The Prevalence of Multidrug Resistance Is Higher among Bovine than Human Salmonella enterica Serotype Newport,Typhimurium, and 4,5,12:i:? Isolates in the United States but Differs by Serotype and Geographic Region

Salmonella represents an important zoonotic pathogen worldwide, but the transmission dynamics between humans and animals as well as within animal populations are incompletely understood. We characterized Salmonella isolates from cattle and humans in two geographic regions of the United States, the Pacific Northwest and the Northeast, using three common subtyping methods (pulsed-field gel electrophoresis [PFGE], multilocus variable number of tandem repeat analysis [MLVA], and multilocus sequence typing [MLST]). In addition, we analyzed the distribution of antimicrobial resistance among human and cattle Salmonella isolates from the two study areas and characterized Salmonella persistence on individual dairy farms. For both Salmonella enterica subsp. enterica serotypes Newport and Typhimurium, we found multidrug resistance to be significantly associated with bovine origin of isolates, with the odds of multidrug resistance for Newport isolates from cattle approximately 18 times higher than for Newport isolates from humans. Isolates from the Northwest were significantly more likely to be multidrug resistant than those from the Northeast, and susceptible and resistant isolates appeared to represent distinct Salmonella subtypes. We detected evidence for strain diversification during Salmonella persistence on farms, which included changes in antimicrobial resistance as well as genetic changes manifested in PFGE and MLVA pattern shifts. While discriminatory power was serotype dependent, the combination of PFGE data with either MLVA or resistance typing data consistently allowed for improved subtype discrimination. Our results are consistent with the idea that cattle are an important reservoir of multidrug-resistant Salmonella infections in humans. In addition, the study provides evidence for the value of including antimicrobial resistance data in epidemiological investigations and highlights the benefits and potential problems of combining subtyping methods.Salmonella is an important human and animal pathogen worldwide. In the United States, Salmonella causes an estimated 1.4 million human cases, 15,000 hospitalizations, and more than 400 deaths each year (44, 75). Human infections can be acquired through contact with animals or humans shedding Salmonella or through contaminated environments, but the majority of human infections are food-borne, and a large number of human outbreaks have been linked to foods of animal origin (20). Beef represents one well-recognized source of human infection (71). In addition, a number of human cases have been linked to dairy products or cattle contact, for instance at state fairs or on dairy farms (for example, see references 25, 35, and 61).Salmonella enterica subsp. enterica serotypes Typhimurium and Newport are commonly isolated from human cases, including those linked to cattle (20, 61). In 2006, Salmonella serotypes Typhimurium and Newport were isolated from 17 and 8% of reported human salmonellosis cases in the United States, respectively, making them the first and third most common human disease-associated serotypes in the United States (15). S. enterica serotype 4,5,12:i:− is both genetically and antigenically closely related to Salmonella serotype Typhimurium, of which it represents a monophasic variant (62). Salmonella enterica serotype 4,5,12:i:− is characterized by a deletion of flagellar genes fliA and fliB, which prevents expression of the phase 2 flagellar antigen (60). In the United States, the prevalence of Salmonella serotype 4,5,12:i:− has increased considerably over the past 10 years, and in 2006, Salmonella serotype 4,5,12:i:− represented the sixth most commonly isolated serotype from humans in the United States (15, 60).Salmonella serotype Newport represents two distinct clonal groups or lineages—one predominantly associated with isolates from cattle (i.e., Newport lineage A) and one associated with isolates from birds (i.e., Newport lineage B) (1, 33). Members of both lineages cause human infections (1, 33). The two Newport lineages can be clearly distinguished by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE), and some correlation between genetic lineage and antimicrobial resistance profile seems to exist (1, 33). In general, Newport lineage B isolates are pansusceptible or resistant to only a few antimicrobial drugs. In contrast, lineage A is strongly associated with multidrug resistance and includes a Newport subtype commonly referred to as Newport MDR-AmpC (1, 33).The prevalence of antimicrobial resistance among Salmonella serotype Newport and Typhimurium isolates has increased worldwide during the last 2 decades, predominantly as a result of emerging multidrug-resistant (MDR) strains (14, 52, 65). During the 1990s, Salmonella serotype Typhimurium phage type DT104 with pentaresistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) increased considerably in prevalence around the world, and some isolates acquired resistance to additional antimicrobial agents, including trimethoprim or ciprofloxacin (52). MDR Salmonella serotype Typhimurium DT104 has been isolated from a wide variety of host species and caused numerous large human outbreaks around the world (65). Salmonella serotype Newport MDR-AmpC, characterized by resistance to ACSSuT and carrying a plasmid encoding resistance to amoxicillin-clavulanic acid, cefoxitin, ceftiofur, and cephalothin emerged in the United States during the late 1990s, where it quickly became widespread among humans and cattle, leading to several large human outbreaks (14).Whether antimicrobial drug use in animals facilitates the emergence of MDR human pathogens is still subject to debate. Some studies report a temporal association between the introduction of new antimicrobial agents in veterinary medicine and the emergence of antimicrobial resistance (for instance, see references 22 and 58), but questions regarding the underlying evolutionary mechanisms, the origin and distribution of naturally occurring resistance genes, and the role of antimicrobial usage among humans remain (for example, see references 2 and 66 for reviews on this topic). Moreover, some studies report a higher prevalence of antimicrobial resistance among Salmonella isolates from farm animals than humans. Gebreyes et al. (26), for instance, found a higher prevalence of antimicrobial resistance among Salmonella isolates from pigs than humans, but potential effects attributable to differences in serotype distribution are difficult to assess in this study. In recent years, risk factors for MDR have received considerable attention. Infections with MDR Salmonella strains can lead to treatment failures, may be of longer duration, and may result in more severe clinical disease. Hence, such infections lead more often to hospitalization or death than infections with susceptible Salmonella strains, but serotype or subtype differences between resistant and susceptible Salmonella strains complicate the interpretation of clinical data (34, 41, 68).Subtyping methods allow characterization of Salmonella isolates and include phenotypic methods (e.g., serotyping or phage typing) as well as molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), ribotyping, multilocus variable number of tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) (5). PFGE is widely used and robust, and rigorous standardization allows comparison between laboratories (5). However, the method is time-intensive and laborious, requires careful standardization and analysis, does not allow phylogenetic inference, and can in rare cases be affected by endogenous nucleases or DNA methylation (for a review of this topic, see reference 5). MLVA and MLST are rapid, allow for easy data exchange between laboratories, and provide some phylogenetic information (5). MLVA is highly discriminatory but subject to rapid diversification and therefore most appropriate for the analysis of closely related isolates. While MLST lacks discriminatory power within Salmonella serotypes, it is highly reproducible and allows for phylogenetic analysis of more distantly related isolates (1, 5, 33). PFGE and MLST can be performed regardless of serotype, but MLVA protocols are serotype specific and have so far only been validated for a limited number of Salmonella serotypes. Moreover, MLVA can be complicated by inaccurate sizing of DNA fragments, and the degree of reliability can be considerably influenced by nucleotide composition and fragment length (5). Overall, these subtyping methods differ considerably in discriminatory power and sometimes yield conflicting results, and the most appropriate subtyping method or combination thereof strongly depends on serotype and chosen application (19, 56, 72, 76). Other genetic or phenotypic characteristics, such as antimicrobial resistance patterns or the presence of specific plasmids, have also been used successfully for subtyping in outbreak investigations and other epidemiological studies and can provide valuable additional information (7, 8, 40, 63, 64).Here we describe the distribution and subtype diversity of Salmonella serotypes Newport, 4,5,12:i:−, and Typhimurium among cattle and humans in two geographic regions of the United States, and we assess common risk factors for multidrug resistance. In addition, we utilize three Salmonella subtyping methods (PFGE, MLVA, and MLST), analyze their usefulness for characterizing isolates representing three common human-associated Salmonella serotypes, and compare the combined discriminatory power of PFGE and MLVA to that of PFGE and antimicrobial resistance patterns.     

