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

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

Improved Molecular Detection of Angiostrongylus cantonensis in Mollusks and Other Environmental Samples with a Species-Specific Internal Transcribed Spacer 1-Based TaqMan Assay

Angiostrongylus cantonensis is the most common cause of human eosinophilic meningitis. Humans become infected by ingesting food items contaminated with third-stage larvae that develop in mollusks. We report the development of a real-time PCR assay for the species-specific identification of A. cantonensis in mollusk tissue.Angiostrongylus cantonensis is the most common agent associated with eosinophilic meningitis in humans. Young adult worms develop in the brains of rodents and are carried to pulmonary arteries to reach sexual maturity. Eggs are laid in lung tissues, and first-stage (L1) larvae break into air spaces, migrate to the trachea, are swallowed, and are passed with rodent feces. The L1 larvae must infect mollusks to develop into third-stage (L3) larvae; L3 is the infective stage for rodents and other mammals. Humans become infected by ingesting raw produce contaminated with L3 larvae or infected raw or undercooked mollusks or paratenic hosts. The immature worms remain in the human brain, creating tissue damage and inflammation (2, 19, 21).A. cantonensis is endemic in Southeast Asia, parts of the Caribbean, and the Pacific Islands, including Hawaii (7, 12, 15-17). The worm has been detected in host animals in Louisiana (5, 14) and in one human patient from New Orleans (18), but it is currently unclear to what extent the nematode has spread into other U.S. states (8, 9). Ascertaining the geographic presence of the parasite is important to manage and prevent new cases of eosinophilic meningitis associated with ingestion of infective larvae (12, 18).Detection of A. cantonensis in mollusks can be performed by releasing the larvae from the tissue with pepsin digestion (11). However, that procedure requires access to living mollusks, which complicates analysis of large numbers of samples. After a recent outbreak of angiostrongyliasis in Hawaii (12), we developed a conventional PCR assay and applied it to survey the Hawaiian mollusk population using frozen tissue (20). That PCR assay, as well as morphological identification using pepsin digestion, can only identify the larvae on the superfamily level, so additional molecular work is required for species-specific classification. Here we describe a new real-time PCR assay that allows for a direct detection of A. cantonensis at the species level.The 18S rRNA gene is too conserved among nematode species to allow species-specific detection. The first and second internal transcribed spacers (ITS1 and ITS2) are comparatively more variable than the rRNA coding regions and have thus been used for differentiation of closely related species (1, 4, 6, 10, 22, 23). We PCR amplified and sequenced ITS1 from A. costaricensis (two laboratory strains from Costa Rica and Brazil), A. vasorum (from naturally infected hosts in United Kingdom), and A. cantonensis from three geographical regions (one laboratory strain from Japan plus nine environmental isolates from Hawaii and New Orleans, LA) to assess the variability of this potential PCR target. The oligonucleotide primers used were AngioF1674 (5′-GTCGTAACAAGGTATCTGTAGGTG-3′) and 58SR4 (5′-TAGCTGCGTTTTTCATCGATA-3′). The reaction mixtures contained 0.4 μM each primer and AmpliTaq Gold PCR master mix (Applied Biosystems, Foster City, CA) and were cycled 45 times at 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min. PCR products were cloned into pCR2.1 vectors using the TOPO cloning technique (Invitrogen, Carlsbad, CA) and sequenced on both strands as described elsewhere (20).The sequence analysis revealed high interspecific and low intraspecific variability. A TaqMan assay targeting ITS1 was then designed using Primer Express version 2.3 (Applied Biosystems, Foster City, CA). The real-time PCR assay was performed in a 20-μl total volume containing Platinum qPCR Supermix (Invitrogen, Carlsbad, CA), 0.2 μM (each) primers AcanITS1F1 (5′-TTCATGGATGGCGAACTGATAG-3′) and AcanITS1R1 (5′-GCGCCCATTGAAACATTATACTT-3′), and 0.05 μM the TaqMan probe AcanITS1P1 (5′-6-carboxyfluorescein-ATCGCATATCTACTATACGCATGTGACACCTG-BHQ-3′). The standard cycling conditions for TaqMan assays were used (i.e., 40 cycles of 95°C for 15 s and 60°C for 1 min).We evaluated the real-time PCR assay with a set of 26 Parmarion martensi slugs from Hawaii. Seventeen slugs were positive for L3 larvae as determined by pepsin digestion, and nine slugs were negative. DNA was extracted from approximately 25 mg of tissue of each slug using the DNeasy tissue and blood DNA extraction kit (Qiagen, Inc., Valencia, CA). The real-time PCR performed on this set of samples returned an identical result to the morphological analysis. The real-time PCR amplified only DNA from A. cantonensis and did not react with DNA from other nematode species (Table ​(Table1).1). The detection limit of the assay was determined by serially diluting a recombinant plasmid containing the ITS1 sequence to less than 1 copy per μl of sample. The real-time PCR reliably detected down to 10 plasmid copies in the reaction.

