A Targeted Multilocus Genotyping Assay for Lineage,Serogroup, and Epidemic Clone Typing of Listeria monocytogenes

A 30-probe assay was developed for simultaneous classification of Listeria monocytogenes isolates by lineage (I to IV), major serogroup (4b, 1/2b, 1/2a, and 1/2c), and epidemic clone (EC) type (ECI, ECIa, ECII, and ECIII). The assay was designed to facilitate rapid strain characterization and the integration of subtype data into risk-based inspection programs.Listeria monocytogenes is a facultative intracellular pathogen that can cause serious invasive illness (listeriosis) in humans and other animals. L. monocytogenes is responsible for over 25% of food-borne-disease-related deaths attributable to known pathogens and is a leading cause of food recalls due to microbial adulteration (12, 21). However, not all L. monocytogenes subtypes contribute equally to human illness, and substantial differences in the ecologies and virulence attributes of different L. monocytogenes subtypes have been identified (9, 13, 14, 23, 24, 33, 35, 36). Among the four major evolutionary lineages of L. monocytogenes, only lineages I and II are commonly isolated from contaminated food and human listeriosis patients (19, 27, 29, 33). Lineage I strains are overrepresented among human listeriosis isolates, particularly those associated with epidemic outbreaks, whereas lineage II strains are overrepresented in foods and the environment (13, 14, 24). Lineage III strains account for approximately 1% of human listeriosis cases but are common among animal listeriosis isolates and appear to be a host-adapted group that is poorly adapted to food-processing environments (6, 34-36). The ecological and virulence attributes of lineage IV are poorly understood, as this lineage is rare and was only recently described based on a small number of strains (19, 26, 29, 33).L. monocytogenes is differentiated into 13 serotypes; however, four major serogroups (4b, 1/2b, 1/2a, and 1/2c) from within lineages I and II account for more than 98% of human and food isolates (16, 31). Serogroups refer to evolutionary complexes typified by a predominant serotype but which include very rare serotypes that represent minor evolutionary variants (7, 9, 33). Phylogenetic analyses have indicated that rare serotypes may have evolved recently, or even multiple times, from one of the major serotypes (9), and numerous molecular methods fail to discriminate minor serotypes as independent groups (1, 4, 7, 9, 18, 22, 33, 38, 39). Serotyping is one of the most common methods for L. monocytogenes subtyping, and serogroup classifications are a useful component of strain characterization because ecotype divisions appear largely congruent with serogroup distinctions (16, 34). Serogroup 4b strains are of particular public health concern because contamination with these strains appears to increase the probability that a ready-to-eat (RTE) food will be implicated in listeriosis (16, 28). Serogroup 4b strains account for approximately 40% of sporadic listeriosis and also are responsible for the majority of listeriosis outbreaks despite being relatively rare contaminants of food products (9, 13, 17, 30, 34). In addition, serogroup 4b strains are associated with more severe clinical presentations and higher mortality rates than other serogroups (11, 16, 20, 31, 34). Serogroups 1/2a and 1/2b are overrepresented among food isolates but also contribute significantly to human listeriosis, whereas serogroup 1/2c rarely causes human illness and may pose a lower risk of listeriosis for humans (16). Serogroup-specific differences in association with human listeriosis are consistent with the prevalence of virulence-attenuating mutations in inlA within these serogroups (32, 34); however, a number of additional factors likely contribute to these differences.Four previously described epidemic clones (ECs; ECI, ECIa, ECII, and ECIII) of L. monocytogenes have been implicated in numerous listeriosis outbreaks and have contributed significantly to sporadic illness (15, 34). ECI, ECIa, and ECII are distinct groups within serogroup 4b that were each responsible for repeated outbreaks of listeriosis in the United States and Europe. ECIII is a lineage II clone of serotype 1/2a that persisted in the same processing facility for more than a decade prior to causing a multistate outbreak linked to contaminated turkey (15, 25). While there has been speculation that epidemic clones possess unique adaptations that explain their frequent involvement in listeriosis outbreaks (9, 34, 37), it is not clear that epidemic clones are more virulent than other strains with the same serotype. However, contamination of RTE food with EC strains would be cause for increased concern due to the previous involvement of these clones in major outbreaks of listeriosis (16).As a result of the L. monocytogenes subtype-specific differences in ecology, virulence, and association with human illness, molecular subtyping technologies have the potential to inform assessments of relative risk and to improve risk-based inspection programs. The objective of the present study was to develop a single assay for rapid and accurate classification of L. monocytogenes isolates by lineage, major serogroup, and epidemic clone in order to facilitate strain characterization and the integration of subtype data into inspection programs that are based on assessment of relative risk.A database of more than 5.3 Mb of comparative DNA sequences from 238 L. monocytogenes isolates (9, 33-35) was scanned for single nucleotide polymorphisms that could be used to differentiate lineages, major serogroups, and epidemic clones via a targeted multilocus genotyping (TMLGT) approach. The acronym TMLGT is used to distinguish this approach from previously published multilocus genotyping (MLGT) assays that were lineage specific and designed for haplotype discrimination (9, 33). To provide for simultaneous interrogation of the selected polymorphisms via TMLGT, six genomic regions (Table ​(Table1)1) were coamplified in a multiplex PCR. While the previous MLGT assays were based on three lineage-specific multiplexes and required prior identification of lineage identity, TMLGT was designed to target variation across all of the lineages simultaneously and is based on a unique set of amplicons. PCR was performed in 50-μl volumes with 1× High Fidelity PCR buffer (Invitrogen Life Technologies), 2 mM MgSO₄, 100 μM deoxynucleoside triphosphate (dNTP), 300 nM primer, 1.5 U Platinum Taq high-fidelity DNA polymerase (Invitrogen Life Technologies), and 100 ng of genomic DNA. PCR consisted of an initial denaturation of 90 s at 96°C, followed by 40 cycles of 30 s at 94°C, 30 s at 50°C, and 90 s at 68°C. Amplification products were purified using Montage PCR cleanup filter plates (Millipore) and served as a template for allele-specific primer extension (ASPE) reactions utilizing subtype-specific probes.