Role of Ceftiofur in Selection and Dissemination of blaCMY-2-Mediated Cephalosporin Resistance in Salmonella enterica and Commensal Escherichia coli Isolates from Cattle

Third-generation cephalosporin resistance of Salmonella and commensal Escherichia coli isolates from cattle in the United States is predominantly conferred by the cephamycinase CMY-2, which inactivates β-lactam antimicrobial drugs used to treat a wide variety of infections, including pediatric salmonellosis. The emergence and dissemination of bla_CMY-2--bearing plasmids followed and may in part be the result of selection pressure imposed by the widespread utilization of ceftiofur, a third-generation veterinary cephalosporin. This study assessed the potential effects of ceftiofur on bla_CMY-2 transfer and dissemination by (i) an in vivo experimental study in which calves were inoculated with competent bla_CMY-2-bearing plasmid donors and susceptible recipients and then subjected to ceftiofur selection and (ii) an observational study to determine whether ceftiofur use in dairy herds is associated with the occurrence and frequency of cephalosporin resistance in Salmonella and commensal E. coli. The first study revealed bla_CMY-2 plasmid transfer in both ceftiofur-treated and untreated calves but detected no enhancement of plasmid transfer associated with ceftiofur treatment. The second study detected no association (P = 0.22) between ceftiofur use and either the occurrence of ceftiofur-resistant salmonellosis or the frequency of cephalosporin resistance in commensal E. coli. However, herds with a history of salmonellosis (including both ceftiofur-resistant and ceftiofur-susceptible Salmonella isolates) used more ceftiofur than herds with no history of salmonellosis (P = 0.03) These findings fail to support a major role for ceftiofur use in the maintenance and dissemination of bla_CMY-2-bearing plasmid mediated cephalosporin resistance in commensal E. coli and in pathogenic Salmonella in these dairy cattle populations.The major mechanism of third-generation cephalosporin resistance among U.S. human and veterinary clinical isolates of Salmonella enterica subsp. enterica is the beta-lactamase CMY-2 (12, 17, 43, 44, 46). bla_CMY-2, which likely originated from the chromosomal AmpC locus of Citrobacter freundii, is disseminated among a group of similar plasmids harbored by diverse Enterobacteriaceae species (1, 2, 20, 26, 30, 31, 42, 45). In Salmonella, bla_CMY-2-bearing plasmids have been observed in more than 30 serovars, notably including serovar Newport, which has gained specific attention from public health officials as a rapidly emerging threat (2, 6, 31).Commensal Escherichia coli frequently harbors bla_CMY-2-bearing plasmids (15, 33, 44), and these plasmids may be transferable to pathogens, since bla_CMY-2 plasmids isolated from E. coli and S. enterica share extensive sequence similarity in addition to the bla_CMY-2 open reading frame (5, 12, 42, 44). This transfer may occur in the gastrointestinal tracts of cattle, where these bacterial species periodically coexist and where transconjugants may be subjected to specific antimicrobial selection pressure. In fact, in vivo transfer of bla_CMY-2 in the gastrointestinal tract has been reported between a Klebsiella pneumoniae bla_CMY-2 plasmid donor and a Salmonella enterica serovar Typhimurium isolate in cattle and goats (29).Ceftiofur is the only third-generation cephalosporin antimicrobial drug that is used in cattle production systems and is labeled for the treatment of pneumonia, postpartum metritis, necrotizing pododermatitis, and mastitis. Two ceftiofur preparations, ceftiofur sodium (Naxcel) and ceftiofur hydrochloride (Excenel) (Pfizer Animal Health, New York, NY), are unique in the veterinary pharmacopeia because they require no withholding and discard of milk collected from treated cows, making them frequent therapeutic choices in lactating animals (19, 35). Ceftiofur was licensed in 1988 (41) and its resistance in Salmonella spp. isolated from U.S. cattle, presumably conferred by bla_CMY-2, was first documented in 1998 (6).The effects of ceftiofur use on selection of bla_CMY-2-bearing commensal E. coli has been examined for cattle both epidemiologically and experimentally. Tragesser et al. studied 18 Ohio dairy herds and determined that the 11 herds that used ceftiofur in any capacity (labeled indications and/or extralabel use) were 25 times more likely to have E. coli with reduced susceptibility to ceftriaxone (an expected bla_CMY-2 phenotype) than the seven herds that reported no ceftiofur use (40). Interestingly, however, within eight herds that had detailed treatment records, no association was detected between the prevalence of E. coli with reduced susceptibility to ceftriaxone and use of ceftiofur on an individual-animal basis (40). In an experimental study by Jiang et al., ceftiofur administered to dairy calves was correlated with a 14% increase in ceftriaxone-resistant fecal E. coli compared to untreated controls (21). Together, these studies show a correlation between selection pressure within the gastrointestinal tracts at the individual-animal level and show that ceftiofur use may promote the dissemination of resistance in commensal E. coli at the whole-herd level.Whether or not ceftiofur treatment directly affects in vivo horizontal transfer of bla_CMY-2-bearing elements among E. coli and Salmonella has yet to be addressed. The diversity of bla_CMY-2 plasmid-bearing bacterial hosts is consistent with wide dissemination of this genetic element. One hypothesis that could explain this wide dissemination is that ceftiofur may itself promote the in vivo horizontal transfer of bla_CMY-2-bearing plasmids. Specifically, due to the relatively slow bactericidal activity of aminothiazolyl cephalosporins such as ceftiofur, it has been suggested that exposure to these compounds promotes filament formation in gram-negative bacteria prior to cell death that may increase the surface area and increase receptiveness of the cells for resistance plasmids (11).Because bla_CMY-2 may be disseminated by horizontal transfer of R plasmids and/or clonal expansion of individual strains, we examined the effect of ceftiofur use on these processes with two approaches; the first approach specifically considered the issue of horizontal transfer in an experimental in vivo calf model, while the second approach, a field study, assessed the overall relationship between ceftiofur use and bla_CMY-2 prevalence in the primary agricultural animal niche where it is used.     

Roles of Small,Acid-Soluble Spore Proteins and Core Water Content in Survival of Bacillus subtilis Spores Exposed to Environmental Solar UV Radiation

