Novel Rhamnosyltransferase Involved in Biosynthesis of Serovar 4-Specific Glycopeptidolipid from Mycobacterium avium Complex

Glycopeptidolipids (GPLs) are one of the major glycolipid components present on the surface of Mycobacterium avium complex (MAC) that belong to opportunistic pathogens distributed in the natural environment. The serovars of MAC, up to around 30 types, are defined by the variable oligosaccharide portions of the GPLs. Epidemiological studies show that serovar 4 is the most prevalent type, and the prognosis of pulmonary disease caused by serovar 4 is significantly worse than that caused by other serovars. However, little is known about the biosynthesis of serovar 4-specific GPL, particularly the formation of the oligosaccharide portion that determines the properties of serovar 4. To investigate the biosynthesis of serovar 4-specific GPL, we focused on one segment that included functionally unknown genes in the GPL biosynthetic gene cluster of a serovar 4 strain. In this segment, a putative hemolytic protein gene, hlpA, and its downstream gene were found to be responsible for the formation of the 4-O-methyl-rhamnose residue, which is unique to serovar 4-specific GPL. Moreover, functional characterization of the hlpA gene revealed that it encodes a rhamnosyltransferase that transfers a rhamnose residue via 1→4 linkage to a fucose residue of serovar 2-specific GPL, which is a key pathway leading to the synthesis of oligosaccharide of serovar 4-specific GPL. These findings may provide clues to understanding the biological role of serovar 4-specific GPL in MAC pathogenicity and may also provide new insights into glycosyltransferase, which generates structural and functional diversity of GPLs.The genus Mycobacterium has a unique feature in the cell envelope that contains a multilayered structure consisting of peptidoglycan, mycolyl-arabinogalactan complex, and surface glycolipids (8, 12). It is known that these components play a role in protection from environmental stresses, such as antimicrobial agents and host immune responses (8, 12). Some of them are recognized as pathogenic factors related to mycobacterial diseases, such as tuberculosis and leprosy (8, 12). In case of nontuberculous mycobacteria that are widely distributed in the natural environment as opportunistic pathogens, glycopeptidolipids (GPLs) are abundantly present on the cell envelope as surface glycolipids (34). GPLs have a core structure in which a fatty acyl-tetrapeptide is glycosylated with 6-deoxy-talose (6-d-Tal) and O-methyl-rhamnose (O-Me-Rha) (2, 5, 13). This structure is common to all types of GPLs, and GPLs with this structure that have not undergone further glycosylation are termed non-serovar-specific GPLs (nsGPLs) (2, 5, 13). Structural diversity generated by further glycosylations, such as rhamnosylation, fucosylation, and glucosylation, is observed for the oligosaccharide portion linked to the 6-d-Tal residue of nsGPLs from Mycobacterium avium complex (MAC), a member of the nontuberculous mycobacteria consisting of two species, M. avium and M. intracellulare (2, 5, 34). Consequently, these nsGPLs with varied oligosaccharides lead to the formation of the serovar-specific GPLs (ssGPLs) that define around 30 types of MAC serovars (10).The properties of MAC serovars are known to be notably different from each other and also to be closely associated with the pathogenicity of MAC (3, 6, 18, 30, 31, 32). Various epidemiological studies indicate that serovar 4 is the most prevalent type and is also one of the serovars frequently isolated from AIDS patients (1, 20, 33, 36). Additionally, pulmonary MAC disease caused by serovar 4 is shown to exhibit a poorer prognosis than that caused by other serovars (23). With respect to host immune responses to MAC infection, serovar 4-specific GPL is reported to have characteristic features that are in contrast to those of other ssGPLs (21, 30). Structurally, serovar 4-specific GPL contains a unique oligosaccharide in which the oligosaccharide of serovar 2-specific GPL is further glycosylated with 4-O-methyl-rhamnose (4-O-Me-Rha) residue through a 1→4 linkage (Table ​(Table1)1) (24). Therefore, it is thought that the presence of 4-O-Me-Rha and its linkage position are important in exhibiting the specificity of biological activities. The biosynthesis of the oligosaccharide portion in several ssGPLs is currently being clarified (15, 16, 17, 25, 26), while that of serovar 4-specific GPL is still unresolved. In this study, we have focused on the genomic region predicted to be associated with GPL biosynthesis in the serovar 4 strain and explored the key genes responsible for the formation of 4-O-Me-Rha that might determine the specific properties of MAC serovar 4.