TABLE 1.

Comparison of conventional and real-time PCR for detection of Angiostrongylus cantonensis in mollusks and nematode samples

Diverse Genetic Markers Concordantly Identify Bovine Origin Escherichia coli O157 Genotypes Underrepresented in Human Disease

Genetic markers previously reported to occur at significantly different frequencies in isolates of Escherichia coli O157:H7 obtained from cattle and from clinically affected humans concordantly delineate at least five genetic groups. Isolates in three of these groups consistently carry one or more markers rarely found among clinical isolates.Escherichia coli serotype O157:H7 is an important zoonotic pathogen that may cause diarrhea, bloody diarrhea, and hemolytic-uremic syndrome (3, 10, 14). E. coli O157 is transmitted to humans by direct contact with the animal reservoir (which includes cattle and other ruminant animals) or indirectly by ingestion of contaminated food or water (3, 10). Genetic analyses of bovine isolates of E. coli O157 from diverse geographic origins have provided evidence for the global dissemination of genotypes and also for significant regional differences in the relative prevalence of some genotypes (5, 8, 18, 19).Several research groups have identified genetic markers that occur at different relative frequencies among E. coli O157 isolates from human clinical cases and from cattle. One group initially used octamer-based genomic scanning to identify two lineages of U.S. origin E. coli O157 (7), of which lineage I was composed mostly (36/44) of clinical isolates and lineage II was composed mostly (25/32) of cattle isolates. Subsequently, a simpler multiplex PCR-based assay (Table ​(Table1),1), the lineage-specific polymorphism assay (LSPA), was developed to indentify these lineages (19). Six LSPA loci with alleles characteristic of lineage I, lineage II, or neither lineage I nor lineage II are, respectively, classified with the digit 1, 2, or 3, and these digits are concatenated to an LSPA code: 111111 indicates lineage I, and 211111 indicates a genetically intermediate group termed lineage I/II (20), whereas all other genotype variations are considered to belong to lineage II. More recently, a typing assay based on Shiga toxin-encoding bacteriophage insertion (SBI) sites grouped 91 of 92 clinical E. coli O157 isolates from the northwestern United States into three clusters, of which clusters 1 and 3 predominated (>90%) (16). SBI consists of six PCRs (Table ​(Table1)1) that amplify the Stx toxin genes and the insertion site junctions of the Stx1- and Stx2-encoding bacteriophages of E. coli O157. In a subsequent study, the predominance (92.6%) of clusters 1 and 3 was confirmed in 190 additional human (clinical) isolates (1). In contrast, many (48.8%) E. coli O157 isolates from cattle in the northwestern United States and western Canada demonstrated SBI patterns rarely found among human (clinical) isolates (1).

TABLE 1.

Oligonucleotides used in this study

Prevalence of Escherichia coli O157:H7 in Organically and Naturally Raised Beef Cattle