Spores of Bacillus subtilis contain a number of small, acid-soluble spore proteins (SASP) which comprise up to 20% of total spore core protein. The multiple α/β-type SASP have been shown to confer resistance to UV radiation, heat, peroxides, and other sporicidal treatments. In this study, SASP-defective mutants of B. subtilis and spores deficient in dacB, a mutation leading to an increased core water content, were used to study the relative contributions of SASP and increased core water content to spore resistance to germicidal 254-nm and simulated environmental UV exposure (280 to 400 nm, 290 to 400 nm, and 320 to 400 nm). Spores of strains carrying mutations in sspA, sspB, and both sspA and sspB (lacking the major SASP-α and/or SASP-β) were significantly more sensitive to 254-nm and all polychromatic UV exposures, whereas the UV resistance of spores of the sspE strain (lacking SASP-γ) was essentially identical to that of the wild type. Spores of the dacB-defective strain were as resistant to 254-nm UV-C radiation as wild-type spores. However, spores of the dacB strain were significantly more sensitive than wild-type spores to environmental UV treatments of >280 nm. Air-dried spores of the dacB mutant strain had a significantly higher water content than air-dried wild-type spores. Our results indicate that α/β-type SASP and decreased spore core water content play an essential role in spore resistance to environmentally relevant UV wavelengths whereas SASP-γ does not.Spores of Bacillus spp. are highly resistant to inactivation by different physical stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and high fluences of UV or ionizing radiation (reviewed in references 33, 34, and 48). Under stressful environmental conditions, cells of Bacillus spp. produce endospores that can stay dormant for extended periods. The reason for the high resistance of bacterial spores to environmental extremes lies in the structure of the spore. Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents, such as the calcium chelate of dipicolinic acid and α/β-type small, acid-soluble spore proteins (α/β-type SASP), the last two of which protect spore DNA (6, 42, 46, 48, 52). DNA damage accumulated during spore dormancy is also efficiently repaired during spore germination (33, 47, 48). UV-induced DNA photoproducts are repaired by spore photoproduct lyase and nucleotide excision repair, DNA double-strand breaks (DSB) by nonhomologous end joining, and oxidative stress-induced apurinic/apyrimidinic (AP) sites by AP endonucleases and base excision repair (15, 26-29, 34, 43, 53, 57).Monochromatic 254-nm UV radiation has been used as an efficient and cost-effective means of disinfecting surfaces, building air, and drinking water supplies (31). Commonly used test organisms for inactivation studies are bacterial spores, usually spores of Bacillus subtilis, due to their high degree of resistance to various sporicidal treatments, reproducible inactivation response, and safety (1, 8, 19, 31, 48). Depending on the Bacillus species analyzed, spores are 10 to 50 times more resistant than growing cells to 254-nm UV radiation. In addition, most of the laboratory studies of spore inactivation and radiation biology have been performed using monochromatic 254-nm UV radiation (33, 34). Although 254-nm UV-C radiation is a convenient germicidal treatment and relevant to disinfection procedures, results obtained by using 254-nm UV-C are not truly representative of results obtained using UV wavelengths that endospores encounter in their natural environments (34, 42, 50, 51, 59). However, sunlight reaching the Earth''s surface is not monochromatic 254-nm radiation but a mixture of UV, visible, and infrared radiation, with the UV portion spanning approximately 290 to 400 nm (33, 34, 36). Thus, our knowledge of spore UV resistance has been constructed largely using a wavelength of UV radiation not normally reaching the Earth''s surface, even though ample evidence exists that both DNA photochemistry and microbial responses to UV are strongly wavelength dependent (2, 30, 33, 36).Of recent interest in our laboratories has been the exploration of factors that confer on B. subtilis spores resistance to environmentally relevant extreme conditions, particularly solar UV radiation and extreme desiccation (23, 28, 30, 34 36, 48, 52). It has been reported that α/β-type SASP but not SASP-γ play a major role in spore resistance to 254-nm UV-C radiation (20, 21) and to wet heat, dry heat, and oxidizing agents (48). In contrast, increased spore water content was reported to affect B. subtilis spore resistance to moist heat and hydrogen peroxide but not to 254-nm UV-C (12, 40, 48). However, the possible roles of SASP-α, -β, and -γ and core water content in spore resistance to environmentally relevant solar UV wavelengths have not been explored. Therefore, in this study, we have used B. subtilis strains carrying mutations in the sspA, sspB, sspE, sspA and sspB, or dacB gene to investigate the contributions of SASP and increased core water content to the resistance of B. subtilis spores to 254-nm UV-C and environmentally relevant polychromatic UV radiation encountered on Earth''s surface.     