TABLE 1.

Oligosaccharide structures of serovar 2- and 4-specific GPLs

Flagellated but Not Hyperfimbriated Salmonella enterica Serovar Typhimurium Attaches to and Forms Biofilms on Cholesterol-Coated Surfaces

The asymptomatic, chronic carrier state of Salmonella enterica serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces in vitro and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of S. enterica serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified Salmonella flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.The serovars of Salmonella enterica are diverse, infect a broad array of hosts, and cause significant morbidity and mortality in impoverished and industrialized nations worldwide. S. enterica serovar Typhi is the etiologic agent of typhoid fever, a severe illness characterized by sustained bacteremia and a delayed onset of symptoms that afflicts approximately 20 million people each year (14, 19). Serovar Typhi can establish a chronic infection of the human gallbladder, suggesting that this bacterium utilizes novel mechanisms to mediate enhanced colonization and persistence in a bile-rich environment.There is a strong correlation between gallbladder abnormalities, particularly gallstones, and development of the asymptomatic Salmonella carrier state (47). Antibiotic regimens are typically ineffective in carriers with gallstones (47), and these patients have an 8.47-fold-higher risk of developing hepatobiliary carcinomas (28, 46, 91). Elimination of chronic infections usually requires gallbladder removal (47), but surgical intervention is cost-prohibitive in developing countries where serovar Typhi is prevalent. Thus, understanding the progression of infection to the carrier state and developing alternative treatment options are of critical importance to human health.The formation of biofilms on gallstones has been hypothesized to facilitate enhanced colonization of and persistence in the gallbladder. Over the past 2 decades, bacterial biofilms have been increasingly implicated as burdens for food and public safety worldwide, and they are broadly defined as heterogeneous communities of microorganisms that adhere to each other and to inert or live surfaces (17, 22, 67, 89, 102). A sessile environment provides selective advantages in natural, medical, and industrial ecosystems for diverse species of commensal and pathogenic bacteria, including Streptococcus mutans (40, 92, 104), Staphylococcus aureus (15, 35, 100), Escherichia coli (21, 74), Vibrio cholerae (39, 52, 107), and Pseudomonas aeruginosa (23, 58, 73, 105). Bacterial biofilms are increasingly associated with many chronic infections in humans and exhibit heightened resistance to commonly administered antibiotics and to engulfment by professional phagocytes (54, 55, 59). The bacterial gene expression profiles for planktonic and biofilm phenotypes differ (42, 90), and the changes are likely regulated by external stimuli, including nutrient availability, the presence of antimicrobials, and the composition of the binding substrate.Biofilm formation occurs in sequential, highly ordered stages and begins with attachment of free-swimming, planktonic bacteria to a surface. Subsequent biofilm maturation is characterized by the production of a self-initiated extracellular matrix (ECM) composed of nucleic acid, proteins, or exopolysaccharides (EPS) that encase the community of microorganisms. Planktonic cells are continuously shed from the sessile, matrix-bound population, which can result in reattachment and fortification of the biofilm or systemic infection and release of the organism into the environment. Shedding of serovar Typhi by asymptomatic carriers can contaminate food and water and account for much of the person-to-person transmission in underdeveloped countries.Our laboratory has previously reported that bile is required for formation of mature biofilms with characteristic EPS production by S. enterica serovars Typhimurium, Enteritidis, and Typhi on human gallstones and cholesterol-coated Eppendorf tubes (18, 78). Cholesterol is the primary constituent of human cholesterol gallstones, and use of cholesterol-coated tubes creates an in vitro uniform surface that mimics human gallstones (18). It was also demonstrated that Salmonella biofilms that formed on different surfaces had unique phenotypes and required expression of specific EPS (18, 77), yet the factors mediating Salmonella binding to gallstones and cholesterol-coated surfaces during the initiation of biofilm formation remain unknown. Here, we show that the presence of serovar Typhimurium flagella promotes binding specifically to cholesterol in the early stages of biofilm development and that the FliC subunit is a critical component. Bound salmonellae expressing intact flagella provided a scaffold for other cells to bind to during later stages of biofilm growth. Elucidation of key mechanisms that mediate adherence to cholesterol during Salmonella bile-induced biofilm formation on gallstone surfaces promises to reveal novel drug targets for alleviating biofilm formation in chronic cases.     