We determined the prevalence of Escherichia coli O157:H7 in organically and naturally raised beef cattle at slaughter and compared antibiotic susceptibility profiles of the isolates to those of isolates from conventionally raised beef cattle. The prevalences of E. coli O157:H7 were 14.8 and 14.2% for organically and naturally raised cattle, respectively. No major difference in antibiotic susceptibility patterns among the isolates was observed.Many cattle producers have adopted production methods termed niche marketing to meet consumer demand for safe and healthy beef. The two main niches for beef cattle producers are organic and natural production (3). Organic beef cattle production, regulated by the U.S. Department of Agriculture, requires feeding with certified organic feed (16) and raising cattle without the use of antibiotics, hormones, and other veterinary products (3). Guidelines for producers to label the product as “natural” differ among natural beef programs, and such programs are administered and regulated by the company or organization that owns the brand name rather than the U.S. Department of Agriculture (11). Natural production guidelines often include a complete restriction on the use of antibiotics and growth-promoting hormones, but unlike guidelines for organic production, they allow feed from nonorganic sources (11). Escherichia coli O157:H7 is a major food-borne pathogen that causes outbreaks of hemorrhagic enteritis, which often leads to hemolytic uremic syndrome in children and the elderly (10). Cattle are major reservoirs of E. coli O157:H7, which colonizes the hindgut, specifically the rectoanal mucosal region. Cattle feces are the major source of food and water contamination (10). The impact of organic production methods on the prevalence of food-borne pathogens, including E. coli O157:H7 and Campylobacter spp. in dairy cattle (7, 14) and Campylobacter and Salmonella spp. in chickens (6, 19), has been studied previously. However, there is no published study on the prevalence of E. coli O157:H7 in organically and naturally raised beef cattle. Additionally, nothing is known regarding the effects of organic and natural production methods on the antibiotic susceptibilities of E. coli O157:H7 in beef cattle. Our objectives were to determine the prevalence of E. coli O157:H7 in the feces of organically and naturally raised beef cattle at slaughter and compare the antibiotic susceptibilities of isolates from organically, naturally, and conventionally raised beef cattle.Cattle included in this study were from three types of production systems, organic, natural, and conventional. Organically raised beef cattle were from farms that were certified by the National Organic Program (17). The naturally raised beef cattle were from farms that were certified by the All Natural Source Verified Beef Program (17). The collection of samples from these cattle occurred in an abattoir. Samples from conventionally raised cattle from two feedlots were collected in a different abattoir so that the antibiotic susceptibilities of their isolates could be compared with those of isolates from organically and naturally raised cattle. Fecal samples were obtained by cutting open the rectum and spooning out the contents. The mucosa of the rectum was then rinsed with water until free of visible fecal material and swabbed with a sterile foam-tipped applicator (4). The isolation and identification of E. coli O157 and PCR detection of major virulence genes (eae, stx₁, stx₂, hlyA, and fliC) were carried out as described by Reinstein et al. (13). A subset of 60 isolates, 20 (10 from fecal samples and 10 from rectoanal mucosal swabs [RAMS]) from each production system, was randomly chosen to determine the antibiotic susceptibility patterns by the broth microdilution method (9). The antibiotics (all from Sigma-Aldrich) tested were amikacin, amoxicillin (amoxicilline), ampicillin, apramycin, bacitracin, cefoxitin, ceftazidime, ceftriaxone, cephalothin (cefalotin), chloramphenicol, chlortetracycline, ciprofloxacin, enrofloxacin, erythromycin, florfenicol, gentamicin, kanamycin, lincomycin, monensin, nalidixic acid, neomycin, norfloxacin, novobiocin, oxytetracycline, penicillin, rifampin (rifampicin), spectinomycin, streptomycin, tetracycline, tilmicosin, trimethoprim, tylosin, and vancomycin. The MIC was defined as the lowest concentration of an antibiotic that prevented visible growth of the organism. Each concentration of the antibiotic compound was duplicated in the microtiter plate, and the MIC determination was repeated with a different inoculum preparation. Logistic regression was performed using the PROC GENMOD procedure in the SAS system (SAS Institute, Cary, NC) to compare the prevalences of E. coli O157:H7 (with binomial distribution of outcomes) in fecal samples, RAMS samples, and fecal or RAMS samples (overall animal level prevalence). The MICs of antibiotics for E. coli O157:H7 isolates were analyzed using a nonparametric survival test in the PROC LIFETEST program of SAS to determine the effects of the production system (natural, organic, or conventional). Data were right censored when necessary (when the organism was resistant to the highest concentration evaluated). The Wilcoxon test was utilized to determine the effect of the production system on MICs.Samples from a total of 553, 506, and 322 organically, naturally, and conventionally raised cattle, respectively, were collected. In organically raised cattle, the prevalence of E. coli O157:H7 in fecal samples ranged from 0 to 24.4% across sampling days, with an average of 9.3%, and the prevalence in RAMS ranged from 0 to 30.9%, with an average