Recent Emergence of Clonal Group O25b:K1:H4-B2-ST131 ibeA Strains among Escherichia coli Poultry Isolates,Including CTX-M-9-Producing Strains,and Comparison with Clinical Human Isolates

To discern the possible spread of the Escherichia coli O25b:H4-ST131 clonal group in poultry and the zoonotic potential of avian strains, we made a retrospective search of our strain collection and compared the findings for those strains with the findings for current strains. Thus, we have characterized a collection of 19 avian O25b:H4-ST131 E. coli strains isolated from 1995 to 2010 which, interestingly, harbored the ibeA gene. Using this virulence gene as a criterion for selection, we compared those 19 avian strains with 33 human O25b:H4-ST131 ibeA-positive E. coli strains obtained from patients with extraintestinal infections (1993 to 2009). All 52 O25b:H4-ST131 ibeA-positive E. coli strains shared the fimH, kpsMII, malX, and usp genes but showed statistically significant differences in nine virulence factors, namely, papGIII, cdtB, sat, and kpsMII K5, which were associated with human strains, and iroN, kpsMII K1, cvaC, iss, and tsh, which were associated with strains of avian origin. The XbaI macrorestriction profiles of the 52 E. coli O25b:H4-ST131 ibeA-positive strains revealed 11 clusters (clusters I to XI) of >85% similarity, with four clusters including strains of human and avian origin. Cluster VII (90.9% similarity) grouped 10 strains (7 avian and 3 human strains) that mostly produced CTX-M-9 and that also shared the same virulence profile. Finally, we compared the macrorestriction profiles of the 12 CTX-M-9-producing O25b:H4-ST131 ibeA strains (7 avian and 5 human strains) identified among the 52 strains with those of 15 human O25b:H4-ST131 CTX-M-14-, CTX-M-15-, and CTX-M-32-producing strains that proved to be negative for ibeA and showed that they clearly differed in the level of similarity from the CTX-M-9-producing strains. In conclusion, E. coli clonal group O25b:H4-ST131 ibeA has recently emerged among avian isolates with the new acquisition of the K1 capsule antigen and includes CTX-M-9-producing strains. This clonal group represents a real zoonotic risk that has crossed the barrier between human and avian hosts.Strains of the extensively antimicrobial-resistant Escherichia coli clonal group of sequence type (ST) 131 (ST131) belonging to serotype O25b:H4 have recently been recognized to be important human pathogens worldwide (9, 33). Although it is commonly associated with the dissemination of CTX-M-15 extended-spectrum cephalosporin resistance, E. coli O25b:H4-ST131 also occurs as a fluoroquinolone (FQ)-resistant but cephalosporin-susceptible pathogen (5, 22, 26, 27). Currently, it is assumed that O25b:H4-ST131 strains circulate not only among humans but also among animal hosts (13, 21, 37), which would contribute to the ongoing global emergence of O25b:H4-ST131, in the case of regular transmission between animals and humans. Even though CTX-M-15 is the most widely distributed extended-spectrum beta-lactamase (ESBL) linked to this clonal group, other, different variants of CTX-M have recently been reported, such as CTX-M-9, CTX-M-14, and CTX-M-32 (4, 34, 36, 39). Noteworthy was the detection, for the first time on poultry farms, of this clonal group producing CTX-M-9 that had macrorestriction profiles and virulence genes very similar to those observed in clinical human isolates (10).Extraintestinal pathogenic E. coli (ExPEC) strains, which include avian pathogenic E. coli (APEC) and human uropathogenic E. coli (UPEC), septicemic E. coli, and newborn meningitis-causing E. coli (NMEC) strains, exhibit considerable genome diversity and have a wide range of virulence-associated factors (12, 18). While infections caused by APEC strains initially start as a respiratory tract disease which evolves to a systemic infection of the internal organs and, finally, to sepsis, the most frequent origin of human sepsis is urinary tract infection (UTI), especially pyelonephritis (2, 3, 11). However, APEC strains have been recognized to share common traits with human isolates (29, 30, 31), including the K1 capsule antigen (23, 24, 29) and the ibeA gene (14). In addition, retail chicken products have been found to carry nalidixic-resistant ExPEC strains (17, 19), and although it is drug susceptible, an E. coli strain belonging to the O25b:H4-ST131 clonal group has even recently been detected in retail chicken (41), supporting the urgent necessity for the implementation of food control measures.The aim of the present study was to discern the possible spread of the O25b:H4-ST131 clonal group, especially CTX-M-9-producing strains, in poultry and the zoonotic potential of avian isolates. For this purpose, we made a retrospective search of our human and avian strain collections and compared the findings for those strains with the findings for current strains. Identification of this emerging clone among avian sources and comparison of the clone with clinical human isolates will shed new light on the epidemiology of the O25b:H4-ST131 clonal group.     

Absence of Escherichia coli Phylogenetic Group B2 Strains in Humans and Domesticated Animals from Jeonnam Province,Republic of Korea

