Correlation between Environmental Factors and Prevalence of Vibrio parahaemolyticus in Oysters Harvested in the Southern Coastal Area of Sao Paulo State,Brazil

The presence of Vibrio parahaemolyticus in 123 oyster samples collected from an estuary on the southern coast of Sao Paulo state, Brazil, was investigated. Of the 123 samples, 99.2% were positive with densities ranging from <3 to 10⁵ most probable number (MPN)/g. Densities correlated significantly with water temperature (r = 0.48; P < 0.001) but not with salinity (r = −0.09; P = 0.34). The effect of harvest site on counts was not significant (P > 0.05). These data provide information for the assessment of exposure of V. parahaemolyticus in oysters at harvest.Infections caused by Vibrio parahaemolyticus have been reported in several countries (1, 3-5, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26). Among other pathogenic features, V. parahaemolyticus strains produce a thermostable hemolysin, known as thermostable direct hemolysin (TDH), as well as TRH (a TDH-related hemolysin) (25, 29). However, not all strains are pathogenic, as less than 1% of food or environmental strains produce TDH or TRH (2, 7, 9, 10, 11).The most important vehicle for this microorganism is raw or partially cooked shellfish (8, 13, 25, 29). In this study, the densities of V. parahaemolyticus in oysters collected in six oyster bed sites in the estuary of Cananeia (25°S; 48°W) in the southern coastal area of Sao Paulo state, Brazil (Fig. ​(Fig.1)1) between May 2004 and June 2005 were determined using the most probable number (MPN) technique by the method of De Paola and Kaysner (12). Each sample consisted of 15 oysters, pooled in a plastic bag, and transported in a cold box to the laboratory located in the city of Sao Paulo, Brazil. The temperature during transportation did not exceed 13°C, and the travel time was around 5 h. In the laboratory, the oysters were kept under refrigeration (4 to 8°C) and analyzed within 24 h of collection. Oysters were cleaned and shucked by the method of Cook et al. (6). Identification of V. parahaemolyticus was based on traditional and API 20E strip biochemical tests (bioMérieux, France), using V. parahaemolyticus ATCC 17802 as the reference strain. The observed prevalence of this bacterium was high, as the microorganism was detected in 99.2% (122/123) of the samples and the densities varied between 0.78 and 5.04 log MPN/g.Open in a separate windowFIG. 1.Locations of oyster bed sites in the Cananeia estuary on the southern coast of Sao Paulo state, Brazil. (Courtesy of E. E. de Miranda and A. C. Coutinho [Embrapa Monitoramento por Satélite] [http://www.cdbrasil.cnpm.embrapa.br].)Strategies for the control of V. parahaemolyticus in oysters depend on understanding the seasonal and geographical distribution and the effects of environmental parameters on the growth of this pathogen. To verify the influence of salinity and temperature of seawater on the density of V. parahaemolyticus, samples of water (n = 123) were collected from the same depth of oyster beds using 250-ml plastic flasks. Salinity was determined using a salinometer (model RS10; Rosemount Analytical, Cedar Grove, NJ), and the temperature was determined at the time of collection using a digital thermometer (Hanna Instruments). The results are shown in Fig. ​Fig.22.Open in a separate windowFIG. 2.Total densities of Vibrio parahaemolyticus in oysters from the southern coast of Sao Paulo state, Brazil. Each bar or point represents the arithmetic mean of six sites, and each error bar represents the standard deviation.Total V. parahaemolyticus densities did not correlate significantly with water salinity, as determined by Pearson coefficient (r = −0.09; P = 0.34). However, the mean salinity varied significantly according to the sampling site and season (P < 0.05) (Table ​(Table1).1). The highest mean salinity (24.2 ppt) was detected at site 5 and was 1.4 times higher than at site 2 (17.3 ppt), the lowest mean salinity detected in this study.

TABLE 1.