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.     

Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 × 10⁹ ± 5 × 10⁸ CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 × 10⁷ ± 5 × 10⁶ CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4′,6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.Salmonella spp. are enteropathogenic bacteria that cause diseases that range from a mild gastroenteritis to systemic infections (5, 18) The disease severity is determined by the virulence characteristics of the Salmonella strain, host species, and host health condition. Phylogenetic analysis has demonstrated that the genus Salmonella includes two species: Salmonella bongori and Salmonella enterica. Salmonella strains are conventionally identified and classified according to the Kauffmann-White serotyping scheme, which is based on antigenic variation in the outer membrane (23). To date, more than 2,500 Salmonella serovars have been identified, and most of them are capable of infecting a wide variety of animal species and humans (33). Salmonella can be transmitted directly by person to person via the fecal-oral route or by contact with external reservoirs if fecal contamination of soil, water, and foods occurs. It is therefore necessary to develop robust detection methods for all of these sample types.The diagnostic method currently used for Salmonella detection is bacterial culture (International Organization for Standardization [ISO] method 6579:2002), a time-consuming and laborious process (40). A rapid and reliable tool to assist disease control management should aim to reduce salmonellosis in both people and animals. For this purpose a number of assays, such as the enzyme-linked immunosorbent assay (ELISA), PCR, and fluorescence in situ hybridization (FISH), have been developed to decrease the time required to identify Salmonella in food, feces, water, and other clinical samples (8, 10, 14, 15, 25, 26, 31, 41).Several authors have compared some of these approaches, especially culture-based, ELISA, and PCR methods, for Salmonella detection. Some authors found that PCR and ELISA-based methods failed to detect some samples that were positive by culture method (12, 13, 36, 39, 40). Even so, PCR-based methods have proved to be more accurate. Other work showed that when a selective enrichment step was performed before PCR, all Salmonella samples recovered by the culture method were detected. Moreover, the presence of Salmonella that was not recovered by the culture method could be detected by PCR (13, 35). These studies revealed that the enrichment step could increase the molecular assay sensitivity by eliminating problems such as the low numbers of bacteria and the presence of inhibitory substances in certain types of samples, such as food and fecal matter (11, 28, 36). However, PCR-based methods usually require a DNA extraction step, and none of the methods referred to above allows a direct, in situ visualization of the bacterium within the sample.FISH is a molecular assay widely applied for bacterial identification and localization within samples (2, 3). The method is usually based on the specific binding of nucleic acid probes to particular RNAs, due to their higher numbers of copies in the cells. There are already some studies reporting Salmonella detection by FISH using DNA probes (21, 29). A recently developed synthetic DNA analogue, named peptide nucleic acid (PNA), capable of hybridizing to complementary nucleic acid targets, has made FISH procedures easier and more efficient (38, 42). PNA-FISH methods have been successfully applied to the detection of several pathogenic microorganisms (6, 16, 17, 19, 22, 30, 34, 37, 42). For Salmonella, a PNA probe, designated Sal23S10, that targets the 23S rRNA of both Salmonella species has been already developed (31). However, the probe is also complementary to Actinobacillus actinomycetemcomitans, Buchnera aphidicola, and Haemophilus influenzae 23S rRNAs.In this paper, we identify and describe the design of a new fluorescently labeled PNA probe for the specific identification of the Salmonella genus. A novel, rapid, and reliable PNA-FISH method that can be easily applied to a great variety of sample types, either clinical or environmental, has consequently been developed and optimized.     

