Apparent Virulence Research Articles

Genomic characterization and virulence gene profiling of Erysipelothrix rhusiopathiae isolated from widespread muskox mortalities in the Canadian Arctic Archipelago

BackgroundMuskoxen are important ecosystem components and provide food, economic opportunities, and cultural well-being for Indigenous communities in the Canadian Arctic. Between 2010 and 2021, Erysipelothrix rhusiopathiae was isolated from carcasses of muskoxen, caribou, a seal, and an Arctic fox during multiple large scale mortality events in the Canadian Arctic Archipelago. A single strain (‘Arctic clone’) of E. rhusiopathiae was associated with the mortalities on Banks, Victoria and Prince Patrick Islands, Northwest Territories and Nunavut, Canada (2010–2017). The objectives of this study were to (i) characterize the genomes of E. rhusiopathiae isolates obtained from more recent muskox mortalities in the Canadian Arctic in 2019 and 2021; (ii) identify and compare common virulence traits associated with the core genome and mobile genetic elements (i.e. pathogenicity islands and prophages) among Arctic clone versus other E. rhusiopathiae genomes; and iii) use pan-genome wide association studies (GWAS) to determine unique genetic contents of the Arctic clone that may encode virulence traits and that could be used for diagnostic purposes.ResultsPhylogenetic analyses revealed that the newly sequenced E. rhusiopathiae isolates from Ellesmere Island, Nunavut (2021) also belong to the Arctic clone. Of 17 virulence genes analysed among 28 Arctic clone isolates, four genes – adhesin, rhusiopathiae surface protein-A (rspA), choline binding protein-B (cbpB) and CDP-glycerol glycerophosphotransferase (tagF) – had amino acid sequence variants unique to this clone when compared to 31 other E. rhusiopathiae genomes. These genes encode proteins that facilitate E. rhusiopathiae to attach to the host endothelial cells and form biofilms. GWAS analyses using Scoary found several unique genes to be overrepresented in the Arctic clone.ConclusionsThe Arctic clone of E. rhusiopathiae was associated with multiple muskox mortalities spanning over a decade and multiple Arctic islands with distances over 1000 km, highlighting the extent of its spatiotemporal spread. This clone possesses unique gene content, as well as amino acid variants in multiple virulence genes that are distinct from the other closely related E. rhusiopathiae isolates. This study establishes an essential foundation on which to investigate whether these differences are correlated with the apparent virulence of this specific clone through in vitro and in vivo studies.

Deformed wing virus prevalence in solitary bees put to the test: an experimental transmission study

Virus spillover from managed to unmanaged bees and vice versa may be one mechanism driving colony losses of the former and declines of the latter. There is clear evidence that the ubiquitous Deformed wing virus (DWV) is a major driver of honey bee (Apis mellifera) colony mortality. Although DWV has been detected in the solitary bee Osmia bicornis, data on DWV infectivity and virulence from solitary bees are scarce. Here, we used microinjection to investigate whether DWV genotype A (DWV-A) obtained from honey bees can replicate in O. bicornis. DWV-A titers and intermediate strand analyses suggest that DWV-A does not replicate in O. bicornis and thus is probably not infectious for this solitary bee species. Interestingly, the data demonstrate that DWV-A recovered from O. bicornis 16 days post-microinjection remains infectious for A. mellifera. Therefore, despite the lack of apparent virulence of DWV in this solitary bee species, O. bicornis has the potential to act as a virus spillover host and may contribute to increased colony losses of managed honey bees and declines in populations of other managed or unmanaged bee species.

Salmonella enterica serovar Cerro displays a phylogenetic structure and genomic features consistent with virulence attenuation and adaptation to cattle.