Multiplex PCR analyses of DNAs from genotypically unique Escherichia coli strains isolated from the feces of 138 humans and 376 domesticated animals from Jeonnam Province, South Korea, performed using primers specific for the chuA and yjaA genes and an unknown DNA fragment, TSPE4.C2, indicated that none of the strains belonged to E. coli phylogenetic group B2. In contrast, phylogenetic group B2 strains were detected in about 17% (8 of 48) of isolates from feces of 24 wild geese and in 3% (3 of 96) of isolates obtained from the Yeongsan River in Jeonnam Province, South Korea. The distribution of E. coli strains in phylogenetic groups A, B1, and D varied depending on the host examined, and there was no apparent seasonal variation in the distribution of strains in phylogenetic groups among the Yeongsan River isolates. The distribution of four virulence genes (eaeA, hlyA, stx₁, and stx₂) in isolates was also examined by using multiplex PCR. Virulence genes were detected in about 5% (38 of 707) of the total group of unique strains examined, with 24, 13, 13, and 9 strains containing hlyA, eaeA, stx₂, and stx₁, respectively. The virulence genes were most frequently present in phylogenetic group B1 strains isolated from beef cattle. Taken together, results of these studies indicate that E. coli strains in phylogenetic group B2 were rarely found in humans and domesticated animals in Jeonnam Province, South Korea, and that the majority of strains containing virulence genes belonged to phylogenetic group B1 and were isolated from beef cattle. Results of this study also suggest that the relationship between the presence and types of virulence genes and phylogenetic groupings may differ among geographically distinct E. coli populations.Escherichia coli is a normal inhabitant of the lower intestinal tract of warm-blooded animals and humans. While the majority of E. coli strains are commensals, some are known to be pathogenic, causing intestinal and extraintestinal diseases, such as diarrhea and urinary tract infections (42). Phylogenetic studies done using multilocus enzyme electrophoresis and 72 E. coli strains in the E. coli reference collection showed that E. coli strains can be divided into four phylogenetic groups (A, B1, B2, and D) (20, 41, 48). Recently, a potential fifth group (E) has also been proposed (11). Since multiplex PCR was developed for analysis of phylogenetic groups (6), a number of studies have analyzed a variety of E. coli strains for their phylogenetic group association (10, 12, 17, 18, 23, 54). Duriez et al. (10) reported the possible influence of geographic conditions, dietary factors, use of antibiotics, and/or host genetic factors on the distribution of phylogenetic groups among 168 commensal E. coli strains isolated from human stools from three geographically distinct populations in France, Croatia, and Mali. Random-amplified polymorphic DNA analysis of the intraspecies distribution of E. coli in pregnant women and neonates indicated that there was a correlation between the distribution of phylogenetic groups, random-amplified polymorphic DNA groups, and virulence factors (54). Moreover, based on comparisons of the distribution of E. coli phylogenetic groups among humans of different sexes and ages, it has been suggested that E. coli genotypes are likely influenced by morphological, physiological, and dietary differences (18). In addition, climate has also been proposed to influence the distribution of strains within E. coli phylogenetic groups (12). There are now several reports indicating that there is a potential relationship between E. coli phylogenetic groups, age, and disease. For example, E. coli isolates belonging to phylogenetic group B2 have been shown to predominate in infants with neonatal bacterial meningitis (27) and among urinary tract and rectal isolates (55). Also, Nowrouzian et al. (39) and Moreno et al. (37) reported that strains belonging to phylogenetic group B2 persisted among the intestinal microflora of infants and were more likely to cause clinical symptoms.Boyd and Hartl (2) reported that among the E. coli strains in the E. coli reference and the diarrheagenic E. coli collections, strains in phylogenetic group B2 carry the greatest number of virulence factors, followed by those in group D. Virulence factors carried by group B2 strains are thought to contribute to their strong colonizing capacity; a greater number of virulence genes have been detected in resident strains than in transient ones (38). Moreover, a mouse model of extraintestinal virulence showed that phylogenetic group B2 strains killed mice at greater frequency and possessed more virulence determinants than strains in other phylogenetic groups, suggesting a link between phylogeny and virulence genes in E. coli extraintestinal infection (45). In contrast, Johnson and Kuskowski (25) suggested that a group B2 ancestral strain might have simply acquired virulence genes by chance and that these genes were vertically inherited by group members during clonal expansion. However, numerous studies published to date suggest that there is a relationship between the genomic background of phylogenetic group B2 and its association with virulence factors (12, 28, 35, 39, 45).Both enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC, respectively) strains are among the most important food-borne pathogens worldwide, often causing severe gastrointestinal disease and fatal infections (13). While EPEC strains cause diarrhea and generally do not produce enterotoxin, they possess an adherence factor which is controlled by the chromosomal gene eaeA, encoding intimin (8). Unlike the EPEC strains, however, the EHEC strains typically contain the hlyA, stx₁, and stx₂ virulence genes, encoding hemolysins and Shiga-like type 1 and 2 toxins, respectively, and eaeA. The ability to detect EHEC has been greatly facilitated by the use of multiplex PCR (13, 44, 53). Several studies have shown that strains producing Shiga-like toxin 2 are more frequently found in cases of hemolytic-uremic syndrome than are those containing Shiga-like toxin 1 (30, 43, 46, 49).In the study reported here, we examined the distribution of phylogenetic groups and the prevalence of virulence genes in 659 genotypically unique E. coli strains isolated from humans and domestic animals in South Korea. In addition, we also tested 48 and 96 nonunique E. coli isolates from wild geese and the Yeongsan River, respectively, for phylogenetic distribution and virulence gene profiles. Here, we report that contrary to what has been previously reported in other parts of the world, no E. coli strains belonging to phylogenetic group B2 were found in domesticated animals and in humans from Jeonnam Province, South Korea. We also report that among the strains we examined, virulence genes were mainly found in phylogenetic group B1 strains isolated from beef cattle. Results of these studies may prove to be useful for the development of risk management strategies to maintain public health.     

Prevalence and Persistence of Escherichia coli Strains with Uropathogenic Virulence Characteristics in Sewage Treatment Plants

We investigated the prevalence and persistence of Escherichia coli strains in four sewage treatment plants (STPs) in a subtropical region of Queensland, Australia. In all, 264 E. coli strains were typed using a high-resolution biochemical fingerprinting method and grouped into either a single or a common biochemical phenotype (S-BPT and C-BPT, respectively). These strains were also tested for their phylogenetic groups and 12 virulence genes associated with intestinal and extraintestinal E. coli strains. Comparison of BPTs at various treatment stages indicated that certain BPTs were found in two or all treatment stages. These BPTs constituted the highest proportion of E. coli strains in each STP and belonged mainly to phylogenetic group B2 and, to a lesser extent, group D. No virulence genes associated with intestinal E. coli were found among the strains, but 157 (59.5%) strains belonging to 14 C-BPTs carried one or more virulence genes associated with uropathogenic strains. Of these, 120 (76.4%) strains belonged to seven persistent C-BPTs and were found in all four STPs. Our results indicate that certain clonal groups of E. coli with virulence characteristics of uropathogenic strains can survive the treatment processes of STPs. These strains were common to all STPs and constituted the highest proportion of the strains in different treatment tanks of each STP.Community sewage treatment plants (STPs) receive waste from diverse sources, including residential, industrial, and recreational facilities (31). Waste generated from these facilities contains the liquid and fecal discharges of humans and animals, household wastes, industry-specific materials, and storm water runoff (31). These materials are treated through primary, secondary, and tertiary sedimentation processes (18). Following these processes, effluent is normally clear and thus often recycled for nonpotable use (20), with excess water released into receiving waterways. However, due to possible malfunctions or poor management of wastewater systems (1), effluent containing pathogenic bacteria can be discharged into receiving waterways (11, 34). It has been speculated that waters contaminated with feces are a great risk to human health, as they are likely to contain human-specific enteric pathogens, including Salmonella spp. (30), Shigella spp. (10), enteroviruses (12), hepatitis A virus (13), and pathogenic Escherichia coli (30).E. coli, while widely used as an indicator bacterium (30, 35), can actually be pathogenic and be responsible for both intestinal and extraintestinal diseases (16). Intestinal pathogenic strains of E. coli are rarely encountered in the fecal flora of healthy hosts. Extraintestinal pathogenic E. coli (ExPEC) strains commonly cause infections of any organ or anatomical site (28). The ability of these pathogenic bacteria to cause disease is due to their acquisition of specialized virulence factors, which commensal E. coli strains typically lack. These specialized virulence factors allow them to cause a broad spectrum of diseases (17, 28), such as gastroenteritis (34), diarrhea (16), urinary tract infections and meningitis (29), and soft tissue infections and bacteremia (28). E. coli strains belong to four main phylogenetic groups (A, B1, B2, and D) (2), with pathogenic strains belonging mostly to phylogenetic group B2 and, to a lesser extent, group D. Another phylogenetic group (group E) has also been identified; however, it is uncommon and is not widely used (5).Presently, chlorination is an extremely widespread practice aimed at reducing the pathogen load in the final effluent to levels low enough to ensure that the organisms will not cause disease when the wastewater is discharged (31). Despite this, some pathogenic strains of E. coli may survive to become a significant public health risk (14, 35). The aim of this study was to investigate the presence and survival of these pathogenic E. coli strains during the treatment processes of four community STPs with different capacities in South East Queensland, Australia.     

Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H?/Hnt Isolates from Cattle and Clonal Relationship Analysis

Since enterohemorrhagic Escherichia coli (EHEC) isolates of serogroup O156 have been obtained from human diarrhea patients and asymptomatic carriers, we studied cattle as a potential reservoir for these bacteria. E. coli isolates serotyped by agglutination as O156:H25/H−/Hnt strains (n = 32) were isolated from three cattle farms during a period of 21 months and characterized by rapid microarray-based genotyping. The serotyping by agglutination of the O156 isolates was not confirmed in some cases by the results of DNA-based serotyping as only 25 of the 32 isolates were conclusively identified as O156:H25. In the multilocus sequence typing (MLST) analysis, all EHEC O156:H25 isolates were characterized as sequence type 300 (ST300) and ST688, which differ by a single-nucleotide exchange in the purA gene. Oligonucleotide microarrays allow simultaneous detection of a wider range of EHEC-associated and other E. coli virulence markers than other methods. All O156:H25 isolates showed a wide spectrum of virulence factors typical for EHEC. The stx₁ genes combined with the EHEC hlyA (hlyA_EHEC) gene, the eae gene of the ζ subtype, as well as numerous other virulence markers were present in all EHEC O156:H25 strains. The behavior of eight different cluster groups, including four that were EHEC O156:H25, was monitored in space and time. Variations in the O156 cluster groups were detected. The results of the cluster analysis suggest that some O156:H25 strains had the genetic potential for a long persistence in the host and on the farm, while other strains did not. As judged by their pattern of virulence markers, E. coli O156:H25 isolates of bovine origin may represent a considerable risk for human infection. Our results showed that the miniaturized E. coli oligonucleotide arrays are an excellent tool for the rapid detection of a large number of virulence markers.Shiga toxin-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (45). In humans, infections with some STEC serotypes may result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by the hemolytic uremic syndrome (HUS) (32). These STEC strains are also designated enterohemorrhagic Escherichia coli (EHEC). Consequently, EHEC strains represent a subgroup of STEC with high pathogenic potential for humans. Although E. coli O157:H7 is the most frequent EHEC serotype implicated in HUS, other serotypes can also cause this complication. Non-O157:H7 EHEC strains including serotypes O26:H11/H−, O103:H2/H−, O111:H8/H10/H−, and O145:H28/H25/H− and sorbitol-fermenting E. coli O157:H− isolates are present in about 50% of stool cultures from German HUS patients (10, 42). However, STEC strains that cause human infection belong to a large number of E. coli serotypes, although a small number of STEC isolates of serogroup O156 were associated with human disease (7). Strains of the serotypes O156:H1/H8/H21/H25 were found in human cases of diarrhea or asymptomatic infections (9, 22, 25, 26). The detection of STEC of serogroup O156 from healthy and diseased ruminants such as cattle, sheep, and goats was reported by several authors (1, 11-13, 21, 39, 46, 50, 52). Additional EHEC-associated virulence genes such as stx, eae, hlyA_EHEC, or nlaA were found preferentially in the serotypes O156:H25 and O156:H− (11-13, 21, 22, 50, 52).Numerous methods exist for the detection of pathogenic E. coli, including genotypic and phenotypic marker assays for the detection of virulence genes and their products (19, 47, 55, 57). All of these methods have the common drawback of screening a relatively small number of determinants simultaneously. A diagnostic DNA microarray based on the ArrayTube format of CLONDIAG GmbH was developed as a viable alternative due to its ability to screen multiple virulence markers simultaneously (2). Further microarray layouts working with the same principle but different gene targets were developed for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria (5) and for the rapid DNA-based serotyping of E. coli (4). In addition, a protein microarray for E. coli O serotyping based on the ArrayTube format was described by Anjum et al. (3).The aim of our study was the molecular genotyping of bovine E. coli field isolates of serogroup O156 based on miniaturized E. coli oligonucleotide arrays in the ArrayStrip format and to combine the screening of E. coli virulence markers, antimicrobial resistance genes, and DNA serotyping targets, some of which were partially described previously for separate arrays (2, 4, 5). The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O156 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.     

ICEEc2, a New Integrative and Conjugative Element Belonging to the pKLC102/PAGI-2 Family,Identified in Escherichia coli Strain BEN374

The diversity of the Escherichia coli species is in part due to the large number of mobile genetic elements that are exchanged between strains. We report here the identification of a new integrative and conjugative element (ICE) of the pKLC102/PAGI-2 family located downstream of the tRNA gene pheU in the E. coli strain BEN374. Indeed, this new region, which we called ICEEc2, can be transferred by conjugation from strain BEN374 to the E. coli strain C600. We were also able to transfer this region into a Salmonella enterica serovar Typhimurium strain and into a Yersinia pseudotuberculosis strain. This transfer was then followed by the integration of ICEEc2 into the host chromosome downstream of a phe tRNA gene. Our data indicated that this transfer involved a set of three genes encoding DNA mobility enzymes and a type IV pilus encoded by genes present on ICEEc2. Given the wide distribution of members of this family, these mobile genetic elements are likely to play an important role in the diversification of bacteria.The fantastic diversity of the Escherichia coli species has been known for a long time. With modern sequencing strategies, the molecular bases of this diversity are now being unraveled (49). Analyzing the genome of 20 E. coli strains, Touchon et al. recently showed that only a minority of genes, approximately 1,900 genes, were shared by all E. coli strains and constituted the core genome of the E. coli species (50). Additionally, the total number of genes found in all E. coli strains, the pan-genome, is an order of magnitude larger than this core genome (50). The non-core genome of a strain, also called flexible gene pool, is therefore made of a wide diversity of genes. This genetic diversity of the E. coli species translates into a diversity of phenotypic properties. While most E. coli strains are commensal of the gastrointestinal tract of humans and warm-blooded animals, a significant number are responsible for different diseases in humans and animals (22), including extraintestinal infections in chickens; strains isolated from such cases are designated by the term APEC for avian pathogenic E. coli (10).This diversity arises from frequent horizontal gene transfers of mobile genetic elements such as transposons, plasmids, phages, genomic islands, or integrative and conjugative elements (ICEs) (11, 21, 34). Among these mobile genetic elements, ICEs have a particular place as they share properties with both plasmids, genomic islands, and transposons; they can be defined as elements that encode all the necessary machineries that allow their excision from the chromosome, their transfer to a recipient strain, and their integration into the recipient strain''s genome (5, 6, 46, 54). Well-known representatives of this class of genetic elements include Tn916 discovered in Enterococcus faecalis, the conjugative transposon CTnDOT in Bacteroides thetaiotaomicron, ICEKp1 in Klebsiella pneumoniae, SXT/R391-related elements, PFGI-1 in Pseudomonas fluorescens, and the clc element in Pseudomonas sp. strain B13 as well as ICEBs1 in Bacillus subtilis and ICEEc1 in the E. coli strain ECOR31 (1, 39, 44, 46, 54). Typically, ICEs contain at least three modules that are required for key steps in the ICE''s life cycle: an excision/integration module, a transfer module, and a regulation module (54). Besides these, ICEs often contain cargo regions that confer on their host a diverse array of properties, such as virulence properties (ICEEc1), antibiotic resistance (SXT), or degradation of chemical compounds (clc). Because of their self-transfer abilities and their diverse accessory gene repertoires, ICEs are very likely to play a major role in bacteria evolution (46).A new family of ICEs has recently gained interest and was named the pKLC102/PAGI-2 family. The first element of this family, the clc element, was discovered in Pseudomonas sp. strain B13 and confers on the bacteria the possibility to degrade aromatic compounds (42). The transfer of this element was discovered long before its complete sequence was characterized (16). Other members of this family include several elements present in Pseudomonas strains such as PAGI-1 and PAGI-2 as well as the pKLC102 element first considered to be a plasmid but later on shown to be an ICE because of its ability to integrate into the chromosome of its host (23, 52). pKLC102/PAGI-2 elements share a set of core genes (33) and, like most ICEs and genomic islands, are all integrated downstream of tRNA genes (26, 52). The transfer between strains has been demonstrated, albeit with different frequencies, for only a few members, such as the clc element, Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1), and ICEHin1056 from Haemophilus influenzae (20, 37, 41); this transfer involves the type IV pilus (20), the integrase (40), and in some cases the formation of a circular intermediate of the excised ICE (24).In order to identify new accessory genes of APEC strains, we previously described tRNA loci in the E. coli genome that could represent potential insertion sites for new genomic islands (18). We had already used this strategy to characterize the AGI-3 region that is involved in the virulence of an avian pathogenic E. coli strain and that confers the ability to grow on fructooligosaccharides (7, 43). During this tRNA screening, we showed that genomic islands might potentially be present downstream of the tRNA genes argW, leuX, pheU, pheV, selC, serU, and thrW in several APEC strains.In this report, we describe the identification of a new genomic island located downstream of pheU in the APEC strain BEN374. This region, which we named ICEEc2, was fully sequenced, and its properties were analyzed in detail; ICEEc2 is a new ICE found in E. coli and belongs to the pKLC102/PAGI-2 family described above.     

