Commensal and pathogenic microorganisms must resist the deleterious actions of bile in order to survive in the human gastrointestinal tract. Herein we review the current knowledge on the mechanisms by which Gram-positive and Gram-negative bacteria contend with bile stress. We describe the antimicrobial actions of bile, assess the variations in bile tolerance between bacterial genera and examine the interplay between bile stress and other stresses. The molecular mechanisms underlying bile tolerance are investigated and the relationship between bile and virulence is examined. Finally, the potential benefits of bile research are briefly discussed.

The interaction between bacteria and bile

The List of Bacterial Names with Standing in Nomenclature includes, alphabetically and chronologically, the official names of bacteria as published or validated in the International Journal of Systematic Bacteriology. It encompasses 5,569 taxa (as of 31 December 1996) and is available on the Internet (URL: ftp://ftp.cict.fr/pub/bacterio/).

List of Bacterial Names with Standing in Nomenclature: a Folder Available on the Internet

Multilocus sequence typing (MLST) was proposed in 1998 as a portable, universal, and definitive method for characterizing bacteria, using the human pathogen Neisseria meningitidis as an example. In addition to providing a standardized approach to data collection, by examining the nucleotide sequences of multiple loci encoding housekeeping genes, or fragments of them, MLST data are made freely available over the Internet to ensure that a uniform nomenclature is readily available to all those interested in categorizing bacteria. At the time of writing, over thirty MLST schemes have been published and made available on the Internet, mostly for pathogenic bacteria, although there are schemes for pathogenic fungi and some nonpathogenic bacteria. MLST data have been employed in epidemiological investigations of various scales and in studies of the population biology, pathogenicity, and evolution of bacteria. The increasing speed and reduced cost of nucleotide sequence determination, together with improved web-based databases and analysis tools, present the prospect of increasingly wide application of MLST.

Multilocus Sequence Typing of Bacteria

The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds.

The Virulence Plasmid of Yersinia, an Antihost Genome

Deoxyribonucleic acid from 41 different bacterial species (including Gram-negative and Gram-positive species) were cleaved by different restriction endonucleases, electrophoresed in agarose and transferred to nylon filters. The DNA fragments carrying rRNA genes (rDNA) were localized by hybridization with a 32P-labelled Escherichia coli 16 + 23S rRNA probe. A pattern of hybridized fragments was obtained for each DNA tested. Within a bacterial species (defined as a DNA hybridization group), one or several rDNA restriction patterns were observed. When DNA hybridization data were available, strains showing identical patterns were highly related with insignificant divergence. In a species, different patterns corresponded to significant divergence, as evaluated by thermal stability studies of DNA/DNA hybrids. Sets of rDNA restriction fragment sizes might constitute useful data for inclusion in species and type strain descriptions. Such data might later prove useful in identification of bacteria when biochemical characteristics are poor or atypical.

Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools.

Since the publication of the Approved Lists of Bacterial Names, the type species of the genus Salmonella has been S. choleraesuis. At the time of publication of the Approved Lists, five Salmonella species had standing in the nomenclature, and the description of S. choleraesuis was the same as that of the serotype of that name. Several studies have shown that the genus Salmonella consists of only one species, and the strict application of the Bacteriological Code would recognize S. choleraesuis (the type species) as the single Salmonella species. This can lead to confusion and hazards since the specific epithet is also the name of a serovar (serovar Choleraesuis). This confusion is increased by the common practice of using serovar names as if they represented species names (e.g., S. typhi, S. choleraesuis, and S. typhimurium). Some serovars (e.g., Salmonella choleraesuis subsp. choleraesuis serovar Typhi) are highly pathogenic and cause a disease different from that caused by other serovars (e.g. S. choleraesuis subsp. choleraesuis serovar Choleraesuis). To avoid further confusion, it is proposed to use for the single Salmonella species a name which has not been used earlier for a serotype. It is thus requested that the type species of the genus Salmonella be Salmonella enterica sp. nov., nom. rev. with strain CIP 60.62 (a H2S-producing clone of strain LT2) as the type strain. Since the name S. choleraesuis is not proposed for rejection, bacteriologists who do not accept the single species concept of the genus Salmonella will be free to use the name S. choleraesuis as a synonym of Salmonella enterica subsp. enterica serotype Choleraesuis.