Salmonella enterica subsp. enterica (S.) serovar Cerro is rarely isolated from human clinical cases of salmonellosis but represents the most common serovar isolated from cattle without clinical signs of illness in the United States. In this study, using a large, diverse set of 316 isolates, we utilized genomic methods to further elucidate the evolutionary history of S. Cerro and to identify genomic features associated with its apparent virulence attenuation in humans. Phylogenetic analyses showed that within this polyphyletic serovar, 98.4% of isolates (311/316) represent a monophyletic clade within section Typhi and the remaining 1.6% of isolates (5/316) form a monophyletic clade within subspecies enterica Clade A1. Of the section Typhi S. Cerro isolates, 93.2% of isolates (290/311) clustered into a large clonal clade comprised of predominantly sequence type (ST) 367 cattle and environmental isolates, while the remaining 6.8% of isolates (21/311), primarily from human clinical sources, clustered outside of this clonal clade. A tip-dated phylogeny of S. Cerro ST367 identified two major clades (I and II), one of which overwhelmingly consisted of cattle isolates that share a most recent common ancestor that existed circa 1975. Gene presence/absence and rarefaction curve analyses suggested that the pangenome of section Typhi S. Cerro is open, potentially reflecting the gain/loss of prophage; human isolates contained the most open pangenome, while cattle isolates had the least open pangenome. Hypothetically disrupted coding sequences (HDCs) displayed clade-specific losses of intact speC and sopA virulence genes within the large clonal S. Cerro clade, while loss of intact vgrG, araH, and vapC occurred in all section Typhi S. Cerro isolates. Further phenotypic analysis suggested that the presence of a premature stop codon in speC does not abolish ornithine decarboxylase activity in S. Cerro, likely due to the activity of the second ornithine decarboxylase encoded by speF, which remained intact in all isolates. Overall, our study identifies specific genomic features associated with S. Cerro's infrequent isolation from humans and its apparent adaptation to cattle, which has broader implications for informing our understanding of the evolutionary events facilitating host adaptation in Salmonella.

Differential Growth of Francisella tularensis, Which Alters Expression of Virulence Factors, Dominant Antigens, and Surface-Carbohydrate Synthases, Governs the Apparent Virulence of Ft SchuS4 to Immunized Animals.

The gram-negative bacterium Francisella tularensis (Ft) is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize Ft grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to Ft grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select Ft virulence factors by an unknown mechanism. Here, we tested the hypotheses that Ft grown in BHI (BHI-Ft) accurately displays a full protein composition more similar to that reported for infection-derived Ft and that this similarity would make BHI-Ft more susceptible to pre-existing, vaccine-induced immunity than MHB-Ft. We performed comprehensive proteomic analysis of Ft grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published “omics” data derived from Ft grown in vivo. Based on the abundance of ~1,000 proteins, the fingerprint of BHI-Ft is one of nutrient-deprived bacteria that—through induction of a stringent-starvation-like response—have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI-Ft would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown Ft S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent.

Highly potent host external immunity acts as a strong selective force enhancing rapid parasite virulence evolution.

Virulence is often under selection during host-parasite coevolution. In order to increase fitness, parasites are predicted to circumvent and overcome host immunity. A particular challenge for pathogens are external immune systems, chemical defence systems comprised of potent antimicrobial compounds released by prospective hosts into the environment. We carried out an evolution experiment, allowing for coevolution to occur, with the entomopathogenic fungus, Beauveria bassiana, and the red flour beetle, Tribolium castaneum, which has a well-documented external immune system with strong inhibitory effects against B. bassiana. After just seven transfers of experimental evolution we saw a significant increase in parasite induced host mortality, a proxy for virulence, in all B. bassiana lines. This apparent virulence increase was mainly the result of the B. bassiana lines evolving resistance to the beetles' external immune defences, not due to increased production of toxins or other harmful substances. Transcriptomic analyses of evolved B. bassiana implicated the up-regulation of oxidative stress resistance genes in the observed resistance to external immunity. It was concluded that external immunity acts as a powerful selective force for virulence evolution, with an increase in virulence being achieved apparently entirely by overcoming these defences, most likely due to elevated oxidative stress resistance.