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.     

Chickens and Cattle as Sources of Sporadic Domestically Acquired Campylobacter jejuni Infections in Finland

A substantial sampling among domestic human campylobacter cases, chicken process lots, and cattle at slaughter was performed during the seasonal peak of human infections. Campylobacter jejuni isolates (n = 419) were subtyped using pulsed-field gel electrophoresis with SmaI, and isolates representing overlapping types (n = 212) were further subtyped using KpnI for restriction. The SmaI/KpnI profiles of 55.4% (97/175) of the human isolates were indistinguishable from those of the chicken or cattle isolates. The overlapping SmaI/KpnI subtypes accounted for 69.8% (30/43) and 15.9% (32/201) of the chicken and cattle isolates, respectively. The occurrence of identical SmaI/KpnI subtypes with human C. jejuni isolates was significantly associated with animal host species (P < 0.001). A temporal association of isolates from chickens and patients was possible in 31.4% (55/175) of the human infections. Besides chickens as sources of C. jejuni in the sporadic infections, the role of cattle appears notable. New approaches to restrict the occurrence of campylobacters in other farm animals may be needed in addition to hygienic measures in chicken production. However, only about half of the human infections were attributable to these sources.The incidence of human enteric infections caused by campylobacters is highest in the summer months, showing a consistent peak at the end of July in Finland (www.ktl.fi/attachments/suomi/julkaisut/julkaisusarja_b/2008/2008b09.pdf), as well as in other Nordic countries (16, 33). Almost 70% of campylobacter infections detected in July and August in Finland are domestically acquired, whereas the annual average proportion of domestic cases is about 30%, and most of them are caused by Campylobacter jejuni (30). The prevalence of campylobacters in Finnish broiler flocks peaks simultaneously with the human cases (7), and similar sero- and genotypes have been reported among human and poultry strains isolated in Finland and in other countries (5, 8, 21-23). Several epidemiological studies have identified the handling and consumption of raw or undercooked poultry meat as a major risk factor for campylobacter enteritis (for example, see references 18, 20, and 41), whereas opposite conclusions about the significance of the consumption of chicken meat were drawn from the Swedish case-control study among young children (2) and an extensive Danish register-based study (6).Data derived from the genotyping studies of C. jejuni isolates from human infections and animals support the current suggestion that poultry is the most important single source of sporadic campylobacteriosis (12, 22, 29). However, several reports on genotype comparisons suggest that poultry may be a less significant source of campylobacters than generally thought, and other animal reservoirs should also be considered notable sources of campylobacters pathogenic to humans (3, 8, 17, 27, 31). Studies of the temporal occurrence of campylobacters in human infections and poultry flocks have revealed that the peak in prevalence, as well as some of the overlapping sero- and genotypes, is detected in humans prior to being detected in poultry (21, 28).Although cattle are well-known carriers of campylobacters, the survival of these fragile organisms in beef is poor (39, 42). In recent years, some authors (1, 4, 10) have raised the question of an indirect association between cattle and human cases. In a Finnish study combining data from the multilocus sequence typing of campylobacters isolated from production animals and from epidemiological studies of human cases, significant associations emerged between certain sequence-type complexes from human infections and contact with cattle, the consumption of unpasteurized milk, or the tasting or consumption of raw minced meat (23).The aim of this study was to investigate the contributions of poultry and cattle as sources of human C. jejuni infections in Finland by comparing over a limited time frame the macrorestriction profiles obtained from pulsed-field gel electrophoresis (PFGE) analysis of a geographically representative collection of C. jejuni isolates from domestically acquired sporadic human infections, chicken process lots, and cattle.     