Thioredoxin 1 Participates in the Activity of the Salmonella enterica Serovar Typhimurium Pathogenicity Island 2 Type III Secretion System

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium relies on its Salmonella pathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in a trxA mutant. Complementation analyses using wild-type trxA or a genetically engineered trxA coding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections.In Escherichia coli, thioredoxin 1 (TrxA, encoded by trxA) is an evolutionary conserved 11-kDa cytosolic highly potent reductase that supports the activities of various oxidoreductases and ribonucleotide reductases (1, 29) and interacts with a number of additional cytoplasmic proteins through the formation of temporary covalent intermolecular disulphide bonds (32). Consequently, as trxA mutants of E. coli (51), Helicobacter pylori (13), and Rhodobacter sphaeroides (34) show increased sensitivity to hydrogen peroxide, TrxA has been defined as a significant oxidoprotectant. In addition, TrxA possess a protein chaperone function that is disconnected from cysteine interactions (30, 32).Salmonella enterica serovar Typhimurium is closely related to E. coli. During divergent evolution, the Salmonella genome acquired a number of virulence-associated genes (20). Many of these genes are clustered on genetic regions termed Salmonella pathogenicity islands (or SPIs). Of these, SPI1 and SPI2 code for separate type III secretion systems (T3SSs). T3SSs are supramolecular virulence-associated machineries that, in several pathogenic gram-negative bacterial species, enable injection of effector proteins from the bacteria into host cells (22, 57). The effector proteins, in turn, manipulate intrinsic host cell functions to facilitate the infection.The SPI1 T3SS of S. serovar Typhimurium is activated for expression in the intestine in response to increased osmolarity and decreased oxygen tension (22, 57). SPI1 effector proteins are primarily secreted into cells that constitute the epithelial layer and interfere with host cell Cdc42 and Rac-1 signaling and actin polymerization. This enables the bacteria to orchestrate their own actin-dependent uptake into nonphagocytic cells (57). SPI1 effector proteins also induce inflammatory signaling and release of interleukin-1β from infected cells (25, 26).Subsequent systemic progression of S. serovar Typhimurium from the intestinal tissue relies heavily on an ability to survive and replicate in phagocytic cells (18, 46, 53, 54). S. serovar Typhimurium uses an additional set of effector proteins secreted by the SPI2 T3SS for replication inside host cells and for coping with phagocyte innate responses to the infection (10, 11, 54). The functions of SPI2 effectors include diversion of vesicular trafficking, induction of apoptotic responses, and manipulation of ubiquitination of host proteins (28, 40, 45, 53). Hence, SPI2 effector proteins create a vacuolar environment that sustains intracellular replication of S. serovar Typhimurium (28).In addition to pathogenicity islands, the in vivo fitness of Salmonella spp. relies on selected functions shared with other enterobacteria. Thus, many virulence genes are integrated into “housekeeping” gene regulatory networks, coded for by a core genome, which steer bacterial stress responses (12, 17, 27, 55). Selected anabolic pathways also contribute to virulence of S. serovar Typhimurium (18, 27), evidently by providing biochemical building blocks for bacterial replication (36).In S. serovar Typhimurium, TrxA is a housekeeping protein that strongly contributes to virulence in cell culture and mouse infection models (8). However, the mechanism by which TrxA activity adds to virulence has not been defined. Here we show that the contribution of TrxA to virulence of S. serovar Typhimurium associates with its functional integration with the SPI2 T3SS under conditions that prevail in the intracellular vacuolar compartment of the host cell. These findings ascribe a novel role to TrxA in bridging environmental adaptations with virulence gene expression and illuminate a new aspect of the interaction between evolutionary conserved and horizontally acquired gene functions in bacteria.     