Designation of Salmonella enterica sp. nov., nom. rev., as the Type and Only Species of the Genus Salmonella: Request for an Opinion

In this study we examined the taxonomic relationships of strains variously labeled Achromobacter species biotypes 1 and 2, Achromobacter group A, and Centers for Disease Control (CDC) groups Vd-1 and Vd-2. Previous studies of ribosomal ribonucleic acid cistron similarities placed these organisms on the Brucella ribosomal ribonucleic acid branch of ribosomal ribonucleic acid superfamily IV; their closest neighbors were Brucella, Phyllobacterium, and the Agrobacterium-Rhizobium complex. We performed a numerical taxonomic analysis of 284 phenotypic features (69 conventional tests, 147 API assimilation tests, 68 API ZYM tests) carried out on 95 strains. These organisms comprised 56 strains thought to correspond to CDC group Vd (including 3 strains originally labeled “Pseudomonas arsenoxydans”) and 39 strains (included for reference purposes) representing the genera Achromobacter, Agrobacterium, Alcaligenes, Brucella, Mycoplana, Phyllobacterium, and Rhizobium. A phenotypic analysis showed that group Vd bacteria are most similar to Phyllobacterium. However, strains of Group Vd were shown to be distinct by deoxyribonucleic acid (DNA)-DNA hybridization and by several phenotypic tests from Phyllobacterium and other related taxa. The CDC group Vd strains formed essentially a single taxon in the numerical taxonomic analysis of phenotypic characters and as determined by DNA-DNA hybridization. This taxon could be subdivided into three biotypes (biotypes A, C, and D), but none of these corresponded to the two biotypes originally described among the group Vd strains. For CDC group Vd we propose a new genus and new species, Ochrobactrum anthropi: The type strain is strain CIP 82.115 (= CIP 14970 = NCTC 12168 = LMG 3331). O. anthropi strains are rod shaped, aerobic, gram negative, nonpigmented, and motile by means of peritrichous flagella, produce acid from several carbohydrates, and reduce both nitrate and nitrite. The guanine-plus-cytosine contents of the DNAs of 29 strains ranged from 56 to 59 mol%. Almost all 56 group Vd strains were originally isolated from various human clinical specimens, commonly from blood cultures.

Ochrobactrum anthropi gen. nov., sp. nov. from Human Clinical Specimens and Previously Known as Group Vd

Synthesis of Vi antigen, a capsular polysaccharide expressed by Salmonella typhi, is controlled by the viaA and viaB chromosomal loci. It was previously shown that Vi antigen expression was regulated by a system similar to the rcs regulatory system involved in colanic acid synthesis in Escherichia coli. We have cloned the rcsA, rcsB, and rcsC genes from S. typhi. The predicted amino sequences of the RcsA and RcsB proteins showed a high degree of similarity to their E. coli homologs. The nucleotide sequence of the rcsC gene was partially determined and was shown to be homologous to that of its E. coli counterpart. Complementation experiments indicated that rcsB and rcsC were encompassed within the viaA locus. The RcsA protein was not involved in Vi antigen synthesis. In contrast, the RcsB protein acted as a positive regulator of Vi polysaccharide expression. By mRNA and gene fusion analyses, we studied the role of RcsB and TviA, a via-B-encoded regulatory protein characterized previously, in regulating Vi antigen synthesis. The transcriptional start point of tviA mRNA was not influenced by RcsB or TviA. In the absence of RcsB or TviA protein, transcription of tviA gave rise to only a monocistronic tviA-specific mRNA. The presence of RcsB and TriA not only increased the amount of monocistronic tviA-specific mRNA but also resulted in countranscription of tviA and tviB, which is located immediately downstream of tviA on the viaB locus. In addition, TviA protein did not appear to be subject to degradation by the Lon protease. These results strongly suggest that TviA might act in concert with RcsB at the tviA promoter to activate transcription of the genes involved in Vi polymer synthesis in S. typhi in a Lon-independent manner.

Characterization of the rcsA and rcsB genes from Salmonella typhi: rcsB through tviA is involved in regulation of Vi antigen synthesis.