Seasonal distribution of the total density of Vibrio parahaemolyticus in oysters, water temperature, and salinity in the southern coastal area of Sao Paulo state, Brazil

Growth of Arthrobacter sp. Strain JBH1 on Nitroglycerin as the Sole Source of Carbon and Nitrogen

Arthrobacter sp. strain JBH1 was isolated from nitroglycerin-contaminated soil by selective enrichment. Detection of transient intermediates and simultaneous adaptation studies with potential intermediates indicated that the degradation pathway involves the conversion of nitroglycerin to glycerol via 1,2-dinitroglycerin and 1-mononitroglycerin, with concomitant release of nitrite. Glycerol then serves as the source of carbon and energy.Nitroglycerin (NG) is manufactured widely for use as an explosive and a pharmaceutical vasodilator. It has been found as a contaminant in soil and groundwater (7, 9). Due to NG''s health effects as well as its highly explosive nature, NG contamination in soils and groundwater poses a concern that requires remedial action (3). Natural attenuation and in situ bioremediation have been used for remediation in soils contaminated with certain other explosives (16), but the mineralization of NG in soil and groundwater has not been reported.To date, no pure cultures able to grow on NG as the sole carbon, energy, and nitrogen source have been isolated. Accashian et al. (1) observed growth associated with the degradation of NG under aerobic conditions by a mixed culture originating from activated sludge. The use of NG as a source of nitrogen has been studied in mixed and pure cultures during growth on alternative sources of carbon and energy (3, 9, 11, 20). Under such conditions, NG undergoes a sequential denitration pathway in which NG is transformed to 1,2-dinitroglycerin (1,2DNG) or 1,3DNG followed by 1-mononitroglycerin (1MNG) or 2MNG and then glycerol, under both aerobic and anaerobic conditions (3, 6, 9, 11, 20), and the enzymes involved in denitration have been characterized in some detail (4, 8, 15, 21). Pure cultures capable of completely denitrating NG as a source of nitrogen when provided additional sources of carbon include Bacillus thuringiensis/cereus and Enterobacter agglomerans (11) and a Rhodococcus species (8, 9). Cultures capable of incomplete denitration to MNG in the presence of additional carbon sources were identified as Pseudomonas putida, Pseudomonas fluorescens (4), an Arthobacter species, a Klebsiella species (8, 9), and Agrobacterium radiobacter (20).Here we describe the isolation of bacteria able to degrade NG as the sole source of carbon, nitrogen, and energy. The inoculum for selective enrichment was soil historically contaminated with NG obtained at a facility that formerly manufactured explosives located in the northeastern United States. The enrichment medium consisted of minimal medium prepared as previously described (17) supplemented with NG (0.26 mM), which was synthesized as previously described (18). During enrichment, samples of the inoculum (optical density at 600 nm [OD₆₀₀] ∼ 0.03) were diluted 1/16 in fresh enrichment medium every 2 to 3 weeks. Isolates were obtained by dilution to extinction in NG-supplemented minimal medium. Cultures were grown under aerobic conditions in minimal medium at pH 7.2 and 23°C or in tryptic soy agar (TSA; 1/4 strength).Early stages of enrichment cultures required extended incubation with lag phases of over 200 h and exhibited slow degradation of NG (less than 1 μmol substrate/mg protein/h). After a number of transfers over 8 months, the degradation rates increased substantially (2.2 μmol substrate/mg protein/h). A pure culture capable of growth on NG was identified based on 16S rRNA gene analysis (504 bp) as an Arthrobacter species with 99.5% similarity to Arthrobacter pascens (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU246730","term_id":"289474572","term_text":"GU246730"}}GU246730). Purity of the cultures was confirmed microscopically and by formation of a single colony type on TSA plates. 16S gene sequencing and identification were done by MIDI Labs (Newark, DE) and SeqWright DNA Technology Services (Houston, TX). The Arthrobacter cells stained primarily as Gram-negative rods with a small number of Gram-positive cocci (data not shown); Gram variability is also a characteristic of the closely related Arthrobacter globiformis (2, 19). The optimum growth temperature is 30°C, and the optimum pH is 7.2. Higher pH values were not investigated because NG begins to undergo hydrolysis above pH 7.5 (data not shown). The isolated culture can grow on glycerol, acetate, succinate, citrate, and lactate, with nitrite as the nitrogen source. Previous authors described an Arthrobacter species able to use NG as a nitrogen source in the presence of additional sources of carbon. However, only dinitroesters were formed, and complete mineralization was not achieved (9).To determine the degradation pathway, cultures of the isolated strain (5 ml of inoculum grown on NG to an OD₆₀₀ of 0.3) were grown in minimal medium (100 ml) supplemented with NG at a final concentration of 0.27 mM. Inoculated bottles and abiotic controls were continuously mixed, and NG, 1,2DNG, 1,3DNG, 1MNG, 2MNG, nitrite, nitrate, CO₂, total protein, and optical density were measured at appropriate intervals. Nitroesters were analyzed with an Agilent high-performance liquid chromatograph (HPLC) equipped with an LC-18 column (250 by 4.6 mm, 5 μm; Supelco) and a UV detector at a wavelength of 214 nm (13). Methanol-water (50%, vol/vol) was used as the mobile phase at a flow rate of 1 ml/min. Nitrite and nitrate were analyzed with an ion chromatograph (IC) equipped with an IonPac AS14A anion-exchange column (Dionex, CA) at a flow rate of 1 ml/min. Carbon dioxide production was measured with a Micro Oxymax respirometer (Columbus Instruments, OH), and total protein was quantified using the Micro BCA protein assay kit (Pierce Biotechnology, IL) according to manufacturer''s instructions. During the degradation of NG the 1,2DNG concentration was relatively high at 46 and 72 h (Fig. ​(Fig.1).1). 1,3DNG, detected only at time zero, resulted from trace impurities in the NG stock solution. Trace amounts of 1MNG appeared transiently, and trace amounts of 2MNG accumulated and did not disappear. Traces of nitrite at time zero were from the inoculum. The concentration of NG in the abiotic control did not change during the experiment (data not shown).Open in a separate windowFIG. 1.Growth of strain JBH1 on NG. ×, NG; ▵, 1,2DNG; ⋄, 1MNG; □, 2MNG; ○, protein.Results from the experiment described above were used to calculate nitrogen and carbon mass balances (Tables ​(Tables11 and ​and2).2). Nitrogen content in protein was approximated using the formula C₅H₇O₂N (14). Because all nitrogen was accounted for throughout, we conclude that the only nitrogen-containing intermediate compounds are 1,2DNG and 1MNG, which is consistent with previous studies (6, 9, 20). The fact that most of the nitrogen was released as nitrite is consistent with previous reports of denitration catalyzed by reductase enzymes (4, 8, 21). The minor amounts of nitrate observed could be from abiotic hydrolysis (5, 12) or from oxidation of nitrite. Cultures supplemented with glycerol or other carbon sources assimilated all of the nitrite (data not shown).