Genetic Diversity and Multihost Pathogenicity of Clinical and Environmental Strains of Burkholderia cenocepacia

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.The betaproteobacterium Burkholderia cenocepacia, 1 of now 17 classified species belonging to the Burkholderia cepacia complex (BCC), is ubiquitous and extremely versatile in its metabolic capabilities and interactions with other organisms (38, 40, 57, 58). Strains of B. cenocepacia are pathogens of onion and banana plants, opportunistic pathogens of humans, symbionts of numerous plant rhizospheres, contaminants of pharmaceutical and industrial products, and inhabitants of soil and surface waters (14, 29, 33, 34, 37, 45). Originally described as a pathogen of onions (8), organisms of the BCC emerged in the past 3 decades as serious human pathogens, capable of causing devastating chronic lung infections in persons with cystic fibrosis (CF) or chronic granulomatous disease (21, 24, 28). Infections due to BCC are a serious concern to CF patients due to their inherent antibiotic resistance and high potential for patient-to-patient transmission (23). Although 16 of the BCC species have been recovered from respiratory secretions of CF patients in many countries (46, 58), B. cenocepacia has been the most common species isolated in North America, detected in 50% of 606, 83% of 447, and 45.6% of 1,218 patients in recent studies (35, 46, 52).The epidemiology of infectious disease caused by B. cenocepacia appears to involve patient-to-patient spread of genetically distinct lineages. B. cenocepacia lineages, such as ET12, Midwest, and PHDC, have been identified from large numbers of individuals in disease outbreaks in North America and Europe (11, 32, 54). A recently developed multilocus sequence typing (MLST) scheme has been shown to be a reliable epidemiologic tool for differentiating between the five subgroups (IIIA to IIIE) of B. cenocepacia, and strains representing three of these subgroups (IIIA, IIIB, and IIID) have been recovered from CF patients (2). Outside of the patient-to-patient transmission of clonal lineages, the mode of acquisition of strains causing sporadic cases of B. cenocepacia in CF patients remains unclear, although environmental sources are a logical reservoir for infection. Previously, an isolate of B. cenocepacia indistinguishable from the PHDC epidemic clonal lineage by using standard typing methods (e.g., repetitive-sequence-based PCR, randomly amplified polymorphic DNA, pulsed-field gel electrophoresis) was detected in an agricultural soil sample (34). Similarly, three distinct MLST sequence types containing both clinical and environmental (plant and soil) B. cenocepacia isolates were identified (1). These findings suggest that natural populations of B. cenocepacia in soil or associated with plants are a potential reservoir for the emergence of new human pathogenic lineages.Experimental models for the study of virulence potential and traits of B. cenocepacia include mouse and rat models with genetic defects allowing chronic lung infections to be established (e.g., see reference 48). Nematode (Caenorhabditis elegans), alfalfa (Medicago sativa), and onion (Allium cepa) models have also been routinely utilized for the identification of virulence factors (5, 29, 31). C. elegans has been extensively used to study the pathogenesis and virulence factors of a wide variety of bacterial and fungal pathogens (9, 15, 42, 51, 56). In several pathogens, including Pseudomonas (56) and Burkholderia (20), putative virulence factors important for the pathogenesis in mammalian systems (15, 51) have been identified using the C. elegans model. The C. elegans model might be limited in the detection of host-specific virulence factors; however, several attributes, such as small size and rapid development, make it an excellent whole animal model for pathogenesis research (16, 51).The evidence that individual strains of B. cenocepacia can be pathogenic to both plants and humans and are prevalent in various environmental niches has provoked particular interest in elucidating the clinical pathogenic potential of environmental isolates. The basis of this study was to examine whether genetically related B. cenocepacia strains exhibit shared characteristics that contribute to their pathogenicity in multiple hosts and to examine the potential for circulating environmental isolates to emerge as new clinical pathogens. Here, we tested the degree of virulence in animal (nematode) and plant (onion) infection models, the production of antifungal activity, and the genetic relatedness of clinical and environmental B. cenocepacia subgroup IIIB strains predominantly isolated from Michigan.     

Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from Klebsiella pneumoniae and Escherichia coli

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.The most common type of adhesive organelle in the Enterobacteriaceae is the type 1 fimbria, which has been most extensively studied in Escherichia coli. The corresponding structures of Klebsiella pneumoniae are similar to those of E. coli with regard to genetic composition and regulation (15). Type 1 fimbriae are composed primarily of the structural subunit FimA, with minor amounts of three ancillary subunits, FimF, FimG, and the mannose-specific adhesin FimH. The FimH adhesin is an allosteric protein that mediates the catch bond mechanism of adhesion where the binding is increased under increased shear stress (48).It has been demonstrated in E. coli that FimH has two domains, the mannose-binding lectin domain (from amino acid [aa] 1 through 156) and the fimbria-incorporating pilin domain (from aa 160 through 279), connected via a 3-aa-long linker chain (6). A mannose-binding site is located at the top of the lectin domain, at the opposite end from the interdomain linker (17).Several studies have demonstrated that type 1 fimbriae play an important role in E. coli urinary tract infection (UTI) (7, 21, 23, 35). In addition, in urinary E. coli isolates, the FimH adhesin accumulates amino acid replacements which increase tropism for the uroepithelium and various components of basement membranes (21, 30, 35, 37, 49). Most of the replacements increase the monomannose binding capability of FimH under low shear, by altering allosteric catch bond properties of the protein (48). The mutated FimH variants were shown to provide an advantage in colonization of the urinary tract in the mouse model (35) and correlate with the overall extraintestinal virulence of E. coli (16). Thus, FimH mutations are pathoadaptive in nature.Klebsiella pneumoniae is recognized as an important opportunistic pathogen frequently causing UTIs, septicemia, or pneumonia in immunocompromised individuals (29). It is responsible for up to 10% of all nosocomial bacterial infections (18, 41). K. pneumoniae is ubiquitous in nature, and it has been shown that environmental isolates are phenotypically indistinguishable from clinical isolates (22, 26, 27, 29, 33). Furthermore, it has been demonstrated that environmental isolates of K. pneumoniae are as virulent as clinical isolates (28, 45).K. pneumoniae possesses a number of known virulence factors, including a pronounced capsule, type 3 fimbriae, and type 1 fimbriae (29, 44). Type 1 fimbriae produced by K. pneumoniae are described as functionally and structurally similar to type 1 fimbriae from E. coli (25) and have been shown to play a significant role in K. pneumoniae UTI (32, 43).We have previously shown that mature FimH from 54 isolates of K. pneumoniae (isolated from urine, blood, liver, and the environment) is represented by seven protein variants due to point amino acid replacements. (42) When K. pneumoniae FimH was aligned with the FimH of E. coli, they showed ∼85% similarity at the amino acid level. Furthermore, a majority (14 out of 21 isolates) of the K. pneumoniae strains isolated from patients with UTI grouped into a single clonal group based on multilocus sequence typing, but fimH in one isolate in the group differed from the others by a single nucleotide mutation resulting in an amino acid change, serine to alanine, in position 62 (42). The same mutation has been found in FimH of a highly uropathogenic clone of E. coli and significantly increases the adhesin''s ability to adhere to monomannose under low or no shear (19, 39, 50).In this study, we describe the extent and pattern of structural variability of the FimH protein from K. pneumoniae and perform comparative analyses of the functional properties of FimH from both K. pneumonae and E. coli.     

Phylogenetic Groups and Pathogenicity Island Markers in Fecal Escherichia coli Isolates from Asymptomatic Humans in China

The study of phylogenetic groups and pathogenicity island (PAI) markers in commensal Escherichia coli strains from asymptomatic Chinese people showed that group A strains are the most common and that nearly half of all fecal strains which were randomly selected harbor PAIs.Escherichia coli is a well-diversified commensal species in the intestine of healthy humans but also includes intestinal or extraintestinal pathogens. It has been reported that pathogenic E. coli may be derived from fecal strains by acquisition of virulence determinants (11). The relationship between the E. coli genetic background and the acquisition of virulence factors is now better understood (1, 5). Extraintestinal E. coli strains may harbor several virulence factors, such as adhesins, fimbriae, and hemolysin, which can contribute to bacterial pathogenesis. These traits are usually encoded on pathogenicity islands (PAIs), which have been studied in pathogenic E. coli previously (15). The E. coli population includes 4 major phylogroups (A, B₁, B₂, and D) (2). Pathogenic strains belong mainly to groups B₂ and D, while most fecal isolates belong to groups A and B₁. Strains of groups B₂ and D often carry virulence factors that are lacking in group A and B₁ strains (3, 9, 13).In this study, we examined the distribution of phylogroups and the prevalence of PAIs in commensal E. coli strains isolated from asymptomatic persons in one region of China.     

Characterization of Environmentally Persistent Escherichia coli Isolates Leached from an Irish Soil

Soils are typically considered to be suboptimal environments for enteric organisms, but there is increasing evidence that Escherichia coli populations can become resident in soil under favorable conditions. Previous work reported the growth of autochthonous E. coli in a maritime temperate Luvic Stagnosol soil, and this study aimed to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached from the soil. Molecular analysis (16S rRNA sequencing, enterobacterial repetitive intergenic consensus PCR, pulsed-field gel electrophoresis, and a multiplex PCR method) established the genetic diversity of the isolates (n = 7), while physiological methods determined the metabolic capability and environmental fitness of the isolates, relative to those of laboratory strains, under the conditions tested. Genotypic analysis indicated that the leached isolates do not form a single genetic grouping but that multiple genotypic groups are capable of surviving and proliferating in this environment. In physiological studies, environmental isolates grew well across a broad range of temperatures and media, in comparison with the growth of laboratory strains. These findings suggest that certain E. coli strains may have the ability to colonize and adapt to soil conditions. The resulting lack of fecal specificity has implications for the use of E. coli as an indicator of fecal pollution in the environment.Escherichia coli is a well-established indicator of fecal contamination in the environment. The organism''s validity as an indicator of water pollution is dependent, among other factors, on its fecal specificity and its inability to multiply outside the primary host, the gastrointestinal tracts of humans and warm-blooded animals (9). While many pathogens and indicator organisms are considered to be poorly adapted for long-term survival, or proliferation, outside their primary hosts (24), there is increasing evidence that this view needs to be reconsidered with respect to E. coli (17, 38). In particular, questions remain about its fate and survival capacity in environmental matrices, such as soil. While the habitat within the primary host is characterized by constant warm temperature conditions and a ready availability of nutrients and carbon, that of soil is often characterized by oligotrophic and highly dynamic conditions, temperature and pH variation, predatory populations, and competition with environmentally adapted indigenous microflora (39). Soils are thus typically considered to be suboptimal environments for enteric organisms, and growth is thought to be negligible, with die-off of organisms at rates reported to be a function of the interaction of numerous factors, including the type and physiological state of the microorganism, the physical, chemical, and biological properties of the soil, atmospheric conditions (including sunlight, moisture, and temperature), and organism application method (10).In recent years, the growth of E. coli in soils, sediments, and water in tropical and subtropical regions has been widely documented, and the organism is considered to be an established part of the soil biota within these regions (4, 5, 7, 12, 14, 19, 25, 32). The integration of E. coli as a component of the indigenous microflora in soils of tropical and subtropical regions may be attributable to the nutrient-rich nature and warm temperatures of these habitats (21, 39), combined with the metabolic versatility of the organism and its simple nutritional requirements (21). In addition to tropical and subtropical regions, the presence of autochthonous E. coli populations in the cooler soils of temperate and northern temperate regions has also been reported (6, 20, 22, 37), with one report on an alpine soil (34) and, most recently, a report on a maritime temperate grassland soil (3). The growth of E. coli within soils can act as a reservoir for the further contamination of bodies of water (20, 31, 32), compromising the indicator status of E. coli within these regions. As such, an understanding of the ecological characteristics of E. coli in soil is critical to its validation as an indicator organism. With respect to the input of pathogenic E. coli into the environment, this knowledge becomes essential for assessing the potential health risk to human and animal hosts from agricultural activities such as landspreading of manures and slurries (24).It has been suggested that E. coli can sustain autochthonous populations within soils in temperate regions, wherever favorable conditions exist (21). The phenotypic traits of the organism (including its metabolic diversity and its ability to grow both aerobically and anaerobically in a broad temperature range) may assist the persistence, colonization, and growth of E. coli when conditions permit. The challenging nature of the soil environment and the disparity of conditions between the primary host and the secondary habitat raises the question of how these E. coli populations survive and compete for niche space among the highly competitive and diverse coexisting populations of the indigenous microflora (15, 21). There is some evidence that naturalized E. coli may form genetically distinct populations in the environment (17, 20, 34, 36). This suggests that autochthonous E. coli populations in soil may have increased environmental fitness, facilitating their residence in soil (20, 34, 38). Little is known, however, of the physiology of these organisms, and their capacity for survival in soil remains poorly understood (21).Previous work (3) recorded continuous low-level leaching of viable E. coli from lysimeters of a poorly drained Luvic Stagnosol soil type, more than 9 years after the last application of fecal material. This finding was indicative of the growth of E. coli within the soil and suggested the presence of autochthonous E. coli populations within the soil that could be leached subsequently. To our knowledge, prior to this report, naturalized autochthonous E. coli populations persisting under the relatively oligotrophic, low-temperature conditions of maritime temperate soil environments had not been described previously. Growth within this soil was attributed chiefly to favorable characteristics of the soil, which include high clay and moisture contents, nutrient retention, and the presence of anaerobic zones. The objective of this work was to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached. In particular, we were interested in determining if the isolates possessed phenotypic characteristics that may enhance their capacity to survive and occupy niche space within the soil. This study tested the hypothesis that E. coli clones persisting in lysimeters of this soil form a genetically distinct grouping and possess a physiology tailored to the soil environment.     