Differential Growth of Francisella tularensis, Which Alters Expression of Virulence Factors, Dominant Antigens, and Surface-Carbohydrate Synthases, Governs the Apparent Virulence of Ft SchuS4 to Immunized Animals

Differential Growth of Francisella tularensis, Which Alters Expression of Virulence Factors, Dominant Antigens, and Surface-Carbohydrate Synthases, Governs the Apparent Virulence of Ft SchuS4 to Immunized Animals

Streptococcal group B integrative and mobilizable element IMESag-rpsI encodes a functional relaxase involved in its transfer.

Streptococcus agalactiae or Group B Streptococcus (GBS) are opportunistic bacteria that can cause lethal sepsis in children and immuno-compromised patients. Their genome is a reservoir of mobile genetic elements that can be horizontally transferred. Among them, integrative and conjugative elements (ICEs) and the smaller integrative and mobilizable elements (IMEs) primarily reside in the bacterial chromosome, yet have the ability to be transferred between cells by conjugation. ICEs and IMEs are therefore a source of genetic variability that participates in the spread of antibiotic resistance. Although IMEs seem to be the most prevalent class of elements transferable by conjugation, they are poorly known. Here, we have studied a GBS-IME, termed IMESag-rpsI, which is widely distributed in GBS despite not carrying any apparent virulence trait. Analyses of 240 whole genomes showed that IMESag-rpsI is present in approximately 47% of the genomes, has a roughly constant size (approx. 9 kb) and is always integrated at a single location, the 3′-end of the gene encoding the ribosomal protein S9 (rpsI). Based on their genetic variation, several IMESag-rpsI types were defined (A–J) and classified in clonal complexes (CCs). CC1 was the most populated by IMESag-rpsI (more than 95%), mostly of type-A (71%). One CC1 strain (S. agalactiae HRC) was deep-sequenced to understand the rationale underlying type-A IMESag-rpsI enrichment in GBS. Thirteen open reading frames were identified, one of them encoding a protein (MobSag) belonging to the broadly distributed family of relaxases MOBV1. Protein MobSag was purified and, by a newly developed method, shown to cleave DNA at a specific dinucleotide. The S. agalactiae HRC-IMESag-rpsI is able to excise from the chromosome, as shown by the presence of circular intermediates, and it harbours a fully functional mobilization module. Further, the mobSag gene encoded by this mobile element is able to promote plasmid transfer among pneumococcal strains, suggesting that MobSag facilitates the spread of IMESag-rpsI and that this spread would explain the presence of the same IMESag-rpsI type in GBS strains belonging to different CCs.

White spot syndrome virus (WSSV) genome stability maintained over six passages through three different penaeid shrimp species.

White spot syndrome virus (WSSV) replicates rapidly, can be extremely pathogenic and is a common cause of mass mortality in cultured shrimp. Variable number tandem repeat (VNTR) sequences present in the open reading frame (ORF)94, ORF125 and ORF75 regions of the WSSV genome have been used widely as genetic markers in epidemiological studies. However, reports that VNTRs might evolve rapidly following even a single transmission through penaeid shrimp or other crustacean hosts have created confusion as to how VNTR data is interpreted. To examine VNTR stability again, 2 WSSV strains (PmTN4RU and LvAP11RU) with differing ORF94 tandem repeat numbers and slight differences in apparent virulence were passaged sequentially 6 times through black tiger shrimp Penaeus monodon, Indian white shrimp Feneropenaeus indicus or Pacific white leg shrimp Litopenaeus vannamei. PCR analyses to genotype the ORF94, ORF125 and ORF75 VNTRs did not identify any differences from either of the 2 parental WSSV strains after multiple passages through any of the shrimp species. These data were confirmed by sequence analysis and indicate that the stability of the genome regions containing these VNTRs is quite high at least for the WSSV strains, hosts and number of passages examined and that the VNTR sequences thus represent useful genetic markers for studying WSSV epidemiology.