TABLE 1.

Primers used in multiplex amplification for the TMLGT assay

Comparative Analysis of Myxococcus Predation on Soil Bacteria

Predator-prey relationships among prokaryotes have received little attention but are likely to be important determinants of the composition, structure, and dynamics of microbial communities. Many species of the soil-dwelling myxobacteria are predators of other microbes, but their predation range is poorly characterized. To better understand the predatory capabilities of myxobacteria in nature, we analyzed the predation performance of numerous Myxococcus isolates across 12 diverse species of bacteria. All predator isolates could utilize most potential prey species to effectively fuel colony expansion, although one species hindered predator swarming relative to a control treatment with no growth substrate. Predator strains varied significantly in their relative performance across prey types, but most variation in predatory performance was determined by prey type, with Gram-negative prey species supporting more Myxococcus growth than Gram-positive species. There was evidence for specialized predator performance in some predator-prey combinations. Such specialization may reduce resource competition among sympatric strains in natural habitats. The broad prey range of the Myxococcus genus coupled with its ubiquity in the soil suggests that myxobacteria are likely to have very important ecological and evolutionary effects on many species of soil prokaryotes.Predation plays a major role in shaping both the ecology and evolution of biological communities. The population and evolutionary dynamics of predators and their prey are often tightly coupled and can greatly influence the dynamics of other organisms as well (1). Predation has been invoked as a major cause of diversity in ecosystems (11, 12). For example, predators may mediate coexistence between superior and inferior competitors (2, 13), and differential trajectories of predator-prey coevolution can lead to divergence between separate populations (70).Predation has been investigated extensively in higher organisms but relatively little among prokaryotes. Predation between prokaryotes is one of the most ancient forms of predation (27), and it has been proposed that this process may have been the origin of eukaryotic cells (16). Prokaryotes are key players in primary biomass production (44) and global nutrient cycling (22), and predation of some prokaryotes by others is likely to significantly affect these processes. Most studies of predatory prokaryotes have focused on Bdellovibrionaceae species (e.g., see references 51, 55, and 67). These small deltaproteobacteria prey on other Gram-negative cells, using flagella to swim rapidly until they collide with a prey cell. After collision, the predator cells then enter the periplasmic space of the prey cell, consume the host cell from within, elongate, and divide into new cells that are released upon host cell lysis (41). Although often described as predatory, the Bdellovibrionaceae may also be considered to be parasitic, as they typically depend (apart from host-independent strains that have been observed [60]) on the infection and death of their host for their reproduction (47).In this study, we examined predation among the myxobacteria, which are also deltaproteobacteria but constitute a monophyletic clade divergent from the Bdellovibrionaceae (17). Myxobacteria are found in most terrestrial soils and in many aquatic environments as well (17, 53, 74). Many myxobacteria, including the model species Myxococcus xanthus, exhibit several complex social traits, including fruiting body formation and spore formation (14, 18, 34, 62, 71), cooperative swarming with two motility systems (64, 87), and group (or “wolf pack”) predation on both bacteria and fungi (4, 5, 8, 9, 15, 50). Using representatives of the genus Myxococcus, we tested for both intra- and interspecific variation in myxobacterial predatory performance across a broad range of prey types. Moreover, we examined whether prey vary substantially in the degree to which they support predatory growth by the myxobacteria and whether patterns of variation in predator performance are constant or variable across prey environments. The latter outcome may reflect adaptive specialization and help to maintain diversity in natural populations (57, 59).Although closely related to the Bdellovibrionaceae (both are deltaproteobacteria), myxobacteria employ a highly divergent mode of predation. Myxobacteria use gliding motility (64) to search the soil matrix for prey and produce a wide range of antibiotics and lytic compounds that kill and decompose prey cells and break down complex polymers, thereby releasing substrates for growth (66). Myxobacterial predation is cooperative both in its “searching” component (6, 31, 82; for details on cooperative swarming, see reference 64) and in its “handling” component (10, 29, 31, 32), in which secreted enzymes turn prey cells into consumable growth substrates (56, 83). There is evidence that M. xanthus employs chemotaxis-like genes in its attack on prey cells (5) and that predation is stimulated by close contact with prey cells (48).Recent studies have revealed great genetic and phenotypic diversity within natural populations of M. xanthus, on both global (79) and local (down to centimeter) scales (78). Phenotypic diversity includes variation in social compatibility (24, 81), the density and nutrient thresholds triggering development (33, 38), developmental timing (38), motility rates and patterns (80), and secondary metabolite production (40). Although natural populations are spatially structured and both genetic diversity and population differentiation decrease with spatial scale (79), substantial genetic diversity is present even among centimeter-scale isolates (78). No study has yet systematically investigated quantitative natural variation in myxobacterial predation phenotypes across a large number of predator genotypes.Given the previous discovery of large variation in all examined phenotypes, even among genetically extremely similar strains, we anticipated extensive predatory variation as well. Using a phylogenetically broad range of prey, we compared and contrasted the predatory performance of 16 natural M. xanthus isolates, sampled from global to local scales, as well as the commonly studied laboratory reference strain DK1622 and representatives of three additional Myxococcus species: M. flavescens (86), M. macrosporus (42), and M. virescens (63) (Table ​(Table1).1). In particular, we measured myxobacterial swarm expansion rates on prey lawns spread on buffered agar (31, 50) and on control plates with no nutrients or with prehydrolyzed growth substrate.