of 8.7% (Fig. ​(Fig.1).1). In naturally raised cattle, the prevalence of E. coli O157:H7 in fecal samples ranged from 0 to 20.3%, with an average of 7.2%, and the prevalence in RAMS ranged from 0 to 23.8%, with an average of 8.9% (Fig. ​(Fig.1).1). In both organically and naturally raised cattle, the prevalence (total) detected by both sampling methods together was greater (P < 0.05) than the prevalence detected by either method alone (Fig. ​(Fig.1).1). Samples (either feces or RAMS) from 36 (11.2%) of 322 conventionally raised feedlot cattle were culture positive for E. coli O157:H7. The fecal prevalence of E. coli O157:H7 was 6.5%, and the prevalence determined by the RAMS sampling method was 7.1%. Most isolates (66.7% from organically raised beef cattle and 77.8% from naturally raised beef cattle) were positive for eae, stx₂, hlyA, and fliC but negative for stx₁. The stx₂ gene was present in 100 and 95% of isolates from organically and naturally raised cattle, respectively. The prevalences of E. coli O157:H7 that we observed in organically and naturally raised beef cattle were similar to the previously reported prevalence in conventionally raised cattle (1). Our study did not include a statistical comparison of the prevalence data because of a number of differences, particularly in diet, among the organic, natural, and conventional production systems. Organically and naturally raised cattle are either required to graze a pasture or fed a forage-based diet. Although conflicting data exist (1), studies have shown that cattle fed a forage diet have both higher levels and longer durations of fecal shedding of E. coli O157:H7 than cattle fed a grain diet (18).Open in a separate windowFIG. 1.Prevalences of E. coli O157:H7 in organically and naturally raised beef cattle at slaughter. For each production system, bars not labeled with the same letter represent significantly different levels at P of <0.05.None of the tested isolates from the three production systems were susceptible to bacitracin, lincomycin, monensin, novobiocin, tilmicosin, tylosin, and vancomycin (MICs > 50 μg/ml). The MICs of 12 antibiotics (amikacin, apramycin, cefoxitin, ceftriaxone, gentamicin, kanamycin, nalidixic acid, neomycin, penicillin, rifampin, streptomycin, and tetracycline) for isolates collected from different production systems were significantly different (P < 0.05). MICs of gentamicin and neomycin for E. coli O157:H7 isolates from conventionally raised cattle were higher (P < 0.05) than those for isolates from naturally and/or organically raised cattle (Table ​(Table1).1). However, MICs of amikacin, apramycin, cefoxitin, ceftriaxone, kanamycin, nalidixic acid, penicillin, rifampin, and tetracycline for isolates from conventionally fed cattle were lower (P < 0.05) than those for isolates from naturally and/or organically raised cattle (Table ​(Table1).1). Among the 60 isolates tested for antibiotic susceptibilities, 6 isolates (10%) were susceptible to all antibiotics included in the study, excluding the seven antibiotics to which all isolates were resistant. Forty-two isolates (70%) were resistant to one antibiotic (MIC, >50 μg or >50 IU/ml), nine isolates (15%) were resistant to two antibiotics, and two isolates (3%) were resistant to five antibiotics. One isolate from the organically raised cattle group was resistant to 10 (amoxicillin, ampicillin, cefoxitin, cephalothin, chloramphenicol, florfenicol, oxytetracycline, penicillin, streptomycin, and tetracycline) of the 26 antibiotics that were inhibitory to other isolates. We have presented the data as the median MICs for each production system. In some instances, the median values were the same but the actual MIC data differed between production systems. This effect occurred because the data were right censored if isolates were not susceptible at 50 μg or 50 IU/ml. If more isolates from a particular production system than from another are censored, it may lead to statistical differences. This pattern justifies the use of survival analysis for this type of data. There were differences between MICs of many antibiotics (cefoxitin, ceftriaxone, gentamicin, nalidixic acid, neomycin, penicillin, rifampin, and tetracycline) for isolates from organically raised cattle and conventionally raised cattle. Similarly, there were differences between MICs of many antibiotics (amikacin, apramycin, ceftriaxone, kanamycin, nalidixic acid, and rifampin) for isolates from naturally raised cattle and conventionally raised cattle. For many of these antibiotics, MICs for isolates from organically or naturally raised cattle were greater than those for isolates from conventionally raised cattle. Resistance genes can be transferred among the enteric pathogen populations in food animals and humans (8), and it is possible that resistance genes from other bacteria in the gastrointestinal system of cattle may be acquired by E. coli O157:H7. For cattle, heavy metals like copper and zinc, which are also antimicrobial, are included in diets at concentrations in excess of the nutritional requirements, often replacing conventional antibiotics, to achieve growth promotion (5). Feeding with metals also results in the emergence of bacterial populations resistant to metals (5), which in some instances may lead to resistance to antibiotics. Mechanisms of resistance to copper at concentrations above those usually tolerated by normal cellular processes have been found on plasmids linked to resistance to antibiotics in some bacteria (5). Therefore, it is possible that isolates from organically or naturally raised cattle that are not exposed to antibiotics still may become resistant to antibiotics.