Poly-N-Acetylglucosamine Matrix Polysaccharide Impedes Fluid Convection and Transport of the Cationic Surfactant Cetylpyridinium Chloride through Bacterial Biofilms

Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix. Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is a major biofilm matrix component in phylogenetically diverse bacteria. In this study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced in vitro by the gram-negative porcine respiratory pathogen Actinobacillus pleuropneumoniae and the gram-positive device-associated pathogen Staphylococcus epidermidis. The effect of PNAG on bulk fluid flow was determined by measuring the rate of fluid convection through biofilms cultured in centrifugal filter devices. The rate of fluid convection was significantly higher in biofilms cultured in the presence of the PNAG-degrading enzyme dispersin B than in biofilms cultured without the enzyme, indicating that PNAG decreases bulk fluid flow. PNAG also blocked transport of the quaternary ammonium compound cetylpyridinium chloride (CPC) through the biofilms. Binding of CPC to biofilms further impeded fluid convection and blocked transport of the azo dye Allura red. Bioactive CPC was efficiently eluted from biofilms by treatment with 1 M sodium chloride. Taken together, these findings suggest that CPC reacts directly with the PNAG matrix and alters its physical and chemical properties. Our results indicate that PNAG plays an important role in controlling the physiological state of biofilms and may contribute to additional biofilm-associated processes such as biocide resistance.Biofilms are composed of bacterial cells encased in a self-synthesized, extracellular polymeric matrix (7). The main function of the biofilm matrix is to provide a structural framework that holds the cells together in a mass and firmly attaches the bacterial mass to the underlying surface. In addition to having a structural role, the matrix provides biofilm cells with a protected microenvironment containing dissolved nutrients, secreted enzymes, DNA, and phages. The matrix may also contribute to the increased antimicrobial resistance exhibited by biofilm cells, either by providing a diffusion barrier or by directly binding to antimicrobial agents and preventing their penetration into the biofilm (19).Polysaccharides are a major matrix component in most bacterial biofilms (26). Poly-β(1,6)-N-acetyl-d-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm cohesion in numerous gram-negative members of the Proteobacteria family, including Escherichia coli, Yersinia pestis, Pseudomonas fluorescens, Bordetella spp., Xenorhabdus nematophila, Aggregatibacter actinomycetemcomitans, and Actinobacillus pleuropneumoniae (4, 8, 15, 22), and in the gram-positive species Staphylococcus aureus and Staphylococcus epidermidis (3, 17). Specific biofilm-related functions ascribed to PNAG include abiotic surface attachment (1), epithelial cell attachment (23, 28), intercellular adhesion (15, 17), and resistance to killing by antibiotics, detergents, antimicrobial peptides, and mammalian phagocytic cells (9, 10, 16, 27, 29).In the present study we investigated the physical and chemical properties of the PNAG matrix in biofilms produced by the porcine respiratory pathogen A. pleuropneumoniae and the device-associated pathogen S. epidermidis. By using a novel centrifugal filter device assay, we obtained evidence that PNAG significantly inhibits fluid convection and solute transport through A. pleuropneumoniae and S. epidermidis biofilms.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Antibiotic Resistance Patterns and Detection of blaDHA-1 in Salmonella Species Isolates from Chicken Farms in South Korea