TABLE 1.

List of myxobacteria used, with geographical origin

Spontaneous Quinolone Resistance in the Zoonotic Serovar of Vibrio vulnificus

This work demonstrates that Vibrio vulnificus biotype 2, serovar E, an eel pathogen able to infect humans, can become resistant to quinolone by specific mutations in gyrA (substitution of isoleucine for serine at position 83) and to some fluoroquinolones by additional mutations in parC (substitution of lysine for serine at position 85). Thus, to avoid the selection of resistant strains that are potentially pathogenic for humans, antibiotics other than quinolones must be used to treat vibriosis on farms.Vibrio vulnificus is an aquatic bacterium from warm and tropical ecosystems that causes vibriosis in humans and fish (http://www.cdc.gov/nczved/dfbmd/disease_listing/vibriov_gi.html) (33). The species is heterogeneous and has been subdivided into three biotypes and more than eight serovars (6, 15, 33; our unpublished results). While biotypes 1 and 3 are innocuous for fish, biotype 2 can infect nonimmune fish, mainly eels, by colonizing the gills, invading the bloodstream, and causing death by septicemia (23). The disease is rapidly transmitted through water and can result in significant economic losses to fish farmers. Surviving eels are immune to the disease and can act as carriers, transmitting vibriosis between farms. Interestingly, biotype 2 isolates belonging to serovar E have been isolated from human infections, suggesting that serovar E is zoonotic (2). This serovar is also the most virulent for fish and has been responsible for the closure of several farms due to massive losses of fish. A vaccine, named Vulnivaccine, has been developed from serovar E isolates and has been successfully tested in the field (14). Although the vaccine provides fish with long-term protection from vibriosis, at present its use is restricted to Spain. For this reason, in many fish farms around the world, vibriosis is treated with antibiotics, which are usually added to the food or water.Quinolones are considered the most effective antibiotics against human and fish vibriosis (19, 21, 31). These antibiotics can persist for a long time in the environment (20), which could favor the emergence of resistant strains under selective pressure. In fact, spontaneous resistances to quinolones by chromosomal mutations have been described for some gram-negative bacteria (10, 11, 17, 24, 25, 26). Therefore, improper antibiotic treatment of eel vibriosis or inadequate residue elimination at farms could favor the emergence of human-pathogenic serovar E strains resistant to quinolones by spontaneous mutations. Thus, the main objective of the present work was to find out if the zoonotic serovar of biotype 2 can become quinolone resistant under selective pressure and determine the molecular basis of this resistance.Very few reports on resistance to antibiotics in V. vulnificus have been published; most of them have been performed with biotype 1 isolates. For this reason, the first task of this study was to determine the antibiotic resistance patterns in a wide collection of V. vulnificus strains belonging to the three biotypes that had been isolated worldwide from different sources (see Table S1 in the supplemental material). Isolates were screened for antimicrobial susceptibility to the antibiotics listed in Table S1 in the supplemental material by the agar diffusion disk procedure of Bauer et al. (5), according to the standard guideline (9). The resistance pattern found for each isolate is shown in Table S1 in the supplemental material. Less than 14% of isolates were sensitive to all the antibiotics tested, and more than 65% were resistant to more than one antibiotic, irrespective of their biotypes or serovars. The most frequent resistances were to ampicillin-sulbactam (SAM; 65.6% of the strains) and nitrofurantoin (F; 60.8% of the strains), and the least frequent were to tetracycline (12%) and oxytetracycline (8%). In addition, 15% of the strains were resistant to nalidixic acid (NAL) and oxolinic acid (OA), and 75% of these strains came from fish farms (see Table S1 in the supplemental material). Thus, high percentages of strains of the three biotypes were shown to be resistant to one or more antibiotics, with percentages similar to those found in nonbiotyped environmental V. vulnificus isolates from Asia and North America (4, 27, 34). In those studies, resistance to antibiotics could not be related to human contamination. However, the percentage of quinolone-resistant strains found in our study is higher than that reported in other ones, probably due to the inclusion of fish farm isolates, where the majority of quinolone-resistant strains were concentrated. This fact suggests that quinolone resistance could be related to human contamination due to the improper use of these drugs in therapy against fish diseases, as has been previously suggested (18, 20). Although no specific resistance pattern was associated with particular biotypes or serovars, we found certain differences in resistance distribution, as shown in Table ​Table1.1. In this respect, biotype 3 displayed the narrowest spectrum of resistances and biotype 1 the widest. The latter biotype encompassed the highest number of strains with multiresistance (see Table S1 in the supplemental material). Within biotype 2, there were differences among serovars, with quinolone resistance being restricted to the zoonotic serovar (Table ​(Table11).

TABLE 1.