Susceptibility of juvenile Macrobrachium rosenbergii to different doses of high and low virulence strains of white spot syndrome virus (WSSV)

As some literature on the susceptibility of different life stages of Macrobrachium rosenbergii to white spot syndrome virus (WSSV) is conflicting, the pathogenesis, infectivity and pathogenicity of 2 WSSV strains (Thai-1 and Viet) were investigated here in juveniles using conditions standardized for Penaeus vannamei. As with P. vannamei, juvenile M. rosenbergii (2 to 5 g) injected with a low dose of WSSV-Thai-1 or a high dose of WSSV-Viet developed comparable clinical pathology and numbers of infected cells within 1 to 2 d post-infection. In contrast, a low dose of WSSV-Viet capable of causing mortality in P. vannamei resulted in no detectable infection in M. rosenbergii. Mean prawn infectious dose 50% endpoints (PID₅₀ ml⁻¹) determined in M. rosenbergii were in the order of 100-fold higher for WSSV-Thai-1 (105.3 ± 0.4 PID₅₀ ml⁻¹) than for WSSV-Viet (103.2 ± 0.2 PID₅₀ ml⁻¹), with each of these being about 20-fold and 400-fold lower, respectively, than found previously in P. vannamei. The median lethal dose (LD₅₀ ml⁻¹) determined in M. rosenbergii was also far higher (~1000-fold) for WSSV-Thai-1 (105.4 ± 0.4 LD₅₀ ml⁻¹) than for WSSV-Viet (102.3 ± 0.3 LD₅₀ ml⁻¹). Based on these data, it is clear that juvenile M. rosenbergii are susceptible to WSSV infection, disease and mortality. In comparison to P. vannamei, however, juvenile M. rosenbergii appear more capable of resisting infection and disease, particularly in the case of a WSSV strain with lower apparent virulence.

Identification and immunoprotective analysis of a Streptococcus iniae subunit vaccine candidate

Streptococcus iniae is a Gram-positive bacterium and a severe aquaculture pathogen that can infect a wide range of farmed fish species. In the summer of 2006, an epidemic broke out in a fish farm in north China, and examination of moribund fish (Japanese flounder) identified the possible etiological agent of the outbreak as a strain named SF1, which exhibited apparent virulence in a Japanese flounder infection model and conforms to the description of S. iniae by 16S rRNA sequence analysis and API 20 Strep test. Biochemical and random amplified polymorphic DNA analyses indicated that SF1 is of the serotype I. A putative iron-binding protein, Sip11, was identified from SF1 using a previously established molecular trap that selects exported proteins. Recombinant Sip11 was purified from Escherichia coli and found to be protective against SF1 infection when used as an injection vaccine administered intraperitoneally into Japanese flounder. To improve the vaccine potential of Sip11, an E. coli strain was constructed, which expresses and secrets recombinant Sip11 covalently linked to a carrier protein in the form of a chimera. Vaccination of Japanese flounder with live Sip11-secreting E. coli afforded complete protection upon the fish following lethal SF1 challenge. These results indicate that Sip11, especially when delivered by a live bacterial carrier, is an effective vaccine candidate against SF1 infection.

Acyl-homoserine lactone-mediated cross talk among epiphytic bacteria modulates behavior of Pseudomonas syringae on leaves