Contribution of Lipoproteins and Lipoprotein Processing to Endocarditis Virulence in Streptococcus sanguinis

Streptococcus sanguinis is an important cause of infective endocarditis. Previous studies have identified lipoproteins as virulence determinants in other streptococcal species. Using a bioinformatic approach, we identified 52 putative lipoprotein genes in S. sanguinis strain SK36 as well as genes encoding the lipoprotein-processing enzymes prolipoprotein diacylglyceryl transferase (lgt) and signal peptidase II (lspA). We employed a directed signature-tagged mutagenesis approach to systematically disrupt these genes and screen each mutant for the loss of virulence in an animal model of endocarditis. All mutants were viable. In competitive index assays, mutation of a putative phosphate transporter reduced in vivo competitiveness by 14-fold but also reduced in vitro viability by more than 20-fold. Mutations in lgt, lspA, or an uncharacterized lipoprotein gene reduced competitiveness by two- to threefold in the animal model and in broth culture. Mutation of ssaB, encoding a putative metal transporter, produced a similar effect in culture but reduced in vivo competiveness by >1,000-fold. [³H]palmitate labeling and Western blot analysis confirmed that the lgt mutant failed to acylate lipoproteins, that the lspA mutant had a general defect in lipoprotein cleavage, and that SsaB was processed differently in both mutants. These results indicate that the loss of a single lipoprotein, SsaB, dramatically reduces endocarditis virulence, whereas the loss of most other lipoproteins or of normal lipoprotein processing has no more than a minor effect on virulence.Streptococcus sanguinis is a member of the viridans group of streptococci and is a primary colonizer of teeth (8). The viridans species and, in particular, S. sanguinis (15, 18) are a leading cause of infective endocarditis, a serious infection of the valves or lining of the heart (48). Damage to the heart resulting from rheumatic fever or certain congenital heart defects dramatically increases the risk of developing endocarditis (48, 71). The damage is thought to result in the formation of sterile cardiac “vegetations” composed of platelets and fibrin (48) that can be colonized by certain bacteria during periods of bacteremia. This view is supported by animal studies in which formation of sterile vegetation by cardiac catheterization is required for the efficient establishment of streptococcal endocarditis (17). Prevention of infective endocarditis currently relies upon prophylactic administration of antibiotics prior to dental or other surgical procedures that are likely to produce bacteremia. The growing realization that oral bacteria such as S. sanguinis can enter the bloodstream through routine daily activities such as eating has led the American Heart Association (71) and others (57) to question the value of using antibiotic prophylaxis for dental procedures. Clearly, a better understanding of the bacterial virulence factors that contribute to endocarditis could lead to better preventive measures, such as a vaccine that could potentially afford continuous protection to high-risk patients (71).In a previous study, we used the signature-tagged mutagenesis (STM) technique to search for endocarditis virulence factors of S. sanguinis in a rabbit model (53). This study identified a number of housekeeping enzymes that contribute to endocarditis. Because these proteins are not likely to be surface localized, they hold little promise as vaccine candidates. One class of streptococcal surface proteins that is rich in both virulence factors (4, 7, 25, 33, 38, 60) and promising vaccine candidates (6, 39, 42, 51, 70) is the lipoproteins. Lipoprotein activities that have been suggested to contribute to streptococcal virulence include adhesion (4, 7, 63), posttranslational modification (25, 29, 51), and ATP-binding cassette (ABC)-mediated transport (33, 52, 60). In the last instance, lipoproteins anchored to the cell membrane by their lipid tails appear to serve the same transport function as the periplasmic substrate-binding proteins of gram-negative bacteria (66). STM studies performed with Streptococcus pneumoniae (26, 41, 55) and Streptococcus agalactiae (34) have identified multiple lipoprotein mutants among collections of reduced virulence mutants. In an attempt to determine the cumulative contribution of streptococcal lipoproteins to virulence, some investigators have created mutations in the lgt or lspA genes, encoding lipoprotein-processing enzymes (12, 25, 27, 36). The lgt gene encodes prolipoprotein diacylglyceryl transferase, which catalyzes the transfer of a diacylglycerol lipid unit to a cysteine in the conserved N-terminal “lipobox” of lipoproteins, while lspA encodes the signal peptidase II enzyme that cleaves the signal peptide of the prolipoprotein just prior to the conserved cysteine (59, 65). While mutation of these genes has been shown to be lethal in gram-negative bacteria (21, 73), many gram-positive bacterial species have been shown to tolerate such mutations, often with only minor effects on growth (3, 12, 13, 25, 27, 36, 54). Some of these studies indicated a deleterious effect on the virulence of the lgt (25, 54) or lspA (36) mutation, but others found no effect (12) or an enhancement of virulence (27). It is clear from these and other studies (3, 13) that neither the loss of acylation due to lgt inactivation nor the loss of signal peptidase II-mediated cleavage completely eliminates lipoprotein function, necessitating alternative approaches for assessing the global contribution of lipoproteins to virulence.We have used bioinformatic approaches to identify every putative lipoprotein encoded by S. sanguinis strain SK36. To determine the contribution of these lipoproteins to the endocarditis virulence of S. sanguinis, we have systematically mutagenized each of these genes, as well as the lgt and lspA genes, and evaluated these mutants for virulence by using STM in an animal model. Selected mutants were further examined for virulence in competitive index (CI) assays. A strain with a disrupted ssaB gene, which encodes a putative metal transport protein, was found to exhibit a profound defect in virulence that was far greater than that of any other strain tested, including the lgt or lspA mutant.     

