Fish monocytes as a model for mycobacterial host-pathogen interactions.

Mycobacterium marinum, a relatively rapid-growing fish and human pathogen, has become an important model for the investigation of mycobacterial pathogenesis. M. marinum is closely related to the Mycobacterium tuberculosis complex and causes a disease in fish and amphibians with pathology similar to tuberculosis. We have developed an in vitro model for the study of M. marinum virulence mechanisms using the carp monocytic cell line CLC (carp leukocyte culture). We found that fish monocytes can differentiate between pathogenic and nonpathogenic mycobacterial species. Interestingly, M. marinum enters fish monocytes at a 40- to 60-fold-higher rate than Mycobacterium smegmatis. In addition, M. marinum survives and replicates in fish monocytes while M. smegmatis is killed. We also found that M. marinum inhibits lysosomal fusion in fish monocytes, indicating that these cells may be used to dissect the mechanisms of intracellular trafficking in mycobacteria. We conclude from these observations that monocytic cells from fish, a natural host for M. marinum, provide an extremely valuable model for the identification and characterization of mycobacterial virulence determinants in the laboratory.

Entry mechanisms of mycobacteria.

Since many mycobacteria are facultative intracellular pathogens, their ability to cause disease involves entry, survival and replication within host cells. Despite the fact that mycobacteria were first associated with disease more than 125 years ago, the first step in the production of an infection, entry into host cells, is not well understood. Mycobacteria have the ability to enter a number of different cell types, but the primary cell type that they are thought to replicate within during human disease is macrophages. Since macrophages have a large number of receptors that are designed for relatively non-specific uptake of foreign particles, there are multiple routes by which nearly any bacteria can be taken up. The outcome of mycobacterial entry into macrophages via different mechanisms is unclear. Although it is thought that mycobacteria may enter macrophages by a mechanism that allows them to avoid lysosomal fusion, it remains possible that mycobacteria enter by more than one mechanism, yet remain viable and replicate intracellularly through modification of the phagosome. In the current discussion we will review mycobacterial research specifically relating to the mechanisms of entry into host cells. Although much progress has been made in our understanding of entry by mycobacteria, we anticipate that clarification of the role of entry in pathogenesis will require further application of newly developed molecular tools to dissect each of the proposed mechanisms.

Legionella pneumophila entry gene rtxA is involved in virulence.

Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1, enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to beta(2) integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry gene rtxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.

Phenotypic diversity of enterotoxigenic Escherichia coli strains from a community-based study of pediatric diarrhea in periurban Egypt.

No past studies of diarrhea in children of the Middle East have examined in detail the phenotypes of enterotoxigenic Escherichia coli (ETEC) strains, which are important pathogens in this setting. During a prospective study conducted from November 1993 to September 1995 with 242 children under 3 years of age with diarrhea living near Alexandria, Egypt, 125 episodes of diarrhea were positive for ETEC. ETEC strains were available for 98 of these episodes, from which 100 ETEC strains were selected and characterized on the basis of enterotoxins, colonization factors (CFs), and O:H serotypes. Of these representative isolates, 57 produced heat-stable toxin (ST) only, 34 produced heat-labile toxin (LT) only, and 9 produced both LT and ST. Twenty-three ETEC strains expressed a CF, with the specific factors being CF antigen IV (CFA/IV; 10 of 23; 43%), CFA/II (5 of 23; 22%), CFA/I (3 of 23; 13%), PCFO166 (3 of 23; 13%), and CS7 (2 of 23; 9%). No ETEC strains appeared to express CFA/III, CS17, or PCFO159. Among the 100 ETEC strains, 47 O groups and 20 H groups were represented, with 59 O:H serotypes. The most common O serogroups were O159 (13 strains) and O43 (10 strains). O148 and O21 were each detected in five individual strains, O7 and O56 were each detected in four individual strains, O73, O20, O86, and O114 were each detected in three individual strains, and O23, O78, O91, O103, O128, and O132 were each detected in two individual strains. The most common H serogroups were H4 (16 strains), 12 of which were of serogroup O159; H2 (9 strains), all of which were O43; H18 (6 strains); H30 (6 strains); and H28 (5 strains); strains of the last three H serogroups were all O148. Cumulatively, our results suggest a high degree of clonal diversity of disease-associated ETEC strains in this region. As a low percentage of these strains expressed a CF, it remains possible that other adhesins for which we either did not assay or that are as yet undiscovered are prevalent in this region. Our findings point out some potential barriers to effective immunization against ETEC diarrhea in this population and emphasize the need to identify additional protective antigens commonly expressed by ETEC for inclusion in future vaccine candidates.

Enteropathogens associated with diarrhea among military personnel during Operation Bright Star 96, in Alexandria, Egypt.