The leaf surface harbors a host of bacterial epiphytes that are capable of influencing the quorum sensing (QS) system of the plant pathogen Pseudomonas syringae pv. syringae (Pss). Pss uses QS to regulate expression of genes conferring extracellular polysaccharide production, motility and factors contributing to virulence to plants. About 7% of bacterial epiphytes isolated in this study produce the Pss cognate signal, 3-oxohexanoyl-homoserine lactone (3OC6HSL), often in amounts more than 10-fold higher than Pss. Premature induction of QS in Pss by these 3OC6HSL-producing epiphytes suppressed swarming motility and subsequent disease of the leaf. Co-inoculation of 3OC6HSL-producing strains with Pss reduced the number of lesions when inoculated together onto leaves compared with that of plants inoculated with Pss alone. Strains in which 3OC6HSL accumulation was quenched by expression of an N-acyl-homoserine lactonase did not decrease disease when co-inoculated with Pss. Disease incidence caused by a nonmotile mutant of Pss was not affected by 3OC6HSL-producing bacteria, suggesting that exogenous 3OC6HSL signal that altered the motility of Pss was responsible for reducing the apparent virulence of this pathogen. Thus, considerable cross talk involving exogenous 3OC6HSL occurs on leaves and this process can be exploited for disease control.

THE EVOLUTION OF PARASITES IN RESPONSE TO TOLERANCE IN THEIR HOSTS: THE GOOD, THE BAD, AND APPARENT COMMENSALISM

Tolerance to parasites reduces the harm that infection causes the host (virulence). Here we investigate the evolution of parasites in response to host tolerance. We show that parasites may evolve either higher or lower within-host growth rates depending on the nature of the tolerance mechanism. If tolerance reduces virulence by a constant factor, the parasite is always selected to increase its growth rate. Alternatively, if tolerance reduces virulence in a nonlinear manner such that it is less effective at reducing the damage caused by higher growth rates, this may select for faster or slower replicating parasites. If the host is able to completely tolerate pathogen damage up to a certain replication rate, this may result in apparent commensalism, whereby infection causes no apparent virulence but the original evolution of tolerance has been costly. Tolerance tends to increase disease prevalence and may therefore lead to more, rather than less, disease-induced mortality. If the parasite is selected, even a highly efficient tolerance mechanism may result in more individuals in total dying from disease. However, the evolution of tolerance often, although not always, reduces the individual risk of dying from infection.

Entamoeba histolytica: Differences in phagosome acidification and degradation between attenuated and virulent strains

Phagocytosis is the important virulent determinant of the enteric protozoan parasite Entamoeba histolytica. We compared the kinetics of phagosome maturation of attenuated and highly-virulent strains of E. histolytica using video microscopy. Phagosomes of attenuated strains were acidified rapidly within 2 min after phagosome formation (at the rate of 0.96 pH/min), persisted at pH 4.46 ± 0.13, and degraded ingested GFP- Leishmania very efficiently (90–94% GFP fluorescence was lost in 30 min), while phagosomes of highly-virulent strains were acidified slowly (0.69 pH/min), persisted at 5.11 ± 0.23, and degraded GFP less efficiently (60–71% decrease). These results suggest that efficiency of phagosome maturation is most probably inversely correlated with apparent virulence.

THE EVOLUTION OF PARASITES IN RESPONSE TO TOLERANCE IN THEIR HOSTS: THE GOOD, THE BAD, AND APPARENT COMMENSALISM

Tolerance to parasites reduces the harm that infection causes the host (virulence). Here we investigate the evolution of parasites in response to host tolerance. We show that parasites may evolve either higher or lower within-host growth rates depending on the nature of the tolerance mechanism. If tolerance reduces virulence by a constant factor, the parasite is always selected to increase its growth rate. Alternatively, if tolerance reduces virulence in a nonlinear manner such that it is less effective at reducing the damage caused by higher growth rates, this may select for faster or slower replicating parasites. If the host is able to completely tolerate pathogen damage up to a certain replication rate, this may result in apparent commensalism, whereby infection causes no apparent virulence but the original evolution of tolerance has been costly. Tolerance tends to increase disease prevalence and may therefore lead to more, rather than less, disease-induced mortality. If the parasite is selected, even a highly efficient tolerance mechanism may result in more individuals in total dying from disease. However, the evolution of tolerance often, although not always, reduces the individual risk of dying from infection.