Acquisition of Complement Resistance through Incorporation of CD55/Decay-Accelerating Factor into Viral Particles Bearing Baculovirus GP64

A major obstacle to gene transduction by viral vectors is inactivation by human complement in vivo. One way to overcome this is to incorporate complement regulatory proteins, such as CD55/decay accelerating factor (DAF), into viral particles. Lentivirus vectors pseudotyped with the baculovirus envelope protein GP64 have been shown to acquire more potent resistance to serum inactivation and longer transgene expression than those pseudotyped with the vesicular stomatitis virus (VSV) envelope protein G. However, the molecular mechanisms underlying resistance to serum inactivation in pseudotype particles bearing the GP64 have not been precisely elucidated. In this study, we generated pseudotype and recombinant VSVs bearing the GP64. Recombinant VSVs generated in human cell lines exhibited the incorporation of human DAF in viral particles and were resistant to serum inactivation, whereas those generated in insect cells exhibited no incorporation of human DAF and were sensitive to complement inactivation. The GP64 and human DAF were detected on the detergent-resistant membrane and were coprecipitated by immunoprecipitation analysis. A pseudotype VSV bearing GP64 produced in human DAF knockdown cells reduced resistance to serum inactivation. In contrast, recombinant baculoviruses generated in insect cells expressing human DAF or carrying the human DAF gene exhibited resistance to complement inactivation. These results suggest that the incorporation of human DAF into viral particles by interacting with baculovirus GP64 is involved in the acquisition of resistance to serum inactivation.Gene therapy is a potential treatment option for genetic diseases, malignant diseases, and other acquired diseases. To this end, safe and efficient gene transfer into specific target cells is a central requirement, and a variety of nonviral and viral vector systems have been developed (6, 44). Recombinant viruses can be used for efficient gene transfer. Retroviruses, adeno-associated viruses, and lentiviruses are able to integrate foreign genes into host genomes and are suitable for gene therapeutics by virtue of their permanent expression of the therapeutic genes, whereas adenoviruses, herpesviruses, and baculoviruses can transiently express foreign genes (6, 12, 44). Pseudotype particles bearing other viral envelope proteins have been developed to improve transduction efficiency and the safety of viral vectors, including retrovirus (4, 7), lentivirus (25), vesicular stomatitis virus (VSV) (29), and baculovirus (17, 42). Pseudotype retroviruses and lentiviruses bearing the baculovirus envelope protein GP64 of Autographa californica nucleopolyhedrosis virus (AcNPV) have been shown to exhibit efficient gene transduction into a wide variety of cells with a lower cytotoxicity compared to those bearing the VSV envelope protein G (VSVG), which is commonly used for pseudotyping (18, 32, 35, 36).However, a drawback of gene transduction by viral vectors is that human sera inactivate the vectors (11, 40). Complement is a major element of the innate immune response and serves to link innate and adaptive immunity (8). Complement activation can occur via classical, lectin, and alternative pathways (2, 8). All pathways invoke several responses, such as virus opsonization, virolysis, anaphylatoxin, and chemotaxin production, as well as others (2, 8). VSV and baculovirus are inactivated by human sera via the classical pathway (1, 11). Because complement activation also induces potential damage to host cells, the complement system is tightly regulated by the complement regulatory proteins (CRPs), including CD55/decay-accelerating factor (DAF), CD46/membrane cofactor protein (MCP), and CD59 (2, 8, 15). DAF and CD46 inhibit activation of C3/C5-converting enzymes, which regulate the activation of classical and alternative pathways, whereas CD59 regulates the assembly of the membrane attack complex (2, 8, 15).Viral vectors can be manipulated to confer resistance to the complement inactivation. Human immunodeficiency virus (HIV) is known to develop resistance to human complement through the incorporation of DAF, CD46, and CD59 to the viral particles (22, 30, 31, 38). Moloney murine leukemia virus vectors produced in HT1080 cells are resistant to complement inactivation (5). Baculovirus and lentivirus vectors bearing DAF or the fusion protein between the functional domains of human DAF and the GP64 were resistant to complement inactivation (9, 13). It has been shown that lentivirus vectors pseudotyped with the GP64 are more resistant to inactivation in the sera of mice and rats (14, 32) and are capable of executing longer expression of the transgenes in nasal epithelia compared to those pseudotyped with the VSVG (35, 36). However, the precise mechanisms underlying the resistance to complement inactivation by pseudotyping of the GP64 is not known.To clarify the molecular mechanisms underlying the resistance of the viral vectors pseudotyped with the GP64 to the complement inactivation, we produced pseudotype and recombinant VSVs bearing the GP64. The recombinant VSVs carrying the gp64 gene generated in human cells but not in insect cells exhibited incorporation of human DAF on the viral particles and were resistant to the complement inactivation. Furthermore, production of the gp64 pseudotype VSV in the DAF knockdown human cells impaired serum resistance, whereas production of the gp64 recombinant VSV in the CHO cell lines stably expressing human DAF and the recombinant baculoviruses in the insect cells stably expressing human DAF or encoding the DAF gene in the genome conferred resistance to the complement inactivation. These results suggest that DAF incorporation into viral particles bearing baculovirus GP64 confers resistance to serum inactivation.     

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.     