TABLE 1.

MICs of antimicrobials for E. coli O157:H7 isolates from conventionally, naturally, and organically raised beef cattle

Bar-Coded Pyrosequencing of 16S rRNA Gene Amplicons Reveals Changes in Ileal Porcine Bacterial Communities Due to High Dietary Zinc Intake

Feeding high levels of zinc oxide to piglets significantly increased the relative abundance of ileal Weissella spp., Leuconostoc spp., and Streptococcus spp., reduced the occurrence of Sarcina spp. and Neisseria spp., and led to numerical increases of all Gram-negative facultative anaerobic genera. High dietary zinc oxide intake has a major impact on the porcine ileal bacterial composition.Zinc oxide (ZnO) is used as a feed additive for diarrhea prophylaxis in piglets (23). However, the mode of action of ZnO is not fully understood. Besides its effects on the host (10, 30, 31), high dietary zinc levels may affect the diversity of intestinal microbial communities (2, 11, 20). The prevention of postweaning diarrhea in piglets due to high dietary ZnO intake may not be directly related to a reduction of pathogenic E. coli (8) but, rather, to the diversity of the coliform community (15). Studies on the impact of high ZnO levels on the porcine ileal bacterial community are scarce but nevertheless important, as bacterial diarrhea is initiated in the small intestine (9, 17). The small intestine is a very complex habitat with many different factors shaping the bacterial community. Studies on the ecophysiology (22) and maturation of the porcine ileal microbiota (13, 27) indicate a drastic impact directly after weaning and a gradual decline of modifications during the following 2 weeks. Thus, the time point for analysis chosen in this study (14 days postweaning) does reflect a more stable period of the ileal porcine microbiota. In this study, we used bar-coded pyrosequencing of 16S rRNA genes to gain further insight into the mode of action of pharmacological levels of ZnO in the gastrointestinal tract of young pigs.Total DNA was extracted from the ileal digesta of 40- to 42-day-old piglets using a commercial kit (Qiagen stool kit; Qiagen, Hilden, Germany) and PCR amplified with unique bar-coded primer sets targeting the V1-to-V3 and the V6-to-V8 hypervariable regions (see the supplemental material for detailed methods). The rationale behind this approach was derived from the fact that no single “universal” primer pair can completely cover a complex bacterial habitat (4, 24, 32, 33). Furthermore, these studies also show that in silico information on the coverage of selected primer sets diverges from empirical results, and hence, two hypervariable regions were chosen in this study to maximize the detection of phylogenetically diverse bacterial groups.Equimolar dilutions of all samples were combined into one master sample. Pyrosequencing was performed by Agowa (Berlin, Germany) on a Roche genome sequencer FLX system using a Titanium series PicoTiterPlate. The resulting data files were uploaded to the MG-RAST server (http://metagenomics.nmpdr.org/) (19) and processed with its SEED software tool using the RDP database (5) as the reference database. After automated sequence analysis, all sequences with less than five identical reads per sample were deleted in order to increase the confidence of sequence reads and reduce bias from possible sequencing errors (12, 16). Thus, 0.43% of all sequences were not considered (1,882 of 433,302 sequences). These sequences were assigned to a total of 238 genera, of which most only occurred in a few samples (see the supplemental material). Furthermore, all unclassified sequences were removed (8.7%; 41,467 of 474,769 sequences). Due to the use of the RDP reference database, the SEED software incorrectly assigned the majority of unclassified sequences as unclassified Deferribacterales (83%; 34,393 sequences), which were actually identified as 16S soybean or wheat chloroplasts by BLAST or as cyanobacterial chloroplasts by the RDP II seqmatch tool.The pyrosequencing results for the two primer combinations were merged by taking only sequences from the primer combination that yielded the higher number of reads for a specific sequence assignment in a sample. The remaining reads were used to calculate the relative contribution of assigned sequences to total sequence reads in a sample.The Firmicutes phylum dominated the small intestinal bacterial communities in both the control group and the group with high dietary ZnO intake, with 98.3% and 97.0% of total sequence reads, respectively. No significant influence of high dietary ZnO intake was found for the main phyla Proteobacteria (0.92% versus 1.84%), Actinobacteria (0.61% versus 0.75%), Bacteroidetes (0.15% versus 0.17%), and Fusobacteria (0.09% versus 0.12%).On the order level, a total of 20 bacterial orders were detected (data not shown). Lactobacillales dominated bacterial communities in the control and high-dietary-ZnO-intake groups, with 83.37% and 93.24% of total reads. Lactic acid bacteria are well known to dominate the bacterial community in the ileum of piglets (11, 22). No significant difference between the control group and the group with high dietary ZnO intake was observed on the order level, although high dietary ZnO intake led to a strong numerical decrease for Clostridiales (14.4 ± 24.0% [mean ± standard deviation] versus 2.8 ± 1.7%), as well as to numerical increases for Pseudomonadales (0.3 ± 0.3% versus 0.6 ± 0.6%) and Enterobacteriales (0.2 ± 0.2% versus 0.5 ± 0.6%).On the genus level, a total of 103 genera were detected. Table ​Table11 summarizes the main 31 genera which exceeded 0.05% of total reads (see the supplemental material for a complete list). Lactobacilli clearly dominated the bacterial communities in both trial groups, but they also were numerically lower due to high dietary ZnO intake.