Methicillin-resistantStaphylococcus aureusin Cat and Owner

To the Editor: A 3-year-old, neutered male, domestic shorthaired cat was referred for treatment to a veterinary specialty clinic in San Francisco, California, with a 1-year history of multifocal patches of crusted and well-demarcated ulcers on the trunk. Initially, small crusts suspected to be associated with flea allergy and pyoderma were present; however, response was poor to multiple treatments, including repeated corticosteroid therapy and antimicrobial therapy with amoxicillin–clavulanic acid and enrofloxacin. The owner reported having skin abscesses and pneumonia 3 months earlier, although no microbiologic testing was performed. Cytologic evaluation of exudate from the cat's lesions identified neutrophils and eosinophils with engulfed cocci. Leukocytosis with eosinophilia was found on a complete blood cell count. No notable abnormalities were present on thoracic radiograph, abdominal ultrasonograph, urinalysis, and tests for feline leukemia and immunodeficiency virus. Skin biopsy specimens were collected for histologic examination, and swabs of the exudates were submitted for bacterial culture. Histopathologic findings demonstrated ulcers and dermal granulation tissue with linearly arranged eosinophils, mast cells, neutrophils, and plasma cells between dense, homogeneous collagen bundles (sclerosing dermatitis). This pattern of inflammation is distinct from most staphylococcal infections of the skin, and it has been suggested that this uncommon histologic finding in cats is associated with methicillin-resistant staphylococcal infection (1). Methicillin-resistant Staphylococcus aureus (MRSA) was isolated from the skin lesions. Identification was confirmed by detection of penicillin-binding protein 2a (PBP2a) by latex agglutination test (PBP2´ Test Kit, Oxoid, Hants, UK). Typing was performed by SmaI pulsed-field gel electrophoresis as previously described (2), and the isolate was classified as the USA300 clone. Genes encoding production of the Panton-Valentine leukocidin (PVL) were identified by real-time PCR (3). The isolate was susceptible to chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin, but resistant to β-lactams, enrofloxacin, and erythromycin. After identification of MRSA in the cat, swabs of the anterior nares were collected from the owner and the cat, and MRSA was identified in specimens from both. All isolates were indistinguishable. This is the first report of isolation of USA300 MRSA from a household pet. USA300 is a community-associated clone that has disseminated widely throughout North America and Europe (4,5) and is reaching epidemic proportions in many regions. MRSA is becoming an important cause of skin and soft tissue infection in persons in the community (4,5) and has also been implicated in invasive infections such as necrotizing pneumonia (6). This clone possesses genes for PVL production, which may be an important factor in its apparent virulence (4,5). Additional characterization of the isolates from this study was not performed; however, USA300 has previously been reported to carry staphylococcal cassette chromosome mec (SCCmec) type IVa and classified as sequence type 8 (ST8) by multilocus sequence typing (4,5). Reports of MRSA infection and colonization in pets have increased dramatically in the past few years (3,7–9). Although this rise may be partially the result of increased testing and reporting, MRSA is definitely emerging in pet populations throughout the world. The role of pets in transmission of MRSA is still unclear; however, recent evidence suggests that MRSA can be transmitted between persons and their pets, in both directions (9,10). Reports of MRSA infection and colonization in pets have indicated that pets tend to be infected with isolates that are consistent with clones that are predominant in the human population in their area (7–9). Accordingly, USA100 accounted for initial isolations of MRSA in pets in North America (9). The similarity between pet and human isolates has led to speculation that pet MRSA is closely linked to human MRSA and that the source of MRSA in pets may often be colonized humans. If this is the case, it is not surprising that USA300 would emerge as a cause of disease in pets as it increases in prevalence in the human population. Considering the rapid dissemination of USA300 in humans in the United States, particularly in California, where it is the predominant community-associated clone, finding USA300 in a household pet in that state is not unexpected. Because indistinguishable isolates were collected from the owner and the infected cat, MRSA likely was transmitted between species in the household. However, while it is tempting to assume that the owner was the source of infection because of the owner's previous history of a soft tissue infection, this cannot be definitively determined on the basis of the timing of sampling in this case. MRSA appears to be emerging as an important veterinary and zoonotic pathogen, and the epidemiology of MRSA in household pets may take a parallel course to that in humans. Ongoing MRSA surveillance in animals is required, including proper testing of specimens from clinically affected animals and surveillance for colonization. The potential for transmission of this clone between humans and pets should also be evaluated to clarify its epidemiology and to facilitate development of measures to reduce household transmission.