TABLE 1.

Nitrogen mass balance

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

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

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

Structural Characterization of the Dual Glycan Binding Adeno-Associated Virus Serotype 6

The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.Adeno-associated viruses (AAVs) are nonpathogenic single-stranded DNA (ssDNA) parvoviruses that belong to the Dependovirus genus and require helper viruses, such as Adenovirus or Herpesvirus, for lytic infection (4, 8, 22, 67). These viruses package a genome of ∼4.7 kb inside an icosahedral capsid (∼260 Å in diameter) with a triangulation number equal to 1 assembled from a total of 60 copies of their overlapping capsid viral protein (VP) 1 (VP1), VP2, and VP3 in a predicted ratio of 1:1:8/10 (10). The VPs are encoded from a cap open reading frame (ORF). VP3 is 61 kDa and constitutes 90% of the capsid''s protein composition. The less abundant VPs, VP1 (87 kDa) and VP2 (73 kDa), share the same C-terminal amino acid sequence with VP3 but have additional N-terminal sequences. A rep ORF codes for four overlapping proteins required for replication and DNA packaging.To date, more than 100 AAV isolates have been identified (21). Among the human and nonhuman primate AAVs isolated, 12 serotypes (AAV serotype 1 [AAV1] to AAV12) have been described and are classified into six phylogenetic clades on the basis of their VP sequences and antigenic reactivities, with AAV4 and AAV5 considered to be clonal isolates (21). AAV1 and AAV6, which represent clade A, differ by only 6 out of 736 VP1 amino acids (5 amino acids within VP3) and are antigenically cross-reactive. Other clade representatives include AAV2 (clade B), AAV2-AAV3 hybrid (clade C), AAV7 (clade D), AAV8 (clade E), and AAV9 (clade F) (21).The AAVs are under development as clinical gene delivery vectors (e.g., see references 5, 9, 12, 13, 24, 25, 53, and 61), with AAV2, the prototype member of the genus, being the most extensively studied serotype for this application. AAV2 has been successfully used to treat several disorders, but its broad tissue tropism makes it less effective for tissue-specific applications and the prevalence of preexisting neutralizing antibodies in the human population (11, 43) limits its utilization, especially when readministration is required to achieve a therapeutic outcome. Efforts have thus focused on characterizing the capsid-associated tissue tropism and transduction properties conferred by the capsid of representative serotypes of other clades (21). Outcomes of these studies include the observation that AAV1 and AAV6, for example, transduce liver, muscle, and airway epithelial cells more efficiently (e.g., up to 200-fold) than AAV2 (27, 28, 30). In addition, the six residues (Table ​(Table1)1) that differ between the VPs of AAV1 and AAV6 (a natural recombinant of AAV1 and AAV2 [56]) confer functional disparity between these two viruses. For example, AAV6 shows ∼3-fold higher lung cell epithelium transduction than AAV1 (27), and AAV1 and AAV6 bind terminally sialylated proteoglycans as their primary receptor, whereas AAV6 additionally binds to heparan sulfate (HS) proteoglycans with moderate affinity (70, 71). Therefore, a comparison of the AAV1 and AAV6 serotypes and, in particular, their capsid structures can help pinpoint the capsid regions that confer differences in cellular recognition and tissue transduction.