Percentage of resistant strains distributed by biotypes and serovars

Identification and Characterization of Class 1 Integron Resistance Gene Cassettes among Salmonella Strains Isolated from Imported Seafood

A total of 210 Salmonella isolates, representing 64 different serovars, were isolated from imported seafood samples, and 55/210 isolates were found to be resistant to at least one antibiotic. Class 1 integrons from three multidrug-resistant Salmonella enterica strains (Salmonella enterica serovars Newport [strain 62], Typhimurium var. Copenhagen [strain 629], and Lansing [strain 803], originating from Hong Kong, the Philippines, and Taiwan, respectively) were characterized. Southern hybridization of plasmids isolated from these strains, using a class 1 integron probe, showed that trimethoprim-sulfamethoxazole and streptomycin resistance genes were located on a megaplasmid in strain 629. Our study indicates that imported seafood could be a reservoir for Salmonella isolates resistant to multiple antibiotics.Salmonella spp. are recognized as major food-borne pathogens of humans worldwide. In the United States, there are an estimated 800,000 to 4 million Salmonella infections annually, and approximately 500 of the cases are fatal (8, 26). A variety of foods have been implicated as vehicles transmitting salmonellosis to humans, including poultry, beef, pork, eggs, milk, cheese, fish, shellfish, fruits, juice, and vegetables (1, 4, 9, 12, 23). Previous studies by field laboratories of the U.S. Food and Drug Administration have shown the prevalences of Salmonella isolates in imported and domestic seafood as 7.2% and 1.3%, respectively (6, 11, 27).Mobile genetic elements, such as plasmids, transposons, and integrons, which disseminate antibiotic resistance genes by horizontal or vertical transfer, as part of either resistance plasmids or conjugative transposons, play an important role in the evolution and dissemination of multidrug resistance (2, 3, 10, 17). Salmonella genomic island 1 (SGI1), the first genomic island reported to contain an antibiotic resistance gene cluster, was identified in the multidrug-resistant Salmonella enterica serovar Typhimurium strain DT 104 (21).Most studies of the prevalence and characterization of antimicrobial resistance genes and integrons in Salmonella spp. have focused on strains from clinical and veterinary sources. However, little is known about the occurrence of SGI1 and its variants in Salmonella spp. isolated from seafood. We have screened a set of drug-resistant S. enterica strains from seafood belonging to 64 different serovars for SGI1 and class 1 integron conserved sequences (CS). We report the presence of a class I variant integron carrying the dfrXII and aadA2 genes on a megaplasmid in serovar Typhimurium var. Copenhagen and on the chromosome in Salmonella enterica serovar Lansing. We also found the variant class 1 integron carrying the dfrA1 and orfC genes in Salmonella enterica serovar Newport strains from seafood.A total of 210 Salmonella enterica strains isolated from seafood imported into the United States between 2000 and 2005 were identified and serotyped by the Pacific Regional Laboratory-Southwest of the FDA, Irvine, CA. The Salmonella strains represented 20 serogroups (Table ​(Table1)1) from various imported seafood items. The Salmonella strains were tested with 16 antibiotics (14) commonly used in either human or veterinary medicine on Mueller-Hinton agar (Difco Laboratories, Detroit, MI), using a disk diffusion method. The sensitivity and resistance were determined by the criteria of the Clinical and Laboratory Standards Institute (1999).

TABLE 1.

Salmonella serotypes isolated from imported foods

Distribution of Sulfonamide Resistance Genes in Escherichia coli and Salmonella Isolates from Swine and Chickens at Abattoirs in Ontario and Québec,Canada

Sulfonamide-resistant Escherichia coli and Salmonella isolates from pigs and chickens in Ontario and Québec were screened for sul1, sul2, and sul3 by PCR. Each sul gene was distributed differently across populations, with a significant difference between distribution in commensal E. coli and Salmonella isolates and sul3 restricted mainly to porcine E. coli isolates.Resistance to sulfonamides is frequent in bacteria from farm animals (7, 8, 9, 10) and is usually caused by the acquisition of the genes sul1, sul2, and sul3 (20, 22). The objectives of this study were (i) to assess the distribution of these genes in Escherichia coli and Salmonella enterica isolates in swine and chickens from two major provinces in Canada, (ii) to assess whether differences occur in the distribution of these genes among bacterial species found within two different animal host species, and (iii) to assess whether significant differences in the distribution of these genes are present between the commensal E. coli strains used as indicators for surveillance of antimicrobial resistance and the zoonotic Salmonella pathogens found in the same ecological niche. In contrast to previous studies, a multivariable logistic regression model was used to analyze the data, control for confounding factors, and assess the interaction effect between animal and bacterial species in terms of the probability of an isolate carrying a specific sul gene. The distribution of sulfonamide resistance genes among sulfonamide-resistant E. coli (393 isolates from chickens and 311 from swine) and Salmonella (13 isolates from chickens and 221 from swine) isolates was assessed. These isolates were collected by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) between 2003 and 2005 from ceca of apparently healthy animals at abattoirs in Ontario (n = 435) and Québec (n = 503). The methods used by CIPARS are presented in detail elsewhere (8-10). The isolates were screened with a previously published multiplex PCR for sul1, sul2, and sul3 (16). The sul1 and sul2 genes were found in E. coli and Salmonella isolates from both animal species. The sul3 gene was detected in both E. coli and Salmonella isolates from swine but only in E. coli isolates from chickens (Table ​(Table1).1). Three percent of the isolates had no detectable sul gene, 12.5% possessed two genes, and two isolates carried three genes (Table ​(Table1).1). Similar (2, 3, 14, 19) or higher (4, 11, 17) values for multiple genes have been reported by others. The overall higher prevalence of sul1 in Salmonella isolates and of sul2 and sul3 in E. coli isolates was in agreement with the results of previous studies (2-4, 11, 12, 14, 21).

TABLE 1.