Summary The growth pattern of Salmonella enterica subsp. enterica serotype Typhimurium (Typhimurium) was studied in mice to examine the role of the 60-Mdal virulence-associated plasmid in the pathogenesis of mouse typhoid. After repeated subcultures at 45°C, isogenic variants harbouring the virulence-associated plasmid (strains C52, TM122 and TM332) or having lost this large plasmid (strains C53, TM123 and TM333) were obtained from three parental strains (strains C5, TM 12 and TM33, respectively). Plasmid plP1350, present in strain C52, was tagged by Tn10 and transferred by successive conjugations to strains C53, TM123 and TM333. The behaviour of these three Typhimurium lines was studied in C57BL/6, DBA2, 136132 (C57BL/6 x DBA2 F1 hybrid) and OF, mice after oral infection, subcutaneous injection into the hind footpad or intravenous inoculation. The kinetics of organ colonization were followed at intervals after injection by enumeration of viable bacteria in caecum, mesenteric or popliteal lymph node, spleen, liver, kidney and lung depending on the route of infection. Strains harbouring virulence plasmid and their cured derivatives did not differ significantly in their ability to colonize caecal content and to translocate to draining lymph nodes. Elimination of the virulence plasmid was correlated with a significant reduction in the ability of cured variants to colonize spleen and liver. Reintroduction of the virulence plasmid into plasmidless variants restored the virulence to the level originally observed. These data demonstrate that a 60-Mdal plasmid in Typhimurium strains is necessary to ensure colonization of spleen and liver of experimentally infected mice.

Virulence-associated plasmids of Salmonella serotype Typhimurium in experimental murine infection.

Burkholderia pseudomallei, a gram-negative bacterium, is the causative agent of melioidosis in humans and animals (9, 13, 45). Melioidosis is endemic in tropical areas of Southeast Asia and northern Australia, but it is also sporadically found in many other countries. This highly pathogenic microorganism is deemed a potential agent of bioterrorism (listed as category B by the Centers for Disease Control and Prevention). The clinical manifestations of melioidosis vary greatly, ranging from an asymptomatic state to acute septicemia, pulmonary forms, and chronic granulomatous lesions (34). The latency period of the disease is between 2 days and 26 years (21, 34). Successful treatment is difficult because B. pseudomallei is inherently resistant to a wide range of antibiotics and relapse is common (43). Although melioidosis was first described in 1912 (44), the virulence determinants of B. pseudomallei have not been well characterized. Studies of B. pseudomallei pathogenicity have mostly concentrated on exoproducts, which include a protease, a lipase, a phospholipase C, and a hemolysin (3, 34, 45). The regulatory circuits governing the production of exoproducts in B. pseudomallei remain unknown. However, many gram-negative pathogens regulate the production of extracellular virulence factors by quorum sensing, a cell-density-dependent mechanism. Quorum sensing is the process of producing and responding to high intracellular concentrations of N-acylhomoserine lactone (AHL) autoinducers and relies on two proteins: (i) an AHL synthase belonging to the LuxI family, which directs the synthesis of AHL; and (ii) a transcriptional regulator belonging to the LuxR family, which, after binding of AHL, is thought to activate or repress transcription of targeted genes (11, 22, 29). In Burkholderia cepacia, which is phylogenetically related to B. pseudomallei (13), the CepI-CepR quorum-sensing system positively regulates protease production and represses synthesis of the siderophore ornibactin (19).

There has been much interest in the contribution of the B. pseudomallei protease to virulence. This enzyme was purified from culture supernatants as a 36-kDa metalloenzyme and appeared to be necessary for full virulence in a rat model of lung infection (32). In contrast, we found no correlation between virulence and the level of protease activity after intraperitoneal infection of mice with B. pseudomallei (12). These results suggested that the role played by the protease in pathogenesis could be dependent on the route of infection. Recently, the mprA gene of B. pseudomallei was cloned and was found to encode a 50-kDa serine metalloprotease, the only protease produced by B. pseudomallei (17). However, the discrepancy with the size determined by Sexton et al. (32) has not been explained yet; perhaps the difference is due to proteolysis or the use of different techniques (17). Our previous investigations on the B. pseudomallei protease (12, 25) led us to examine whether enzyme production is regulated by quorum sensing.

The role of quorum-sensing systems in the pathogenicity of different gram-negative bacteria has been demonstrated previously (28, 35). The existence of such a system in B. pseudomallei could provide new data on the virulence determinants of this organism.

Here we describe identification of the PmlI-PmlR quorum-sensing system of B. pseudomallei. This system is crucial for full virulence of B. pseudomallei in a murine model of infection and modulates the production of the MprA protease.

M.Y. Popoff

Papers

Designation of Salmonella enterica sp. nov., nom. rev., as the Type and Only Species of the Genus Salmonella: Request for an Opinion

Ochrobactrum anthropi gen. nov., sp. nov. from Human Clinical Specimens and Previously Known as Group Vd

Characterization of the rcsA and rcsB genes from Salmonella typhi: rcsB through tviA is involved in regulation of Vi antigen synthesis.

Virulence-associated plasmids of Salmonella serotype Typhimurium in experimental murine infection.

The PmlI-PmlR Quorum-Sensing System in Burkholderia pseudomallei Plays a Key Role in Virulence and Modulates Production of the MprA Protease