TABLE 1.

Amino acid differences between AAV1 and AAV6 and their reported mutants

Fitness Effects of Replichore Imbalance in Salmonella enterica

A fitness cost due to imbalanced replichores has been proposed to provoke chromosome rearrangements in Salmonella enterica serovars. To determine the impact of replichore imbalance on fitness, the relative fitness of isogenic Salmonella strains containing transposon-held duplications of various sizes and at various chromosomal locations was determined. Although duplication of certain genes influenced fitness, a replichore imbalance of up to 16° did not affect fitness.The bacterial chromosome is a dynamic molecule that can undergo various types of rearrangements, including inversions, translocations, and duplications. These rearrangements can alter gene order and change replichore length. Replichores are defined as the halves of the chromosome between the origin of replication and the terminus region in the vicinity of the dif site (4, 6, 10, 15). In most bacteria, both replichores are approximately the same length, with each comprising 180° around the circular chromosome. In this state, the replichores and DNA replication are balanced. Imbalance is introduced when one replichore becomes longer than the other. For example, if the replichores comprise 200° and 160° around the circular chromosome, the replichores are 20° imbalanced. Various studies over the years have investigated the effect of imbalanced DNA replication on fitness in Escherichia coli (8, 9, 16, 17). In a recent analysis, E. coli strains that contained asymmetrical interreplichore inversions were found to have growth defects when the imbalance was at least 50° (8). While these studies have demonstrated that imbalanced replichores can affect fitness, the approaches used to introduce the imbalance either do not occur or are extremely rare in nature.Duplications play important evolutionary roles because the increase in gene copy number can facilitate adaptation to certain growth conditions (20), as well as being a source for the evolution of new genes (3). Typically duplications occur at frequencies between 10⁻³ and 10⁻⁵ in unselected cultures (2) but are lost at rates of up to 1,000-fold more often if they do not provide a selective advantage (19). Duplications also result in replichore imbalance. However, the effect of duplications on fitness in relation to replichore balance has not been investigated.A major hurdle in studying the effects of duplications on fitness is that if the duplication is detrimental, haploid revertants will outgrow the parental strain containing the duplication. To circumvent this problem, we used a set of 11 isogenic Salmonella enterica strains with transposon-held duplications (5) (Fig. ​(Fig.11 and Table ​Table1).1). Culturing these strains in the presence of chloramphenicol selects for the duplication because if the duplication collapses, the transposon is lost and the cells become chloramphenicol sensitive. As the size of the duplications in these strains varies, so does the amount of introduced replichore imbalance, ranging from 5° to 23°. In addition, fitness effects due to the location of the duplication versus the size of the duplication were also discerned, as the collection includes duplications of similar sizes located in different regions of the chromosome.Open in a separate windowFIG. 1.Genetic map of S. enterica serovar Typhimurium LT2 showing genes used as endpoints in constructing transposon-held duplications of the regions between genes. Balanced replichores are indicated by the symmetry of the oriC-Ter axis. Duplications increase the length of one replichore relative to the other, imbalancing axis symmetry.

TABLE 1.