Frequency of the three resistance genes sul1, sul2, and sul3 in sulfonamide-resistant E. coli and Salmonella isolates from chickens and swine in Ontario and Québec between 2003 and 2005

Wide Variation in the Multiplicity of HIV-1 Infection among Injection Drug Users

Recent studies indicate that sexual transmission of human immunodeficiency virus type 1 (HIV-1) generally results from productive infection by only one virus, a finding attributable to the mucosal barrier. Surprisingly, a recent study of injection drug users (IDUs) from St. Petersburg, Russia, also found most subjects to be acutely infected by a single virus. Here, we show by single-genome amplification and sequencing in a different IDU cohort that 60% of IDU subjects were infected by more than one virus, including one subject who was acutely infected by at least 16 viruses. Multivariant transmission was more common in IDUs than in heterosexuals (60% versus 19%; odds ratio, 6.14; 95% confidence interval [CI], 1.37 to 31.27; P = 0.008). These findings highlight the diversity in HIV-1 infection risks among different IDU cohorts and the challenges faced by vaccines in protecting against this mode of infection.Elucidation of virus-host interactions during and immediately following the transmission event is one of the great challenges and opportunities in human immunodeficiency virus (HIV)/AIDS prevention research (14-16, 31, 34, 45). Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference based on a model of random virus evolution (18-20, 43) have allowed for the identification of transmitted/founder viruses that actually cross from donor to recipient, leading to productive HIV type 1 (HIV-1) infection. Our laboratory and others have made the surprising finding that HIV-1 transmission results from productive infection by a single transmitted/founder virus (or virally infected cell) in ∼80% of HIV-infected heterosexuals and in ∼60% of HIV-infected men who have sex with men (MSM) (1, 13, 18, 24). These studies thus provided a precise quantitative estimate for the long-recognized genetic bottleneck in HIV-1 transmission (6, 11-13, 17, 25, 28, 30, 35, 38, 42, 47-49) and a plausible explanation for the low acquisition rate per coital act and for graded infection risks associated with different exposure routes and behaviors (15, 36).In contrast to sexual transmission of HIV-1, virus transmission resulting from injection drug use has received relatively little attention (2, 3, 29, 42) despite the fact that injection drug use-associated transmission accounts for as many as 10% of new infections globally (26, 46). We hypothesized that SGA strategies developed for identifying transmitted/founder viruses following mucosal acquisition are applicable to deciphering transmission events following intravenous inoculation and that, due to the absence of a mucosal barrier, injection drug users (IDUs) exhibit a higher frequency of multiple-variant transmission and a wider range in numbers of transmitted viruses than do acutely infected heterosexual subjects. We obtained evidence in support of these hypotheses from the simian immunodeficiency virus (SIV)-Indian rhesus macaque infection model, where we showed that discrete low-diversity viral lineages emanating from single or multiple transmitted/founder viruses could be identified following intravenous inoculation and that the rectal mucosal barrier to infection was 2,000- to 20,000-fold greater than with intravenous inoculation (19). However, we also recognized potentially important differences between virus transmission in Indian rhesus macaques and virus transmission in humans that could complicate an IDU acquisition study. For example, in the SIV macaque model, the virus inocula can be well characterized genetically and the route and timing of virus exposure in relation to plasma sampling precisely defined, whereas in IDUs, the virus inoculum is generally undefined and the timing of virus infection only approximated based on clinical history and seroconversion testing (8). In addition, IDUs may have additional routes of potential virus acquisition due to concomitant sexual activity. Finally, there is a paucity of IDU cohorts for whom incident infection is monitored sufficiently frequently and clinical samples are collected often enough to allow for the identification and enumeration of transmitted/founder viruses. To address these special challenges, we proposed a pilot study of 10 IDU subjects designed to determine with 95% confidence if the proportion of multivariant transmissions in IDUs was more than 2-fold greater than the 20% frequency established for heterosexual transmission (1, 13, 18, 24). A secondary objective of the study was to determine whether the range in numbers of transmitted/founder viruses in IDUs exceeded the 1-to-6 range observed in heterosexuals (1, 13, 18, 24). To ensure comparability among the studies, we employed SGA-direct amplicon sequencing approaches, statistical methods, and power calculations identical to those that we had used previously to enumerate transmitted/founder viruses in heterosexual and MSM cohorts (1, 13, 18, 20, 24).We first surveyed investigators representing acute-infection cohorts in the United States, Canada, Russia, and China; only one cohort—the Montreal Primary HIV Infection Cohort (41)—had IDU clinical samples and clinical data available for study. The Montreal cohort of subjects with acute and early-stage HIV-1 infection was established in 1996 and recruits subjects from both academic and private medical centers throughout the city. Injection drug use is an important contributing factor to Montreal''s HIV burden, with IDUs comprising approximately 20% of the city''s AIDS cases and 35% of the cohort (21, 40, 41). A large proportion of Montreal''s IDUs use injection cocaine, with 50 to 69% of subjects reporting cocaine as their injection drug of choice (4, 5, 9, 22, 23).Subjects with documented serological evidence of recent HIV-1 infection and a concurrent history of injection drug use were selected for study. These individuals had few or no reported risk factors for sexual HIV-1 acquisition. Clinical history and laboratory tests of HIV-1 viremia and antibody seroconversion were used to determine the Fiebig clinical stage (8) and to estimate the date of infection (Table ​(Table1).1). One subject was determined to be in Fiebig stage III, one subject was in Fiebig stage IV, five subjects were in Fiebig stage V, and three subjects were in Fiebig stage VI. We performed SGA-direct amplicon sequencing on stored plasma samples and obtained a total of 391 3′ half-genomes (median, 25 per subject; range, 19 to 167). Nine of these sequences contained large deletions or were G-to-A hypermutated and were excluded from subsequent analysis. Sequences were aligned, visually inspected using the Highlighter tool (www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter.html), and analyzed by neighbor-joining (NJ) phylogenetic-tree construction. A composite NJ tree of full-length gp160 env sequences from all 10 subjects (Fig. ​(Fig.1A)1A) revealed distinct patient-specific monophyletic lineages, each with high bootstrap support and separated from the others by a mean genetic distance of 10.79% (median, 11.29%; range, 3.00 to 13.42%). Maximum within-patient env gene diversity ranged from 0.23% to 3.34% (Table ​(Table1).1). Four subjects displayed distinctly lower within-patient maximum env diversities (0.23 to 0.49%) than the other six subjects (1.48% to 3.34%). The lower maximum env diversities in the former group are consistent with infection either by a single virus or by multiple closely related viruses, while the higher diversities can be explained only by transmission of more than one virus based on empirical observations (1, 13, 18, 24) and mathematical modeling (18, 20).Open in a separate windowFIG. 1.NJ trees and Highlighter plots of HIV-1 gp160 env sequences. (A) Composite tree of 382 gp160 env sequences from all study subjects. The numerals at the nodes indicate bootstrap values for which statistical support exceeded 70%. (B) Subject ACT54869022 sequences suggest productive infection by a single virus (V1). (C) Subject HDNDRPI032 sequences suggest productive infection by as many as three viruses. (D) Subject HDNDRPI001 sequences suggest productive infection by at least five viruses with extensive interlineage recombination. Sequences are color coded to indicate viral progeny from distinct transmitted/founder viruses. Recombinant virus sequences are depicted in black. Methods for SGA, sequencing, model analysis, Highlighter plotting, and identification of transmitted/founder virus lineages are described elsewhere (18, 20, 24, 44). The horizontal scale bars represent genetic distance. nt, nucleotide.