Virulence Characteristics and Phylogenetic Background of Multidrug‐Resistant and Antimicrobial‐Susceptible Clinical Isolates ofEscherichia colifrom across the United States, 2000–2001

Increases in antimicrobial resistance in Escherichia coli have been paralleled by an increasing incidence of E. coli sepsis, suggesting a possible link between resistance and virulence. All 76 multidrug-resistant (MDR) E. coli isolates (i.e., those resistant to > or =3 antimicrobial agents, including ampicillin, ceftazidime, trimethoprim-sulfamethoxazole, gentamicin, and ciprofloxacin) reported to the Tracking Resistance in the United States Today studies during 2000-2001 and 76 closely matched pansusceptible control isolates were studied. Extended virulence profiles and E. coli phylogenetic group (A, B1, B2, or D) were compared between groups. The MDR isolates, which represented predominantly non-B2 phylogenetic groups (91%), exhibited significantly reduced molecular virulence, compared with the predominantly group B2-derived control isolates (58%). Only 30% of MDR isolates, compared with 61% of control isolates (P<.001), qualified as extraintestinal pathogenic E. coli (ExPEC), and even these isolates exhibited significantly lower virulence scores than did susceptible ExPEC (7.25 vs. 9.0; P=.001). Phylogenetic differences accounted for the apparent virulence differences between MDR and control isolates. These findings argue against a direct link between virulence traits and antimicrobial resistance in E. coli. Instead, they call into question why non-B2 strains are more commonly MDR, with differential exposure to selection pressure (including in agriculture) as one possible explanation.

Relationship of the glyoxylate pathway to the pathogenesis of Cryptococcus neoformans.

Functional genomics has become a major focus in the study of microbial pathogenesis. This study used a functional genomic tool, differential display reverse transcription-PCR, to identify a transcriptional profile of Cryptococcus neoformans cells as they produced meningitis in an immunosuppressed host. This serial global gene expression during infection allowed for the identification of up- and down-regulated genes during infection. During this profiling, a single gene for the enzyme isocitrate lyase (ICL1) was found to be up regulated at 1 week of infection in a rabbit meningitis model and during a time of maximum host cellular response. The finding suggested that this enzyme and the glyoxylate shunt pathway are important to this yeast's energy production during infection. However, site-directed icl1 mutants had no apparent virulence defect in two animal models and no growth defect within macrophages. These observations suggest that although the yeast responded to a certain environmental cue(s) by an increase in ICL1 expression during infection, this gene was not necessary for progression of a C. neoformans infection. Compounds that specifically target only ICL1 are unlikely to cripple C. neoformans growth in vivo.

Streptococcal M protein enhances TGF-beta production and increases surface IgA-positive B cells in vitro in IgA nephropathy.

High serum levels and enhanced in vitro production of IgA are observed in more than half of patients with IgA nephropathy (IgAN); and transforming forming growth factor-beta (TGF-beta) is certain IgA class switching factor. On the other hand, macroscopic haematuria appears frequently with upper respiratory infection as tonsillitis in IgAN. We compared the lymphocytic response to in-vitro stimulation by group A streptococcal M proteins of apparent virulence factor between IgAN, non-proliferative glomerulonephritis (NPGN), and normal subjects. M proteins were extracted from group A streptococcal strain type 5 and type 12 determined serologically. M protein-induced proliferation of lymphocytes from IgAN was higher than in NPGN but not in healthy control subjects. Flow cytometric analysis indicated that stimulation by M protein extracts derived from type 5 streptococci (M5) increased surface IgA-positive B cells in IgAN, but did not activate the production of soluble IgA. We also showed that M5 induced significant increases in TGF-beta, in culture supernatants of lymphocytes from patients with IgAN. Our results suggest that Streptococcal infection may play an important role in the pathogenesis of IgAN by stimulating IgA production through TGF-beta synthesis.