TABLE 1.

Bacterial genera in the ileum of piglets fed diets supplemented with 200 or 3,000 ppm ZnO

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

Pulsed-Field Gel Electrophoresis Profile Changes Resulting from Spontaneous Chromosomal Deletions in Enterohemorrhagic Escherichia coli O157:H7 during Passage in Cattle

A total of 905 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were recovered from experimentally infected cattle, in addition to the inoculated strain, were analyzed by pulsed-field gel electrophoresis (PFGE). Twelve PFGE profiles other than that of the inoculated strain were observed. We successfully identified five distinct chromosomal deletions that affected the PFGE profiles using whole-genome PCR scanning and DNA sequencing analysis. The changes in PFGE profiles of EHEC O157:H7 isolates after passage through the intestinal tract of cattle were partially generated by deletion of chromosomal regions.Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic-uremic syndrome in humans worldwide (18). Cattle are considered the primary reservoir for this pathogen and play a central role in transmission to humans (6). Healthy cattle transiently carry EHEC O157:H7 and shed the bacteria in their feces (5, 7). Human infections have been associated with the consumption of contaminated meat and milk, direct contact with cattle, and the consumption of vegetables, fruits, and water contaminated with cattle manure (6).Because of its high discriminatory power, pulsed-field gel electrophoresis (PFGE) has been widely employed as a molecular typing method in many epidemiological investigations to identify various outbreaks and routes of transmission of EHEC O157:H7 (1, 12, 15, 17). Simpson''s index of diversity (9) was reported to be >0.985 in previous studies (1, 15), supporting the identification of richness (the number of types among isolates) and evenness (the relative distribution of individual strains among the different types) of molecular typing using PFGE.Instability of the PFGE patterns of EHEC O157:H7 isolates has been reported. Changes in PFGE patterns were observed among strains after repeated subculturing and prolonged storage at room temperature (11). Loss of Shiga toxin genes and a large-scale inversion within the genome were identified as genetic events generating changes in PFGE patterns in vitro (10, 13). Shifts in the genotypes of EHEC O157:H7 clinical isolates from patients and cattle have been reported (3, 14). This phenomenon was also observed in EHEC O157:H7 experimental infections of cattle. Spontaneous curing of a 90-kb plasmid resulted in the loss of two restricted fragments from the PFGE profiles of EHEC O157:H7 isolates obtained from experimentally infected cattle (2). The purpose of the present study was to identify the genetic events affecting the PFGE patterns of EHEC O157:H7 after passage through the intestinal tract of cattle, especially for restriction fragments that are >90 kb long.Four 5-month-old Holstein steers were housed individually in climate-controlled biosafety level 2 containment barns in accordance with the guidelines for animal experimentation defined by the National Institute of Animal Health of Japan. The pens had individual floor drains and were cleaned twice daily with water and disinfectant. All animals were healthy and culture negative for EHEC O157:H7 strains, as determined by a previously described technique (2), prior to inoculation.EHEC O157:H7 strain Sakai-215 (12, 