Properties of the S. enterica strains used in this study

Effect of Growth Phase Feeding Strategies on Succinate Production by Metabolically Engineered Escherichia coli

Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by Escherichia coli AFP111 with knockouts in rpoS, pflAB, ldhA, and ptsG genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.Succinic acid is valued as one of the key basic chemicals used in the preparation of biodegradable polymers or as raw material for chemicals of the C₄ family (8, 19). The fermentative production of succinic acid from renewable resources is environmentally acceptable and sustainable (3). A breakthrough in genetically engineering Escherichia coli (6, 7, 11, 18) for succinate production was the isolation of strain AFP111 (1, 4), a mutant of NZN111 with a spontaneous ptsG mutation (pflAB ldhA double mutant). The process involves a two-stage fermentation, with aerobic cell growth followed by anaerobic conditions for succinate production (16, 21, 22). The aerobically induced enzymes can maintain their activity during the anaerobic phase and significantly affect succinate fermentation (22, 23). Using the best transition time based on the activities of the key enzymes and other physiological states, a two-stage fermentation using the recombinant AFP111 strain harboring pTrc99A-pyc achieved a final succinic acid concentration and productivity of 99.2 g·liter⁻¹ and 1.3 g·liter⁻¹·h⁻¹, respectively (21).Aerobic cell growth is essential for the subsequent anaerobic fermentation. However, few studies have focused on the regulation of aerobic cell growth. As a regulation method, gluconeogenic carbon sources were used instead of glucose for the aerobic growth of Escherichia coli NZN111 and the activities of enzymes that are favorable for the anaerobic synthesis of succinate were enhanced (23, 24). Unfortunately, a gluconeogenic carbon source (e.g., sodium acetate) might increase the osmotic pressure of culture media, which would be detrimental to succinate production (23). As another regulation method, a glucose feeding strategy controlling the glucose concentration at about 0.5 g·liter⁻¹ up to 1 g·liter⁻¹ was reported to prevent excessive formation of acetic acid (16).In this study, we investigated different glucose feeding strategies for the aerobic growth phase of the two-phase process for succinate production by E. coli AFP111. Specifically, we compared several growth rates by using glucose limitation in addition to maximum growth under conditions of excess glucose.E. coli AFP111 [F⁺ λ⁻ rpoS396(Am) rph-1 ΔpflAB::Cam ldhA::Kan ptsG] (4, 16), which was a kind gift from D. P. Clark (Southern Illinois University), was the only strain used in this study. Luria-Bertani (LB) medium (60 ml) was used for inoculum culture in 1,000-ml flasks, and 3 liters of chemically defined medium (13, 14) was used for two-stage culture in a 7-liter fermentor. Two-stage fermentations were divided into three types, based on the glucose feeding strategy used during the aerobic stage. For type I culture, the glucose concentration was maintained at about 20 g·liter⁻¹ during aerobic cell growth. Type II and III cultures comprised a batch process and subsequent glucose-limited fed-batch process (Fig. ​(Fig.1).1). The batch process initially contained 13 g/liter of glucose. The fed-batch process began when the dry cell weight (DCW) reached about 6 g/liter, with type II and type III cultures using a 600 g/liter glucose feed to achieve cell growth rates of 0.15 h⁻¹ and 0.07 h⁻¹, respectively (10). When the DCW reached 12 g·liter⁻¹, the aerobically grown cells were directly transferred to anaerobic conditions (Fig. ​(Fig.1).1). For the anaerobic process, oxygen-free CO₂ was sparged at 0.5 liter·min⁻¹, the pH was controlled between 6.4 and 6.8 with intermittent supplementation of solid magnesium carbonate hydroxide, and the glucose concentration was maintained at about 20 g·liter⁻¹ by supplying glucose in an 800-g·liter⁻¹ solution.Open in a separate windowFIG. 1.Concentrations of glucose (circles), DCW (triangles), and succinic acid (squares) in the three types of two-stage fermentation by AFP111. μ, growth rate.The optical density at 600 nm was used to monitor cell growth, and this value was correlated to DCW. The concentration of glucose was assayed with an enzyme electrode analyzer, and organic acids were quantified by high-performance liquid chromatography (HPLC). The intracellular concentrations of NADH and NAD⁺ were assayed with a cycling method (12). The activities of isocitrate lyase (ICL) (20), pyruvate kinase (PYK) (17), phosphoenolpyruvate (PEP) carboxykinase (PCK) (20, 23), PEP carboxylase (PPC) (23), and malate dehydrogenase (MDH) (23) were measured spectrophotometrically at the end of the aerobic phase and 12 h after the onset of the anaerobic phase.All three types of fermentations were terminated when the succinate concentration increased less than 1 g·liter⁻¹ in 5 h. Type III fermentation was terminated at a final succinic acid concentration of 101.2 g·liter⁻¹ and an anaerobic-phase productivity of 1.89 g·liter⁻¹·h⁻¹ (Fig. ​(Fig.1).1). Trace amounts of by-products (such as acetate, ethanol, and pyruvate) accumulated and did not follow any trend in the anaerobic phase (data not shown).At the end of the aerobic culture phase, the specific enzyme activities of PCK, PYK, and ICL in type III culture were 2.9, 2.5, and 11.4 times higher, respectively, than the activities in type I culture (Table ​(Table1)1) . This phenomenon is consistent with published reports that suggest that the expression of enzymes involved in anaplerotic metabolism and the glyoxylate shunt (5, 15) is elevated in E. coli grown under glucose-limited conditions. These enzymes maintained their activities in the subsequent anaerobic phase (Table ​(Table1)1) and would be central to succinate production (22, 23). The elevated levels of PCK and PPC would provide the reductive branch of the tricarboxylic acid (TCA) cycle with oxaloacetate (OAA) at a higher rate (9), thereby supplying both malate and citrate (Table ​(Table11).