TABLE 1.

Subject demographics and HIV-1 envelope analysis results

Dehalogenation Activities and Distribution of Reductive Dehalogenase Homologous Genes in Marine Subsurface Sediments

Halogenated organic compounds serve as terminal electron acceptors for anaerobic respiration in a diverse range of microorganisms. Here, we report on the widespread distribution and diversity of reductive dehalogenase homologous (rdhA) genes in marine subsurface sediments. A total of 32 putative rdhA phylotypes were detected in sediments from the southeast Pacific off Peru, the eastern equatorial Pacific, the Juan de Fuca Ridge flank off Oregon, and the northwest Pacific off Japan, collected at a maximum depth of 358 m below the seafloor. In addition, significant dehalogenation activity involving 2,4,6-tribromophenol and trichloroethene was observed in sediment slurry from the Nankai Trough Forearc Basin. These results suggest that dehalorespiration is an important energy-yielding pathway in the subseafloor microbial ecosystem.Scientific ocean drilling explorations have revealed that marine subsurface sediments harbor remarkable numbers of microbial cells that account for approximately 1/10 to 1/3 of all living biota on Earth (20, 25, 33). Thermodynamic calculations of pore-water chemistry suggest that subseafloor microbial activities are generally supported by nutrient and energy supplies from the seawater and/or underlying basaltic aquifers (6, 7). Although sulfate, nitrate, Fe(III), Mn(IV), and bicarbonate are known to be potential electron acceptors for anaerobic microbial respiration in marine subsurface sediments (5), the incidence of both the dissimilatory dehalorespiration pathway and microbial activity in halogenated organic substrates remains largely unknown.Previous molecular ecological studies using 16S rRNA gene sequences demonstrated that Chloroflexi is one of the most frequently detected phyla in subseafloor sediments of the Pacific Ocean margins (12-14). Some of the sequences within the Chloroflexi are closely related to sequences in the genus Dehalococcoides, which contains obligatory dehalorespiring bacteria that employ halogenated organic compounds as terminal electron acceptors (21, 29). The frequent detection of Dehalococcoides-related 16S rRNA genes from these environments implies the occurrence of dissimilatory dehalorespiration in marine subsurface sediments.In this study, we detected and phylogenetically analyzed the reductive dehalogenase homologous (rdhA) genes, key functional genes for dehalorespiration pathways, from frozen sediment core samples obtained by Ocean Drilling Program (ODP) Leg 201 (Peru margin and eastern equatorial Pacific) (7, 14); Integrated Ocean Drilling Program (IODP) Expedition 301 (Juan de Fuca Ridge flank) (8, 24); Chikyu Shakedown Expedition CK06-06 (Northwest Pacific off Japan) (20, 23); and IODP Expedition 315 (Nankai Trough Forearc Basin off Japan) (Table ​(Table1).1). DNA was extracted using an ISOIL bead-beating kit (Nippon Gene, Japan) and purified using a MagExtractor DNA fragment purification kit (Toyobo, Japan) according to the manufacturer''s instructions. To increase concentration, DNA was amplified by multiple displacement amplification using the phi29 polymerase supplied with a GenomiPhi kit (GE Healthcare, United Kingdom) (20). Putative rdhA genes were amplified by PCR using Ex Taq polymerase (TaKaRa, Japan) with degenerate primers RRF2 and B1R (17), dehaloF3, dehaloF4, dehaloF5, dehaloR2, dehaloR3, and dehaloR4 (32), and ceRD2S, ceRD2L, and RD7 (26) and the PCR conditions described in those studies. Amplicons of the approximate target size were gel purified and cloned into the pCR2.1 vector (Invitrogen, Japan). Sequence similarity was analyzed using FastGroupII web-based software (34), and sequences with a 95% identity were tentatively assigned to the same phylotype. Amino acid sequences were aligned by ClustalW (31), including known and putative reductive dehalogenase sequences in the genome of Dehalococcoides ethenogenes strain 195 (28), as well as several functionally characterized reductive dehalogenases from other species.

TABLE 1.