Expanded Multilocus Sequence Typing and Comparative Genomic Hybridization of Campylobacter coli Isolates from Multiple Hosts

The purpose of this work was to evaluate the evolutionary history of Campylobacter coli isolates derived from multiple host sources and to use microarray comparative genomic hybridization to assess whether there are particular genes comprising the dispensable portion of the genome that are more commonly associated with certain host species. Genotyping and ClonalFrame analyses of an expanded 16-gene multilocus sequence typing (MLST) data set involving 85 isolates from 4 different hosts species tentatively supported the development of C. coli host-preferred groups and suggested that recombination has played various roles in their diversification; however, geography could not be excluded as a contributing factor underlying the history of some of the groups. Population genetic analyses of the C. coli pubMLST database by use of STRUCTURE suggested that isolates from swine form a relatively homogeneous genetic group, that chicken and human isolates show considerable genetic overlap, that isolates from ducks and wild birds have similarity with environmental water samples and that turkey isolates have a connection with human infection similar to that observed for chickens. Analysis of molecular variance (AMOVA) was performed on these same data and suggested that host species was a significant factor in explaining genetic variation and that macrogeography (North America, Europe, and the United Kingdom) was not. The microarray comparative genomic hybridization data suggested that there were combinations of genes more commonly associated with isolates derived from particular hosts and, combined with the results on evolutionary history, suggest that this is due to a combination of common ancestry in some cases and lateral gene transfer in others.Campylobacter species are a leading bacterial cause of gastroenteritis within the United States and throughout much of the rest of the developed world. According to the CDC, there are an estimated 2 million to 4 million cases of Campylobacter illness each year in the United States (37). Campylobacter jejuni is generally recognized as the predominant cause of campylobacteriosis, responsible for approximately 90% of reported cases, while the majority of the remainder are caused by the closely related sister species Campylobacter coli (27). Not surprisingly, therefore, the majority of research on Campylobacter has centered on C. jejuni, and C. coli is a less studied organism.A multilocus sequence typing (MLST) scheme of C. jejuni was first developed by Dingle et al. (13) on the basis of the genome sequence of C. jejuni NCTC 11168. There have also been a number of studies using the genome sequence data to develop microarrays for gene presence/absence determination across strains of C. jejuni and to identify the core genome components for the species (6, 15, 32, 33, 42, 43, 53, 57). Although C. coli is responsible for fewer food-borne illnesses than C. jejuni, the impact of C. coli is still substantial, and there is also evidence that C. coli may carry higher levels of resistance to some antibiotics (1). C. coli and C. jejuni also tend to differ in their relative prevalences in animal host species and various environmental sources (4, 48, 58), and there is some evidence that both taxa may include groups of host-specific putative ecotype strains (7, 36, 38, 39, 52, 56). At present, there is only a single draft genome sequence available for C. coli, and there are no microarray comparative genomic hybridization data for C. coli strains. Thus, there is no information on intraspecies variability in gene presence/absence in C. coli and how such variability might correlate with host species.The purpose of this work was to develop and apply an expanded 16-locus MLST genotyping scheme to evaluate the evolutionary history of Campylobacter coli isolates derived from multiple host sources and to use microarray comparative genomic hybridization to assess whether there are particular genes comprising the dispensable portion of the genome that are more commonly associated with isolates derived from different host species.     

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.     

Genomic Regions Conserved in Lineage II Escherichia coli O157:H7 Strains

Populations of the food- and waterborne pathogen Escherichia coli O157:H7 are comprised of two major lineages. Recent studies have shown that specific genotypes within these lineages differ substantially in the frequencies with which they are associated with human clinical disease. While the nucleotide sequences of the genomes of lineage I strains E. coli O157 Sakai and EDL9333 have been determined, much less is known about the genomes of lineage II strains. In this study, suppression subtractive hybridization (SSH) was used to identify genomic features that define lineage II populations. Three SSH experiments were performed, yielding 1,085 genomic fragments consisting of 811 contigs. Bacteriophage sequences were identified in 11.3% of the contigs, 9% showed insertions and 2.3% deletions with respect to E. coli O157:H7 Sakai, and 23.2% did not have significant identity to annotated sequences in GenBank. In order to test for the presence of these novel loci in lineage I and II strains, 27 PCR primer sets were designed based on sequences from these contigs. All but two of these PCR targets were found in the majority (51.9% to 100%) of 27 lineage II strains but in no more than one (<6%) of the 17 lineage I strains. Several of these linage II-related fragments contain insertions/deletions that may play an important role in virulence. These lineage II-related loci were also shown to be useful markers for genotyping of E. coli O157:H7 strains isolated from human and animal sources.Enterohemorrhagic Escherichia coli is associated with diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans (31). E. coli serotype O157:H7 predominates in epidemics and sporadic cases of enterohemorrhagic E. coli-related infections in the United States, Canada, Japan, and the United Kingdom (12). Cattle are considered the most important reservoir of E. coli O157:H7 (10, 24, 37, 41), and foods contaminated with bovine feces are thought to be the most common source of human infection with this pathogen (27, 33). The two most important virulence factors of the organism are the production of one or more Shiga toxins (Stx) (6, 20, 32) and the ability to attach to and efface microvilli of host intestinal cells (AE). Stx genes are encoded by temperate bacteriophage inserted in the bacterial chromosome, and genes responsible for the AE phenotype are located on the locus of enterocyte effacement (LEE) as well as other pathogenicity islands (4, 17). All E. coli O157:H7 strains also possess a large plasmid which is thought to play a role in virulence (10, 40, 42).Octamer-based genome scanning (OBGS) was first used to show that E. coli O157 strains from the United States and Australia could be subdivided into two genetically distinct lineages (21, 22, 46). While both E. coli O157:H7 lineages are associated with human disease and are isolated from cattle, there is a bias in the host distribution between the two lineages, with a significantly higher proportion of lineage I strains isolated from humans than lineage II strains. Several recent studies have shown that there are inherent differences in gene content and expression between populations of lineage I and lineage II E. coli O157:H7 strains. Lejeune et al. (26) reported that the antiterminator Q gene of the stx₂-converting bacteriophage 933W was found in all nine OBGS lineage I strains examined but in only two of seven lineage II strains, suggesting that there may be lineage-specific differences in toxin production. Dowd and Ishizaki (9) used DNA microarray analysis to examine expression of 610 E. coli O157:H7 genes and showed that lineage I and lineage II E. coli O157:H7 strains have evolved distinct patterns of gene expression which may alter their virulence and their ability to survive in different microenvironments and colonize the intestines of different hosts (9, 28, 38).The observations of lineage host bias have been supported and extended by studies using a six-locus-based multiplex PCR termed the lineage-specific polymorphism assay (LSPA-6) (46). However, Ziebell et al. (48) have recently shown that not all LSPA-6 types within lineage II are host biased; e.g., LSPA-6 type 211111 isolation rates from humans and cattle were significantly different from those of other lineage II LSPA-6 types. Therefore, a clearer definition is required of not only the differences between lineages but also the differences among clonal groups within lineages.The genome sequences of two E. coli O157:H7 strains, Sakai and EDL933 (14, 36), have been determined; however, both of these strains are of lineage I, and there are presently no completed and fully annotated genome sequences available for lineage II strains. In our laboratory, comparative studies utilizing suppression subtractive hybridization (SSH) and comparative genomic hybridization revealed numerous potential virulence factors that are conserved in lineage I strains and that are rare or absent in lineage II strains (42, 47). In this study, we have used SSH to identify genomic regions present in E. coli O157:H7 lineage II strains that are absent from lineage I strains. We wished to examine the distribution of these novel gene segments in E. coli O157:H7 strains and gain insight into their origins and functions. We also attempted to identify molecular markers specific to lineage II strains as well as other markers that would be useful in the genetic subtyping or molecular fingerprinting of E. coli O157:H7 strains in population and epidemiological studies (25). This information may be helpful in the identification of genotypes of the organism associated with specific phenotypes of both lesser and greater virulence (29).     