Fifteen nonrepetitive ampicillin-resistant Salmonella spp. were identified among 91 Salmonella sp. isolates during nationwide surveillance of Salmonella in waste from 131 chicken farms during 2006 and 2007. Additional phenotyping and genetic characterization of these 15 isolates by using indicator cephalosporins demonstrated that resistance to ampicillin and reduced susceptibility to cefoxitin in three isolates was caused by TEM-1 and DHA-1 β-lactamases. Plasmid profiling and Southern blot analysis of these three DHA-1-positive Salmonella serovar Indiana isolates and previously reported unrelated clinical isolates of DHA-1-positive Salmonella serovar Montevideo, Klebsiella pneumoniae, and Escherichia coli from humans and swine indicated the involvement of the large-size plasmid. Restriction enzyme digestion of the plasmids from the transconjugants showed variable restriction patterns except for the two Salmonella serovar Indiana isolates identified in this study. To the best of our knowledge, this is the first report of the presence of the DHA-1 gene among Salmonella spp. of animal origin.Nontyphoidal Salmonella (NTS) strains are a significant cause of gastrointestinal infections of food origin. These microbes are a heterogeneous group of medically important Gram-negative bacteria and can infect a wide range of animals, including humans (3, 6, 9-11, 25).Currently, no antimicrobial therapies are recommended for the treatment of NTS infection unless a patient is of extreme age, has an underlying disease, or is infected with an invasive Salmonella sp. However, the use of antibiotics in treatment of clinical enteric infection has been heavily compromised by emerging multidrug-resistant microbes (4, 17, 18, 23). In particular, resistance due to extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases is of special concern as these enzymes confer resistance to some of the front-line antibiotics used to treat enteric infection in humans and animals (4, 13, 14, 19).Four classes of β-lactamases are known to confer resistance to β-lactam antibiotics. Among these, plasmid-mediated class A and class C β-lactamases have been frequently reported, whereas class B and class D β-lactamases are relatively rare (4). TEM and SHV enzymes of class A β-lactamases are generally found in Gram-negative bacteria and are derived by one or more amino acid substitutions around the active site of the enzyme that is responsible for the ESBL phenotype (4). Recently, the CTX-M enzyme of class A β-lactamases has been increasingly reported from enteric microbes, like Salmonella and Escherichia coli (4, 5, 9, 15). These have greater activity against cefotaxime than do other oxyimino-β-lactam substrates, like ceftazidime, ceftriaxone, or cefepime (4, 5). Plasmid-mediated AmpC β-lactamases, like DHA and CMY, are not inhibited by clavulanic acid and have been isolated from a wide variety of clinical and community-acquired microbes (2, 4, 13, 14, 16). These β-lactamases are native to the chromosomes of many Gram-negative bacilli but are missing in some genera, like Salmonella (4). The majority of β-lactamases reported in Salmonella to date have been derived from human clinical isolates, and only limited information is available regarding Salmonella spp. derived from farm animals, although isolates from both humans and animals are of clinical and epidemiological importance (4, 15, 25).In light of this knowledge gap, our study focused on assessing the distribution of Salmonella serovars in poultry farms in South Korea. Subsequently, isolates were analyzed for resistance to antibiotics commonly used in farms. Phenotypic and genetic characteristics of ampicillin-resistant Salmonella isolates were tested to gain insight into what β-lactamases were prevalent among these strains. We also characterized DHA-1-associated plasmids in these Salmonella spp. and compared them with clinical isolates of Salmonella, Klebsiella pneumoniae, and Escherichia coli from humans and from swine.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Poultry-Associated Salmonella enterica subsp. enterica Serovar 4,12:d:? Reveals High Clonality and a Distinct Pathogenicity Gene Repertoire