TABLE 1.

Activities of enzymes at the end of the aerobic culture phase and 12 h after the onset of the anaerobic phase

Nonpathogenic Simian Immunodeficiency Virus Infection of Sooty Mangabeys Is Not Associated with High Levels of Autologous Neutralizing Antibodies

Simian immunodeficiency virus (SIV) infection of natural-host species, such as sooty mangabeys (SMs), is characterized by a high level of viral replication and a low level of generalized immune activation, despite evidence of an adaptive immune response. Here the ability of SIV-infected SMs to mount neutralizing antibodies (Nab) against autologous virus was compared to that of human immunodeficiency virus type 1 (HIV-1) subtype C-infected subjects. While high levels of Nab were observed in HIV-1 infection, samples obtained at comparable time points from SM exhibited relatively low titers of autologous Nab. Nevertheless, SM plasma with higher Nab titers also contained elevated peripheral CD4⁺ T-cell levels, suggesting a potential immunologic benefit for SMs. These data indicate that AIDS resistance in these primates is not due to high Nab titers and raise the possibility that low levels of Nab might be an inherent feature of natural-host SIV infections.More than 40 species of African nonhuman primates (NHPs) naturally harbor CD4⁺-tropic lentiviruses that are collectively known as simian immunodeficiency viruses (SIVs) and represent the ancestors of the human pathogens human immunodeficiency virus type 1 (HIV-1) and HIV-2. Interestingly, African NHPs infected with their cognate SIV generally do not progress to AIDS, despite high levels of sustained virus replication, with the only known exception being chimpanzee SIV (SIVcpz)-infected chimpanzees (16). Among the natural hosts for SIV infection, the sooty mangabey ([SM] Cercocebus atys) is of particular interest, because cross-species transmission of SM SIV (SIVsm) from this natural host into humans initiated the HIV-2 epidemic in West Africa (17). In addition, SIVsm (herein referred to as SIV) is the ancestor of the rhesus macaque SIV (SIVmac) viruses that are used in disease pathogenesis and vaccination studies in the rhesus macaque model (17). Both naturally infected and experimentally inoculated SMs remain healthy, maintain CD4⁺ T cells, and do not progress to AIDS-like disease, despite sustained high levels of virus replication (31).Nonpathogenic infection of SMs is characterized by low levels of immune activation during the chronic phase of infection, which are reached after a transient immune activation that occurs during primary infection (reviewed in reference 31). These findings have led to the hypothesis that the absence of generalized immune activation in SIV-infected SMs during the chronic phase of infection is an important feature that favors the preservation of CD4⁺ T-cell homeostasis, thereby avoiding disease progression (31). However, most of these earlier studies focused on T cells and innate immune cells, with a significant gap existing in our understanding of whether humoral immunity might also differ between pathogenic and nonpathogenic infections. In HIV-1-infected patients, B cells produce neutralizing antibodies against the infecting (autologous) virus, which drives viral escape, continuous de novo antibody production (26-28, 32), and B-cell dysfunction (24). The striking differences in both the clinical outcomes of infection and the levels of immune activation between SIV-infected SMs and HIV-1-infected humans prompted us to compare the neutralizing antibody (Nab) response against the autologous virus in these two populations. To this end, we utilized a pseudovirus assay that has been used extensively by our group and others to evaluate Nab against HIV-1 and SIV envelope (Env) glycoproteins (15, 19, 22, 26, 28, 32, 33; also unpublished data). All SMs were housed at the Yerkes National Primate Research Center (Atlanta, GA) and maintained in accordance with National Institutes of Health guidelines. The Emory University Animal Care and Use Committee approved these studies. Details of the Zambia Emory HIV Research