Polymorphic Mutation Frequencies of Clinical and Environmental Stenotrophomonas maltophilia Populations

Mutation frequencies were studied in 174 Stenotrophomonas maltophilia isolates from clinical and nonclinical environments by detecting spontaneous rifampin-resistant mutants in otherwise-susceptible populations. The distribution of mutation frequencies followed a pattern similar to that found for other bacterial species, with a modal value of 1 × 10⁻⁸. Nevertheless, the proportion of isolates showing mutation frequencies below the modal value (hypomutators) was significantly higher for S. maltophilia than those so far reported in other organisms. Low mutation frequencies were particularly frequent among environmental S. maltophilia strains (58.3%), whereas strong mutators were found only among isolates with a clinical origin. These results indicate that clinical environments might select bacterial populations with high mutation frequencies, likely by second-order selection processes. In several of the strong-mutator isolates, functional-complementation assays with a wild-type allele of the mutS gene demonstrated that the mutator phenotype was due to the impairment of MutS activity. In silico analysis of the amino acid changes present in the MutS proteins of these hypermutator strains in comparison with the normomutator isolates suggests that the cause of the defect in MutS might be a H683P amino acid change.Stenotrophomonas maltophilia is a Gram-negative, nonfermenting environmental bacterial species often isolated from the rhizosphere and from water sources (11, 12, 63). Some S. maltophilia strains have been used for bioremediation (13, 24, 73) or bioaugmentation (37). However, besides its environmental origin and potential relevance for biotechnological purposes, S. maltophilia is also a relevant human opportunistic pathogen (44) associated with a broad spectrum of clinical syndromes, such as bacteremia (79, 81), endocarditis (18), infection in cancer patients (1), and respiratory tract infections, including those suffered by cystic fibrosis (CF) patients (72, 77). One of the most problematic characteristics of S. maltophilia is its intrinsic high resistance to several antibiotics (4). This intrinsic antibiotic resistance is at least partly due to the presence in the genome of S. maltophilia (17) of genes encoding antibiotic-inactivating enzymes (6, 9, 30, 39, 42, 58) and multidrug resistance (MDR) efflux pumps (2, 3, 43, 78). More recently, a chromosomally encoded Qnr protein that contributes to the intrinsic resistance to quinolones of S. maltophilia has been described (67, 68).A clear difference between infective (clinical) and environmental (nonclinical) S. maltophilia strains has not been reported (12, 63). However, although the available data fit the concept that opportunistic pathogens have not specifically evolved to infect humans (48), this does not mean that they do not evolve during the infective process. For most acute infections, we can presume that the time of in-host evolution is probably too short to detect relevant adaptive changes. Nevertheless, the situation might be different in chronic infections, such as those involving the bronchial compartment in CF patients. In this case, the same bacterial clone can be maintained and grow inside the host for years (62). This produces strong diversification over time and in different compartments of the lung (25, 71, 80), a process in which the acquisition of a mutator phenotype is important (52). Thus, isolates derived from an initial clone but presenting different morphotypes (47), different phenotypes of susceptibility to antibiotics (26) or in the expression of virulence determinants (14, 15, 36), or with different mutation frequencies (49, 60) are recovered from each individual patient suffering chronic infections. More recently, intraclonal diversification has also been described for Pseudomonas aeruginosa causing acute infections in intubated patients (38). Taken together, this indicates that bacteria can evolve during infection.For different bacterial species, strains isolated from CF patients with chronic lung infections show high mutation frequencies (hypermutable strains) (19, 60, 61, 66), whereas hypermutators have rarely been found in isolates from acute infections (33). An explanation for this difference could be that hypermutable strains tend to be selected for in the highly compartmentalized environment of the infected lung by intensive antibiotic therapy, as well as by the stressful conditions of the habitat. This is a second-order selection process (75, 76), in which mutations are selected because they confer an advantage in clinical environments in such a way that mutator strains are selected because they can produce more mutants (both advantageous and deleterious) for selection. In cases of chronic infections that are treated, strong and maintained selective local processes might occur, either by antibiotic treatment or by the actions of the anti-infective systems of the host. Natural out-of-host open environments obviously might have local stresses. However, the intensity of selection is expected to be lower in these habitats, and a constant replacement of potentially lost organisms by migration of neighbor populations probably mitigates the local selection of mutators and favors the enrichment of bacteria presenting low mutation frequencies. In the case of chronic infections, the replacement of mutators by neighbor normomutators is unlikely, because those infections are produced by a single clone that remains for several years in the host (62). Furthermore, although the infection process presents strong evolutionary bottlenecks for bacterial populations, the human host also provides a constant temperature, reliable nutrient supplies, and a habitat largely free from predators and competitors. Thus, while hypermutation might increase the capability of bacteria to adapt to some specific challenges in the clinical environment, the cost of hypermutation in terms of deleterious mutations might also be diminished, and these effects might be mutually reinforcing.The hypothesis explored in this paper is that S. maltophilia is adapted to deal with out-of-host fluctuating environmental variations but that once the organism enters a patient as an opportunistic pathogen, its adaptive needs significantly increase due to the actions of stressful local environmental conditions, such as the immune response and, when present, antibiotics. This enhanced stress under infective conditions might result in the selection of variants with increased mutation frequencies in a second-order selection process (75, 76). To test this hypothesis, the mutation frequencies of S. maltophilia clinical isolates (obtained from CF and non-CF patients) and from the environment (nonclinical origin) were compared. Most works that have been published on the different mutation frequencies in bacterial populations have focused on the detection of strains showing a high mutation frequency (mutators). In our work, we describe for the first time the presence of mutators in clinical isolates of S. maltophilia and demonstrate that hypermutation in several of those isolates is due to defects in MutS.Nevertheless, our main goal has been the analysis of the global distribution of mutation frequencies in an ample number of samples from clinical and nonclinical environments. Our results indicate not only that mutators are more frequent in clinical S. maltophilia isolates, but also that the overall distribution of mutation frequencies is different in S. maltophilia populations with environmental or clinical origins, with a tendency toward mutation frequencies lower than the modal mutation value (hypomutators) in the environmental isolates.     