A European baseline survey during the years 2005 and 2006 has revealed that the monophasic Salmonella enterica subsp. enterica serovar 4,12:d:− was, with a prevalence of 23.6%, the most frequently isolated serovar in German broiler flocks. In Denmark and the United Kingdom, its serovar prevalences were 15.15% and 2.8%, respectively. Although poultry is a major source of human salmonellosis, serovar 4,12:d:− is rarely isolated in humans (approximately 0.09% per year). Molecular typing studies using pulsed-field gel electrophoresis and DNA microarray analysis show that the serovar is highly clonal and lacks genes with known contributions to pathogenicity. In contrast to other poultry-associated serovars, all strains were susceptible to 17 antimicrobial agents tested and did not encode any resistance determinant. Furthermore, serovar 4,12:d:− lacked the genes involved in galactonate metabolism and in the glycolysis and glyconeogenesis important for energy production in the cells. The conclusion of the study is that serovar 4,12:d:− seems to be primarily adapted to broilers and therefore causes only rare infections in humans.Salmonella spp. are major zoonotic food-borne pathogens which cause outbreaks and sporadic cases of gastroenteritis in humans worldwide (12). Depending on the serovar, cases of salmonellosis can differ substantially in severity (13). The primary sources of salmonellosis are food-producing animals, such as poultry, pigs, and cattle (30). The pathogen is spread by trade in animals and nonheated animal food products (10).A European baseline survey on the prevalence of Salmonella in commercial broiler flocks of Gallus gallus in 2005 and 2006 showed that in the European Union (EU), 23.7% of the broiler flocks were Salmonella positive (8). However, the Salmonella prevalences and serovar distribution varied widely among the EU member states. The five most frequently isolated Salmonella enterica serovars in Europe were those classically observed, like serovar Enteritidis (33.8%), serovar Infantis (22.0%), serovar Mbandaka (8.1%), serovar Hadar (3.7%), and serovar Typhimurium (3.0%). In Germany, the flock prevalence of Salmonella was 15.0% among the 377 broiler flocks investigated. In contrast to the well-known serovars described above, the predominating serovar was monophasic serovar 4,12:d:−, with a prevalence of 23.6%. This serovar was also isolated in Denmark and the United Kingdom, with prevalences of 15.2% and 2.8%, respectively.The German Salmonella National Reference Laboratory (NRL-Salmonella) has received 818 isolates of this serovar between 1998 and 2007, with peaks in 2001 (240 isolates) and 2004 (160 isolates), for diagnosis. Since 2005, the number has doubled annually, and the serovar obviously established itself well in poultry production lines. These isolates were found mostly in broilers (78%), occasionally in turkeys (11.6%) and feedstuff (8.4%), and rarely in pigs (1.3%) and cattle (0.6%). In contrast, infections in humans are only sporadic. During the last 10 years (1998 to 2007), the National Reference Centre for Salmonellae and Other Enterics located at the Robert-Koch Institute, Wernigerode branch, has received 55 strains of this serovar from sporadic human cases of salmonellosis and carriers in Germany (W. Rabsch, personal communication). Similarly, in Denmark, only two isolates in 1993 and in 2002 were isolated from humans (E. Møller Nielsen, Statens Serum Institut, Copenhagen, Denmark, personal communication).Subtyping food-borne pathogens is an approach often applied to facilitate the epidemiological investigation of outbreaks of gastrointestinal disease and to identify the source of entry into the food chain. Several molecular-based tools have been developed to type bacteria genotypically. Pulsed-field gel electrophoresis (PFGE) is currently the method of choice for the molecular subtyping of Salmonella serovars. It has been proven to be a useful discriminatory method which was standardized by the PulseNet Consortium (9). However, although this approach is certainly valuable, it does not reveal data on the gene repertoire and biological properties of a strain. To overcome this weakness, whole-genome DNA microarrays have successfully been applied in comparative genomic hybridizations for Salmonella (7, 24, 25). However, whole-genome arrays reflect only one genome of one strain. Because of many serovar or strain genome variations described for Salmonella, thematic arrays were developed, such as arrays specially targeting genes involved in resistance profiles (2, 17, 32), phage types (23), or serovars (33, 35). A condensed selection of 109 various Salmonella genetic markers comprising the detection of flagellar and somatic antigen-encoding genes, important virulence genes, phage-associated genes, and antibiotic resistance determinants have been used to show the usefulness of DNA microarrays for the discriminative characterization of Salmonella serovars (18).In this study, we elucidate the contradicting situation between the high prevalence in broilers and source attribution of broiler meat for humans and the low infection rates in humans of the serovar 4,12:d:− by genotypic characterization using PFGE and DNA microarray to determine the clonality, the pathogenic gene repertoire, and resistance determinants. These data give basic information to discuss the hazard potential of this serovar for humans. For that purpose, a new prototype of a Salmonella DNA microarray comprising 281 60-mer oligonucleotide probes was developed and validated in house.     