This study investigated the microbial causes of diarrheal disease among U.S. troops deployed near Alexandria, Egypt, during October 1995. Bacterial causes associated with 19 cases of diarrhea included: enterotoxigenic Escherichia coli (ETEC), 42% (21% heat-stable, 11% heat-labile, and 11% heat-stable/ heat-labile producers); enteropathogenic E. coli (5.3%); and enteroadherent E. coli (42%). Four cases of diarrhea were associated with enteroaggregative E. coli based on probe analysis for enteroaggregative heat-stable enterotoxin 1. Protozoan causes included; Entamoeba histolytica (11%), E. hartmanni (5%), E. nana (5%), Blastocystis hominis (5%), Chilomastix mesnili (11%), Dientamoeba fragilis (5%), Entamoeba coli (5%), and Cryptosporidium (5%). Shigella, Aeromonas, Plesiomonas, Vibrio, Campylobacter, and Salmonella were not detected. Of the eight ETEC cases, one was colonization factor antigen (CFA)/I only, one was both CFA/I and CFA/III, three were CFA/II, two were CFA/IV, and two were CFA-negative. Antibiograms of the ETEC and enteroadherent E. coli strains showed that all isolates were susceptible to norfloxacin, ciprofloxacin, and nalidixic acid but resistant to ampicillin, tetracycline, chloramphenicol, and sulfamethoxazole.

Detection of enterotoxigenic Escherichia coli, Shigella and Campylobacter spp. by multiplex PCR assay.

Three oligonucleotide primers were used in a polymerase chain reaction (PCR) assay for the simultaneous amplification of regions of the invasive plasmid antigen (ipaH) of Shigella spp., flagellin gene (flaA) of Campylobacter spp., and heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC). The multiplex assay was performed using DNA extracted by a chaotropic method directly from diarrhoeal stools. The diagnostic efficacy of the assay was analyzed by agarose gel electrophoresis. This assay shows a novel approach for the diagnosis of diarrhoea caused by Shigella spp., ETEC, and Campylobacter spp.

Prevalence of antibiotic resistance among Egyptian Salmonella typhi strains.

This report describes the resistance of 537 Salmonella typhi isolates identified in Egypt between 1990-1994. Results indicated a high isolation rate for multiple resistant S. typhi (> 71% of isolates collected in 1992-93), particularly to the three standard drug regimens of the clinically relevant antibiotics; ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole. This adds to the complexity and difficulty of treating infections caused by these organisms. Resistance of S. typhi was associated with a transferable 120 MD plasmid. The organism was sensitive to amikacin, aztreonam, cephalothin, ceftriaxone, gentamicin and nalidixic acid, suggesting the use of aztreonam and ceftriaxone as alternative therapeutic drugs for the treatment of multidrug-resistant S. typhi. These results may provide a clinically useful evaluation of the spread and acquisition of resistance among S. typhi strains in Egypt.

Colonization factors of enterotoxigenic E. coli (ETEC) from residents of northern Egypt.

Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a serious health problem to children in developing countries. Colonization of the small intestinal mucosa by ETEC strains is mediated by antigenically specific fimbriae, also known as colonization factor antigens (CFA). The importance of this study arises from reports that active and passive immunization with ETEC strains harboring CFAs induced protective immunity against diarrhea in animal models with preformed antibodies. In humans, ETEC containing CFA/I, II, III and IV have been identified. The aim of this study was to define CFAs of ETEC isolated in Alexandria, Egypt. One hundred and seven ETEC isolates from 132 human residents in Alexandria, Egypt were isolated during a birth cohort study. ETEC isolates were screened for heat labile (LT) and heat stable (ST) toxins using a 32P oligonucleotide hybridization probe and a GM1 ELISA. These isolates were examined using monoclonal antibodies against CFA/I, II, III, IV, and against the putative colonization antigens PCF0159 and PCF0166, CS 7 and CS 17. CFAs were found in 48% of ETEC strains. CFA/I was found in 18% of the strains, CFA/II in 10% and CFA/IV in 14%. CFA III was not found. All fifteen strains expressing CFA/IV expressed CS6 and produced ST. CFA/IV was not found in non-ST producing strains, while CFA/I was absent in ST-only producing strains.

A survey of enteropathogens among United States military personnel during Operation Bright Star '94, in Cairo, Egypt.

Acute gastroenteritis is a potential cause of substantial morbidity in U.S. military personnel during deployment. This study was conducted to evaluate enteric pathogens associated with diarrhea in a U.S. military population on deployment in Cairo, Egypt, during November 1993. Enteric pathogens found to be associated with cases of diarrhea included: enterotoxigenic Escherichia coli (ETEC), 27% (22% heat-stable [ST], 3% heat-labile [LT], and 2% ST/LT producers); Campylobacter spp., 3%; and Salmonella spp. 3%. Other enteric pathogens, namely Shigella, Aeromonas, Plesiomonas, Vibrio spp., Bacillus cereus, and enteric parasites, were not found in any of the 36 patients. Of the 8 patients who were ETEC-positive, three expressed colonization factor antigens (CFA)/II, and two expressed putative colonization factor antigen (PCF) 0159. All of the latter isolates produced ST. ETEC with different surface protein antigens were found to have surface hydrophobicity in the range of 0.2 M to greater than 2.0 M. Plasmid profiles of the ETEC strains showed no correlation with toxin production. In vitro susceptibility testing of the ETEC strain showed that 32% of the strains were resistant to three or more antimicrobial agents, whereas 24% showed 100% susceptibility. The enteropathogens tested were susceptible to norfloxacin, ciprofloxacin, and nalidixic acid, suggesting that the quinolones might be useful for the treatment of diarrheic patients.