23), which was isolated from an outbreak in Sakai, Osaka Prefecture, in 1996 was used for inoculation. This strain harbors the genes encoding Stx1 and Stx2. A spontaneous resistant strain was selected with nalidixic acid in order to facilitate the recovery of this strain from fecal samples. All calves were inoculated using a stomach tube with an exponential-phase culture (10⁹ CFU) of the nalidixic acid-resistant Sakai-215 strain. Fecal samples were collected from the four calves daily for 45 days. Fecal culturing was performed as described previously (2). Eight non-sorbitol-fermenting colonies were selected daily from each animal and identified as EHEC O157:H7 colonies by routine diagnostic methods (25).All animals were clinically normal throughout the experimental period. The EHEC O157:H7-inoculated calves (calves 1 to 4) were culture positive for the organism 24 h after inoculation. Intermittent fecal shedding by the calves was observed until 27, 32, 26, and 39 days postinoculation for calves 1, 2, 3, and 4, respectively (Fig. ​(Fig.1).1). The numbers of EHEC O157:H7 isolates recovered from calves 1, 2, 3, and 4 were 200, 224, 200, and 281, respectively.Open in a separate windowFIG. 1.Changes in PFGE profiles of EHEC O157:H7 isolates recovered from calves 1 (A), 2 (B), 3 (C), and 4 (D). The absence of a bar indicates that no EHEC O157:H7 was detected. The open horizontal bars under the vertical bars indicate that the eight isolates obtained on a day were obtained from the enrichment culture.A total of 905 recovered isolates in addition to the inoculated strain were used for PFGE analysis. Genomic DNA from each EHEC O157:H7 isolate was prepared using the method of Persing (Mayo Clinic, Rochester, MN) described by Rice et al. (20). Agarose-embedded chromosomal DNA was cleaved with XbaI by following the manufacturer''s instructions. PFGE was performed in a 0.85% megabase agarose gel, using a CHEF DR III apparatus (Bio-Rad Laboratories). The pulse time was increased from 12 to 35 s for 18 h. The PFGE profiles of all of the EHEC O157:H7 isolates recovered from the four calves were compared with that of the inoculated strain. The number of band differences was determined by enumerating the loss and addition of fragments (22).Two hundred eighty-nine isolates had PFGE profiles different from that of the inoculated strain, and 12 distinct PFGE profiles were identified for these isolates (Table ​(Table1).1). The fact that only one to three band differences were observed for the 12 profiles suggested that these isolates were closely related (22) and were variants of the inoculated strain. In addition, the pens had individual floor drains and were cleaned twice daily with water and disinfectant, which reduced the likelihood of introduction of novel EHEC O157:H7 strains. We designated the PFGE profiles A to L. PFGE profiles A, C, and H were obtained for all four calves and accounted for 30.4% of the 905 isolates recovered. Different PFGE profiles were obtained for all animals at least 2 days postinoculation (Fig. ​(Fig.1).1). All eight isolates from calf 2 collected on day 15 postinoculation and from calf 3 collected on days 22 and 23 postinoculation had PFGE profiles different from that of the original isolate (Fig. ​(Fig.1).1). The isolates that had the same PFGE profile as the inoculated strain were detected again later.

TABLE 1.

Temporal distribution of PFGE profiles of EHEC O157:H7 isolates recovered from experimentally infected cattle