Roles of Minor Pilin Subunits Spy0125 and Spy0130 in the Serotype M1 Streptococcus pyogenes Strain SF370

Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal ∼1/3 and C-terminal ∼2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.The group A Streptococcus (S. pyogenes) is an exclusively human pathogen that commonly colonizes either the pharynx or skin, where local spread can give rise to various inflammatory conditions such as pharyngitis, tonsillitis, sinusitis, or erysipelas. Although often mild and self-limiting, GAS infections are occasionally very severe and sometimes lead to life-threatening diseases, such as necrotizing fasciitis or streptococcal toxic shock syndrome. A wide variety of cell surface components and extracellular products have been shown or suggested to play important roles in S. pyogenes virulence, including cell surface pili (1, 6, 32). Pili expressed by the serotype M1 S. pyogenes strain SF370 mediate specific adhesion to intact human tonsil epithelia and to primary human keratinocytes, as well as cultured keratinocyte-derived HaCaT cells, but not to Hep-2 or A549 cells (1). They also contribute to adhesion to a human pharyngeal cell line (Detroit cells) and to biofilm formation (29).Over the past 5 years, pili have been discovered on an increasing number of important Gram-positive bacterial pathogens, including Bacillus cereus (4), Bacillus anthracis (4, 5), Corynebacterium diphtheriae (13, 14, 19, 26, 27, 44, 46, 47), Streptococcus agalactiae (7, 23, 38), and Streptococcus pneumoniae (2, 3, 24, 25, 34), as well as S. pyogenes (1, 29, 32). All these species produce pili that are composed of a single major subunit plus either one or two minor subunits. During assembly, the individual subunits are covalently linked to each other via intermolecular isopeptide bonds, catalyzed by specialized membrane-associated transpeptidases that may be described as pilin polymerases (4, 7, 25, 41, 44, 46). These are related to the classical housekeeping sortase (usually, but not always, designated SrtA) that is responsible for anchoring many proteins to Gram-positive bacterial cell walls (30, 31, 33). The C-terminal ends of sortase target proteins include a cell wall sorting (CWS) motif consisting, in most cases, of Leu-Pro-X-Thr-Gly (LPXTG, where X can be any amino acid) (11, 40). Sortases cleave this substrate between the Thr and Gly residues and produce an intermolecular isopeptide bond linking the Thr to a free amino group provided by a specific target. In attaching proteins to the cell wall, the target amino group is provided by the lipid II peptidoglycan precursor (30, 36, 40). In joining pilus subunits, the target is the ɛ-amino group in the side chain of a specific Lys residue in the second subunit (14, 18, 19). Current models of pilus biogenesis envisage repeated transpeptidation reactions adding additional subunits to the base of the growing pilus, until the terminal subunit is eventually linked covalently via an intermolecular isopeptide bond to the cell wall (28, 41, 45).The major subunit (sometimes called the backbone or shaft subunit) extends along the length of the pilus and appears to play a structural role, while minor subunits have been detected either at the tip, the base, and/or at occasional intervals along the shaft, depending on the species (4, 23, 24, 32, 47). In S. pneumoniae and S. agalactiae one of the minor subunits acts as an adhesin, while the second appears to act as a linker between the base of the assembled pilus and the cell wall (7, 15, 22, 34, 35). It was originally suggested that both minor subunits of C. diphtheriae pili could act as adhesins (27). However, recent data showed one of these has a wall linker role (26, 44) and may therefore not function as an adhesin.S. pyogenes strain SF370 pili are composed of a major (backbone) subunit, termed Spy0128, plus two minor subunits, called Spy0125 and Spy0130 (1, 32). All three are required for efficient adhesion to target cells (1). Studies employing purified recombinant proteins have shown that both of the minor subunits, but not the major subunit, bind to Detroit cells (29), suggesting both might act as pilus-presented adhesins. Here we report studies employing a combination of recombinant proteins, specific antisera, and allelic replacement mutants which show that only Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role in linking pili to the cell wall.