United States military personnel in Turkey.

BACKGROUND The recommended treatment for travelers diarrhea is the combination of an appropriate antibiotic (usually a fluoroquinolone) and loperamide. Azithromycin compared favorably with fluoroquinolones in trials that did not include the use of loperamide, but combination therapy has not, to our knowledge, been studied to date. METHODS A randomized, double-blind trial was conducted at Incirlik Air Base, Turkey, fromJ une 2003 through August 2004. Adults from the United States with noninflammatory diarrhea were randomized to receive a single dose of azithromycin (1000 mg; 106 persons) or levofloxacin (500 mg; 101 persons) plus loperamide (4 mg initially and as needed thereafter). Volunteers maintained a symptom diary and were evaluated on days 1, 3, and 7 after treatment. RESULTS No differences were noted with respect to pretreatment symptoms or pathogen distribution. Enterotoxigenic Escherichia coli was the most common pathogen isolated (from 45% of patients in the azithromycin group and 42% of patients in the levofloxacin group), and Campylobacter species was the second most common pathogen isolated (from 6% of patients in the azithromycin group and 9% of patients in the levofloxacin group). Median time to last diarrheal stool (azithromycin group, 13 h; levofloxacin group, 3 h), median time to resolution of associated symptoms (2 days), and additional loperamide usage (azithromycin group, 39% of patients; levofloxacin group, 34% of patients) were similar between groups. Azithromycin use was associated with more nausea in the 30 min after dosing (azithromycin group, 8% of patients; levofloxacin group, 1% of patients; Pp.004), but no vomiting or other adverse events were noted in either group. CONCLUSIONS Single-dose treatment with azithromycin (1000 mg) and loperamide is as effective as single-dose treatment with levofloxacin (500 mg) and loperamide for noninflammatory diarrhea. Although nausea after dosing is uncommon, it is more frequently associated with azithromycin than with levofloxacin. Future studies should focus on determining whether lower doses of azithromycin would decrease the frequency of nausea and decrease treatment costs without affecting efficacy.

Seroepidemiologic Survey for Coxiella burnetii Among US Military Personnel Deployed to Southwest and Central Asia in 2005

We used a seroepidemiologic study to estimate Q fever (Coxiella burnetii) seroprevalence, seroincidence, and risk factors for seroconversion in two deployed military populations in 2005. The first study group resided in an area with a known Q fever outbreak history (Al Asad, Iraq). Of this population, 7.2% seroconverted for an incidence rate of 10.6 seroconversions per 1,000 person-months. The second population included personnel transiting through Qatar on mid-deployment leave from southwest/central Asia. In this group, we found 2.1% prevalence with 0.92 seroconversions per 1,000 person-months. However, no significant risk factors for Q fever seroconversion were found in either population.

Genotypic characterization of Egypt enterotoxigenic Escherichia coli isolates expressing coli surface antigen 6

INTRODUCTION One approach to control enterotoxigenic Escherichia coli (ETEC) infections has been to develop vaccines focused on inducing protective immunity against surface expressed antigenic factors. One such factor is coli surface antigen 6 (CS6); ETEC isolates expressing CS6 may also simultaneously co-express surface antigens CS4 or CS5. However, there is little information regarding the inter-relationships of isolates expressing the CS6 antigen alone or in combination with CS4 or CS5. METHODOLOGY A total of 62 CS6-associated ETEC isolates were evaluated for their antimicrobial susceptibility, mechanisms of resistance, toxin genes, colonization factor expression, and XbaI-pulsed-field gel electrophoretic profiles. RESULTS We observed 46 XbaI profiles; 31 were exclusive to ETEC expressing CS6 alone and 15 among the ETEC co-expressing CS4 or CS5. Nearly half (47%) of these isolates were resistant to ampicillin, a third (37%) of the isolates were resistant to trimethoprim-sulfamethoxazole, and 24% of the isolates were tetracycline-resistant. A blaTEM gene was detected in 24 (83%) ampicillin-resistant isolates. Trimethoprim-sulfamethoxazole-resistant isolates (n = 23) carried either sulI (n = 1, 4%), sulII (n = 8, 35%) or both genes (n = 10, 43%); 4 had no detectable sul gene. CONCLUSIONS Our results show a lack of clonality among Egypt CS6 E. coli isolates and supports the use and the further research on vaccines targeting this cell surface antigen.