Population Variability of the FimH Type 1 Fimbrial Adhesin in Klebsiella pneumoniae

FimH is an adhesive subunit of type 1 fimbriae expressed by different enterobacterial species. The enteric bacterium Klebsiella pneumoniae is an environmental organism that is also a frequent cause of sepsis, urinary tract infection (UTI), and liver abscess. Type 1 fimbriae have been shown to be critical for the ability of K. pneumoniae to cause UTI in a murine model. We show here that the K. pneumoniae fimH gene is found in 90% of strains from various environmental and clinical sources. The fimH alleles exhibit relatively low nucleotide and structural diversity but are prone to frequent horizontal-transfer events between different bacterial clones. Addition of the fimH locus to multiple-locus sequence typing significantly improved the resolution of the clonal structure of pathogenic strains, including the K1 encapsulated liver isolates. In addition, the K. pneumoniae FimH protein is targeted by adaptive point mutations, though not to the same extent as FimH from uropathogenic Escherichia coli or TonB from the same K. pneumoniae strains. Such adaptive mutations include a single amino acid deletion from the signal peptide that might affect the length of the fimbrial rod by affecting FimH translocation into the periplasm. Another FimH mutation (S62A) occurred in the course of endemic circulation of a nosocomial uropathogenic clone of K. pneumoniae. This mutation is identical to one found in a highly virulent uropathogenic strain of E. coli, suggesting that the FimH mutations are pathoadaptive in nature. Considering the abundance of type 1 fimbriae in Enterobacteriaceae, our present finding that fimH genes are subject to adaptive microevolution substantiates the importance of type 1 fimbria-mediated adhesion in K. pneumoniae.Klebsiella pneumoniae is recognized as an important opportunistic pathogen that frequently causes urinary tract infections (UTI), septicemia, or pneumonia, particularly in immunocompromised individuals (25). K. pneumoniae is responsible for up to 10% of all nosocomial bacterial infections (12, 35). In recent years, a high incidence of community-acquired K. pneumoniae pyogenic liver abscess with a high mortality rate has been reported, especially from Taiwan, but also from other Asian countries, Europe, and North America (6, 8, 19, 27, 44). Furthermore, 15% to 30% of K. pneumoniae isolates are resistant to broad-spectrum cephalosporins via plasmid-encoded extended-spectrum β-lactamases (5).In contrast to many other bacterial pathogens, K. pneumoniae is ubiquitous in nature. Its nonclinical habitats include environmental locations, such as vegetation, soil, and surface waters, as well as transient commensal colonization of mucosal surfaces in humans and other animals (1). Several studies have reported K. pneumoniae isolates of environmental origin to be nearly identical to clinical isolates with respect to several phenotypic properties (16, 22, 23, 25, 30). It has been suggested that environmental isolates of K. pneumoniae may be as virulent as clinical isolates (24, 39).Several virulence factors have been identified in K. pneumoniae (25, 38). The prominent polysaccharide capsule expressed by most isolates, together with the lipopolysaccharide layer, protects the bacteria against phagocytosis and the bactericidal activity of serum. Fimbrial adhesins expressed by the bacteria are protein structures able to recognize molecular receptors and to facilitate adherence to specific tissue surfaces in the host. K. pneumoniae produces two major fimbrial adhesion organelles, type 1 and type 3 fimbriae (9). Type 1 fimbriae have mannose-sensitive hemagglutinins, while type 3 fimbriae have mannose-resistant hemagglutinins (21).Type 1 fimbriae are the most common adhesive organelle in Enterobacteriaceae and have been most extensively studied in Escherichia coli. The type 1 fimbrial structures of K. pneumoniae are homologous to those of E. coli with regard to genetic composition and regulation (37). Type 1 fimbriae and the adhesive subunit FimH, in particular, play an important role in UTI caused by both K. pneumoniae and E. coli (3, 15, 17, 30, 37). Analysis of E. coli fimH variation at the population level has revealed that the FimH adhesin in urinary E. coli isolates accumulates amino acid replacements that increase its tropism toward the uroepithelium and various components of basement membranes (14, 26, 31, 33, 46). Most of the replacements increase the monomannose binding capability of FimH under low shear by altering allosteric catch bond properties of the protein (40). The natural FimH mutants were shown to provide an advantage in colonization of the urinary tract in a mouse model (32) and correlate with the overall extraintestinal virulence of E. coli (11). Thus, FimH mutations are pathoadaptive in nature. No such population-wide analysis has been performed for K. pneumoniae fimH.Population genetic analysis involves comparison of the nucleotide and structural variability of the locus of interest across multiple bacterial strains of different clonalities and geographic origins. The clonal structure of the strains can be determined by multiple-locus sequence typing (MLST), in which 400- to 500-bp sequences of multiple genetically unlinked loci are determined in order to define the phylogenetic relationship of the strains and the extent of interclonal gene recombination (horizontal gene transfer). MLST has been used to reveal the epidemiological relationship of ceftazidime- and ciprofloxacin-resistant K. pneumoniae isolates of nosocomial origin (4). In addition, the analysis of gene variability enables the determination of the type of selection processes acting on loci of interest, with possible identification of mutational changes of functional significance that could enhance the organism''s ability to cause disease, i.e., that could be of a pathoadaptive nature.In this study, the population dynamics of the K. pneumoniae FimH adhesin were determined by analysis of fimH allelic diversity in strains of environmental and various clinical origins in the context of K. pneumoniae clonal structure based on the allelic diversity of three loci—tonB, mdh and fumC—commonly used for MLST.