Virus Entry via the Alternative Coreceptors CCR3 and FPRL1 Differs by Human Immunodeficiency Virus Type 1 Subtype

Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding first to CD4 and then to a coreceptor (CoR), of which C-C chemokine receptor 5 (CCR5) is the most common (6, 53). CXCR4 is an additional CoR for up to 50% of subtype B and D HIV-1 isolates at very late stages of disease (4, 7, 28, 35). Many other seven-membrane-spanning G-protein-coupled receptors (GPCRs) have been identified as alternative CoRs when expressed on various target cell lines in vitro, including CCR1 (76, 79), CCR2b (24), CCR3 (3, 5, 17, 32, 60), CCR8 (18, 34, 38), GPR1 (27, 65), GPR15/BOB (22), CXCR5 (39), CXCR6/Bonzo/STRL33/TYMSTR (9, 22, 25, 45, 46), APJ (26), CMKLR1/ChemR23 (49, 62), FPLR1 (67, 68), RDC1 (66), and D6 (55). HIV-2 and simian immunodeficiency virus SIVmac isolates more frequently show expanded use of these alternative CoRs than HIV-1 isolates (12, 30, 51, 74), and evidence that alternative CoRs other than CXCR4 mediate infection of primary target cells by HIV-1 isolates is sparse (18, 30, 53, 81). Genetic deficiency in CCR5 expression is highly protective against HIV-1 transmission (21, 36), establishing CCR5 as the primary CoR. The importance of alternative CoRs other than CXCR4 has remained elusive despite many studies (1, 30, 70, 81). Expansion of CoR use from CCR5 to include CXCR4 is frequently associated with the ability to use additional alternative CoRs for viral entry (8, 16, 20, 63, 79) in most but not all studies (29, 33, 40, 77, 78). This finding suggests that the sequence changes in HIV-1 env required for use of CXCR4 as an additional or alternative CoR (14, 15, 31, 37, 41, 57) are likely to increase the potential to use other alternative CoRs.We have used the highly permissive NP-2/CD4 human glioma cell line developed by Soda et al. (69) to classify virus entry via the alternative CoRs CCR1, CCR3, CCR8, GPR1, CXCR6, APJ, CMKLR1/ChemR23, FPRL1, and CXCR4. Full-length molecular clones of 66 env genes from most prevalent HIV-1 subtypes were used to generate infectious virus pseudotypes expressing a luciferase reporter construct (19, 57). Two types of analysis were performed: the level of virus entry mediated by each alternative CoR and linear regression of entry mediated by CCR5 versus all other alternative CoRs. We thus were able to identify patterns of alternative CoR use that were subtype specific and to determine if use of any alternative CoR was correlated or independent of CCR5-mediated entry. The results obtained have implications for the evolution of env function, and the analyses revealed important differences between subtype B Env function and all other HIV-1 subtypes.     

Identification of Diverse Antimicrobial Resistance Determinants Carried on Bacterial,Plasmid, or Viral Metagenomes from an Activated Sludge Microbial Assemblage

Using both sequence- and function-based metagenomic approaches, multiple antibiotic resistance determinants were identified within metagenomic libraries constructed from DNA extracted from bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage. Metagenomic clones and a plasmid that in Escherichia coli expressed resistance to chloramphenicol, ampicillin, or kanamycin were isolated, with many cloned DNA sequences lacking any significant homology to known antibiotic resistance determinants.Activated sludge in wastewater treatment plants is an open system with a dynamic and phylogenetically diverse microbial community (2, 3, 6, 7, 10, 11). Since the activated sludge process promotes cellular interactions among diverse microorganisms, there is great potential for the lateral transfer of antibiotic resistance genes between microbes in activated sludge and in downstream environments. Several studies have previously identified antibiotic resistance determinants from wastewater communities that are carried on bacterial chromosomes (1, 4, 14) and plasmids (9, 12, 13), but to our knowledge, a simultaneous metagenomic survey of antibiotic resistance determinants from all three genetic reservoirs (i.e., chromosomes, plasmids, and viruses) has never been performed within the same environment. To achieve a more comprehensive assessment of antibiotic resistance genes in the activated sludge microbial community, this study used both function- and sequence-based metagenomic approaches to identify antibiotic resistance determinants carried on bacterial chromosomes, plasmids, or viruses within an activated sludge microbial assemblage.