Project (ZEHRP) have been described elsewhere (2, 10, 21). The Emory University Institutional Review Board and the University of Zambia School of Medicine Research Ethics Committee approved informed-consent and human subject protocols. None of the subjects received antiretroviral therapy during the evaluation period.In HIV-1 infection, autologous Nabs develop to relatively high titers against the newly transmitted virus within the first few months (15, 19, 26-28, 32). Here we sought to test whether a similar increase in Nab titer occurs during nonpathogenic SIV infection of SMs. Samples were obtained from five animals that were inoculated intravenously with plasma from a naturally infected SM as part of a previous study (30). Multiple, biologically functional Envs were cloned from plasma collected at day 14 postinoculation (Table ​(Table1),1), and Nab activity was evaluated in plasma collected at 6 months postinoculation. To facilitate comparison with early HIV-1 infection, Nab activity in plasma was also evaluated between 2 and 9 months against Envs that were cloned between 31 and 88 estimated days after infection from four subtype C HIV-1-infected seroconverters in Zambia (Table ​(Table1).1). Figure ​Figure1A1A demonstrates that Nab activity in plasma diluted 1:100 was readily detectable in all HIV-1-infected subjects at levels approaching 100% neutralization. However, Nab activity in the SM plasma was significantly lower than in the human subjects (median, 10% versus 93%, respectively; P = 0.02). Binding antibody was detected in all five SMs at titers greater than 1:51,200 by enzyme-linked immunosorbent assay (ELISA), demonstrating that all monkeys had seroconverted by 6 months and maintained high titers of binding antibody throughout the evaluation period (Fig. ​(Fig.1B).1B). Thus, the low level of Nab was not due to a diminished humoral immune response.Open in a separate windowFIG. 1.Autologous Nab activity and B-cell proliferation during experimental infection of SMs. (A) Neutralization activity levels in plasma from five SMs (filled black circles), which were experimentally inoculated with plasma from a naturally SIV-infected SM, and four HIV-1-infected Zambian subjects (half-filled squares), who were recently infected through heterosexual contact, are shown. The horizontal bars represent the median for each group. To assess neutralizing activity, pseudoviruses were created by expressing each cloned Env with an HIV-1 env-deficient backbone (ΔSG3). JC53-BL (Tzm-bl) cells were infected with each pseudovirus in the presence or absence of serially diluted autologous plasma. Each point represents the average level of neutralization at a 1:100 dilution of plasma for at least two Env clones (see Table ​Table11 for number of Envs tested). Each neutralization assay was performed twice independently, using duplicate wells. Statistical significance between the groups was determined by a Mann-Whitney test, using GraphPad Prism 5. Longitudinal measurements of endpoint antibody ELISA titers in plasma (filled green circles) (23) (B), autologous neutralization activity in plasma (filled blue diamonds) (C), percentages of Ki-67⁺ CD20⁺ cells in blood (filled black triangles) (D), and percentages of CD20⁺ cells in blood (filled red squares) (E) are shown for the five experimentally inoculated SMs combined. In panel C, each point represents average neutralization at a 1:100 dilution of plasma over time for at least two day 14 Env clones from each SM. For panels D and E, PBMCs were gated by forward and side scatter, and the CD3⁻ CD20⁺ population was assessed for Ki-67 staining (D) by flow cytometry. SP34-2 was used to stain CD3, L27 was used for CD20, and B56 was used for Ki-67 (all from BD Biosciences). Error bars represent the standard errors of the means (SEMs). Plasma viral load peaked at day 14 (data not shown). Filled symbols in panels A through E indicate data generated from experimentally infected SMs.

TABLE 1.

Autologous Nab activity in experimentally SIV-infected SM and acutely HIV-1-infected humans

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