Sample locations and results of PCR amplification of rdhA

Biochemical Characterization of Haloalkane Dehalogenases DrbA and DmbC,Representatives of a Novel Subfamily

This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.Haloalkane dehalogenases (EC 3.8.1.5.) acting on halogenated aliphatic hydrocarbons catalyze carbon-halogen bond cleavage, leading to an alcohol, a halide ion, and a proton as the reaction products (7). Haloalkane dehalogenases originating from various bacterial strains have potential for application in bioremediation technologies (4, 6, 22), construction of biosensors (2), decontamination of warfare agents (17), and synthesis of optically pure compounds (19). Recent evolutionary study of haloalkane dehalogenase sequences revealed the existence of three subfamilies, denoted HLD-I, HLD-II, and HLD-III (3). In contrast to subfamilies HLD-I and HLD-II, the subfamily HLD-III is currently lacking experimentally characterized proteins. We have therefore focused on the isolation and study of two selected representatives of the HLD-III subfamily, DrbA and DmbC.The drbA gene was amplified by PCR using the cosmid pircos.a3g10 originating from marine bacterium Rhodopirellula baltica SH1, and the dmbC gene was amplified from DNA originating from obligatory pathogen Mycobacterium bovis 5033/66. Six-histidine tails were added to the C termini of DrbA and DmbC in a cloning step, enabling single-step purification using Ni-nitrilotriacetic acid resin. Haloalkane dehalogenase DrbA was expressed under the T7 promoter and purified, with a resulting yield of 0.1 mg of protein per gram of cell mass. Haloalkane dehalogenase DmbC was obtained by expression in Mycobacterium smegmatis, with a yield of 0.07 mg of purified protein per gram of cell mass.The correct folding and secondary structures of the newly prepared enzymes were verified by circular dichroism (CD) spectroscopy. Far-UV CD spectra were recorded for DrbA and DmbC enzymes and other, related haloalkane dehalogenases. All enzymes tested exhibited CD spectra with two negative features at 208 and 222 nm and one positive peak at 195 nm, which are characteristic of α-helical content (Fig. ​(Fig.1).1). This suggested that both new enzymes, DrbA and DmbC, were folded correctly. However, DmbC exhibited more intense negative maxima which differed from other haloalkane dehalogenases in the θ₂₂₂/θ₂₀₈ ratio. This finding indicated a slight variation in the arrangement of secondary structure elements of the DmbC enzyme. Thermally induced denaturations of DrbA and DmbC were tested in parallel. Both enzymes showed changes in ellipticity during increasing temperature. The melting temperatures calculated from these curves were 45.8 ± 0.4°C for DmbC and 39.4 ± 0.1°C for DrbA. The thermostability results obtained for DrbA and DmbC were in good agreement with the range of melting temperatures determined for other, related haloalkane dehalogenases.Open in a separate windowFIG. 1.Far-UV CD spectra of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Protein concentration used for far-UV CD spectrum measurement was 0.2 mg/ml.The sizes of the purified proteins were estimated by electrophoresis under native conditions conducted using a 10% polyacrylamide gel (Fig. ​(Fig.2).2). More precise determination of the sizes of DrbA and DmbC was achieved by gel filtration chromatography performed on Sephacryl S-500 HR (GE Healthcare, Uppsala, Sweden), calibrated with blue dextran 2000, thyroglobulin (669 kDa), ferritin (440 kDa), catalase (240 kDa), conalbumin (75 kDa), and ovalbumin (43 kDa) (Fig. ​(Fig.3A).3A). Both DrbA and DmbC were eluted from the column in the fraction prior to blue dextran, indicating that both enzymes form oligomeric complexes of a size larger than 2,000 kDa (Fig. 3B and C). The haloalkane dehalogenases which have been biochemically characterized so far form monomers, except for DbjA isolated from Bradyrhizobium japonicum USDA110 (21), which shows monomeric, dimeric, and tetrameric forms according to the pH of the buffer (R. Chaloupkova, submitted for publication).Open in a separate windowFIG. 2.Native protein electrophoresis of DrbA and DmbC. Lane 1, carbonic anhydrase (29 kDa); lane 2, ovalbumin (43 kDa); lane 3, bovine albumin (67 kDa); lane 4, conalbumin (75 kDa); lane 5, catalase (240 kDa); lane 6, ferritin (440 kDa); lane 7, DrbA; lane 8, DmbC.Open in a separate windowFIG. 3.Gel filtration chromatogram of DrbA and DmbC. (A) The following calibration kit samples (0.5 ml of a concentration of 2 mg/ml protein loaded) were analyzed using 50 mM Tris-HCl with 150 mM NaCl, pH 7.5, as elution buffer: blue dextran (line 1, 9.6-ml fraction), thyroglobulin (line 2, 15.95-ml fraction), ferritin (line 3, 16.78-ml fraction), ovalbumin (line 4, 18.55-ml fraction), and RNase A (line 5, 20.08-ml fraction). (B and C) Haloalkane dehalogenase DrbA eluted in the 9.03-ml fraction (B), and haloalkane dehalogenase DmbC in the 9.31-ml fraction (C).The substrate specificities of DrbA and DmbC were investigated with a set of 30 selected chlorinated, brominated, and iodinated hydrocarbons. Standardized specific activities related to 1-chlorobutane (summarized in Table ​Table1)1) were compared with the activity profiles of other haloalkane dehalogenases (Fig. ​(Fig.4).4). DrbA and DmbC displayed similar activity patterns, with catalytic activities approximately two orders of magnitude lower than those of other known haloalkane dehalogenases (1, 5, 8-11, 13-16, 18, 20, 23). HLD-III subfamily enzymes showed a restricted specificity range and a preference for iodinated short-chain hydrocarbons. Both phenomena may be related to the composition of the catalytic pentad Asp-His-Asp+Asn-Trp, which is unique to the members of the HLD-III subfamily (3). The preference for substrates carrying an iodine substituent can be related to a pair of halide-binding residues and their spatial arrangement with the catalytic triad. These residues make up the catalytic pentad, playing a critical role in substrate binding, formation of the transition states, and the reaction intermediates of the dehalogenation reaction (12).Open in a separate windowFIG. 4.Substrate specificity profiles of DrbA, DmbC, and seven different biochemically characterized haloalkane dehalogenases. Activities were determined using a consistent set of 30 halogenated substrates (see Table ​Table1).1). Data were standardized by dividing each value by the sum of all activities determined for individual enzymes in order to mask the differences in absolute activities. Specific activities (in μmol·s⁻¹·mg⁻¹) with 1-chlorobutane are 0.0003 (DrbA), 0.0001 (DmbC), 0.0003 (DatA), 0.0133 (DbjA), 0.0010 (DbeA), 0.0128 (DhaA), 0.0231 (LinB), 0.0171 (DmbA), and 0.0117 (DhlA).

TABLE 1.

Specific activities of haloalkane dehalogenases DrbA and DmbC toward a set of 30 halogenated hydrocarbons^a

Evidence for a New Avian Paramyxovirus Serotype 10 Detected in Rockhopper Penguins from the Falkland Islands

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.Viruses from the Paramyxoviridae family have caused disease in humans and animals for centuries. Over the last 40 years, many paramyxoviruses isolated from animals and people have been newly described (16, 17, 22, 29, 31, 32, 36, 42, 44, 46, 49, 58, 59, 62-64). Viruses from this family are pleomorphic, enveloped, single-stranded, nonsegmented, negative-sense RNA viruses that demonstrate serological cross-reactivity with other paramyxoviruses related to them (30, 46). The subfamily Paramyxovirinae is divided into five genera: Respirovirus, Morbillivirus, Rubulavirus, Henipavirus, and Avulavirus (30). The Avulavirus genus contains nine distinct avian paramyxovirus (APMV) serotypes (Table ​(Table1),1), and information on the discovery of each has been reported elsewhere (4, 6, 7, 9, 12, 34, 41, 50, 51, 60, 68).

TABLE 1.

Characteristics of prototype viruses APMV1 to APMV9 and the penguin virus

Hepatitis C Virus (HCV) Sequence Variation Induces an HCV-Specific T-Cell Phenotype Analogous to Spontaneous Resolution

Hepatitis C virus (HCV)-specific CD8⁺ T cells in persistent HCV infection are low in frequency and paradoxically show a phenotype associated with controlled infections, expressing the memory marker CD127. We addressed to what extent this phenotype is dependent on the presence of cognate antigen. We analyzed virus-specific responses in acute and chronic HCV infections and sequenced autologous virus. We show that CD127 expression is associated with decreased antigenic stimulation after either viral clearance or viral variation. Our data indicate that most CD8 T-cell responses in chronic HCV infection do not target the circulating virus and that the appearance of HCV-specific CD127⁺ T cells is driven by viral variation.Hepatitis C virus (HCV) persists in the majority of acutely infected individuals, potentially leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The cellular immune response has been shown to play a significant role in viral control and protection from liver disease. Phenotypic and functional studies of virus-specific T cells have attempted to define the determinants of a successful versus an unsuccessful T-cell response in viral infections (10). So far these studies have failed to identify consistent distinguishing features between a T-cell response that results in self-limiting versus chronic HCV infection; similarly, the impact of viral persistence on HCV-specific memory T-cell formation is poorly understood.Interleukin-7 (IL-7) receptor alpha chain (CD127) is a key molecule associated with the maintenance of memory T-cell populations. Expression of CD127 on CD8 T cells is typically only observed when the respective antigen is controlled and in the presence of significant CD4⁺ T-cell help (9). Accordingly, cells specific for persistent viruses (e.g., HIV, cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) have been shown to express low levels of CD127 (6, 12, 14) and to be dependent on antigen restimulation for their maintenance. In contrast, T cells specific for acute resolving virus infections, such as influenza virus, respiratory syncytial virus (RSV), hepatitis B virus (HBV), and vaccinia virus typically acquire expression of CD127 rapidly with the control of viremia (5, 12, 14). Results for HCV have been inconclusive. The expected increase in CD127 levels in acute resolving but not acute persisting infection has been found, while a substantial proportion of cells with high CD127 expression have been observed in long-established chronic infection (2). We tried to reconcile these observations by studying both subjects with acute and chronic HCV infection and identified the presence of antigen as the determinant of CD127 expression.Using HLA-peptide multimers we analyzed CD8⁺ HCV-specific T-cell responses and CD127 expression levels in acute and chronic HCV infection. We assessed a cohort of 18 chronically infected subjects as well as 9 individuals with previously resolved infection. In addition, we longitudinally studied 9 acutely infected subjects (5 individuals who resolved infection spontaneously and 4 individuals who remain chronically infected) (Tables ​(Tables11 and ​and2).2). Informed consent in writing was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval from the local institutional review boards. HLA-multimeric complexes were obtained commercially from Proimmune (Oxford, United Kingdom) and Beckman Coulter (CA). The staining and analysis procedure was as described previously (10). Peripheral blood mononuclear cells (PBMCs) were stained with the following antibodies: CD3 from Caltag; CD8, CD27, CCR7, CD127, and CD38 from BD Pharmingen; and PD-1 (kindly provided by Gordon Freeman). Primer sets were designed for different genotypes based on alignments of all available sequences from the public HCV database (http://hcvpub.ibcp.fr). Sequence analysis was performed as previously described (8).

TABLE 1.

Patient information and autologous sequence analysis for patients with chronic and resolved HCV infection