Stages in the recognition of epidemic pellagra in the United States: 1865-1960.

Recognition of the U.S. pellagra "epidemic" in the early part of this century occurred in stages. The recognition process distorted impressions of magnitude, rate of spread, and virulence. Unrecognized cases: Endemic pellagra developed from dietary deficiencies after the Civil War. Initially, cases were misdiagnosed as other more traditional disorders. Tradition and authority inhibited recognition. Recognition of severe cases: Beginning in 1907, outbreaks were reported in asylums. Existing severe cases came rapidly to medical attention, inflating the apparent rate of spread. Recognized cases had a fulminant course and a high case fatality. Expanded spectrum: Milder cases were increasingly recognized, leading to an exaggerated rate of increase in number of cases and a decrease in case fatality and apparent virulence. Greater sensitivity resulted largely from a shift in diagnostic thresholds, with loss of specificity and increase in false positive diagnoses. Standardization of diagnosis: Although no suitable diagnostic marker test was developed, diagnosis was ultimately standardized by development of a workable case definition and by assessment of response to an effective therapy (nicotinic acid) applied to presumptive cases.

Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes

The consensus sequence of the Sindbis virus AR339 isolate, the prototype alphavirus, has been deduced. THe results presented here suggest (i) that a substantial proportion of the sequence divergence evident between the consensus sequence and sequences of laboratory strains of AR339 has resulted from selection for efficient growth in cell culture, (ii) that many of these changes affect the virulence of the virus in animal models, and (iii) that such modified genetic backgrounds present in laboratory strains can exert a significant influence on genetic studies of virus pathogenesis and host range. A laboratory strain of Sindbis virus AR339 was sequenced and cloned as a cDNA (pTRSB) from which infectious virus (TRSB) could be derived. The consensus sequence was deduced from the complete sequences of pTRSB and HRsp (E. G. Strauss, C. M. Rice, and J. H. Strauss, Virology 133:92-110, 1984), from partial sequences of the glycoprotein genes of three other AR339 laboratory strains, and by comparison with the sequences of the glycoprotein genes of three other AR339 sequence. HRsp differed form the consensus sequence by eight coding changes, and TRSB differed by three coding changes. In the 5' untranslated region, HRsp differed from the consensus sequence at nucleotide (nt) 5. These differences were likely the result of cell culture passage of the original AR339 isolate. At three of the difference loci (one in TRSB and two in HRsp), selection of cell-culture-adaptive mutations was documented with Sindbis virus or other alphaviruses. Selection in cell culture often results in attenuation of virulence in animals. Considering the TRSB and HRsp sequences together, one noncoding difference from the consensus (an A-for-G substitution in the 5' untranslated region at nt 5) and six coding differences in the glycoprotein genes (at E2 amino acids 1, 3, 70, and 172 and at E1 amino acids 72 and 237) were at loci which, either individually or in combination, significantly affected alphavirus virulence in mice. Although the levels of virulence of isogenic strains containing either nt 5 A or nt 5 G did not differ significantly in neonatal mice, the presence of nt 5 A greatly enhanced the effect of a second attenuating mutation in the E2 gene. These results suggest that minimal differences in the "wild type" genetic background into which an additional mutation is introduced can have a dramatic effect on apparent virulence and pathogenesis phenotypes. A cDNA clone of the consensus AR339 sequence, a sequence devoid of occult attenuating mutations introduced by cell culture passage, will allow the molecular genetic examination of cell culture and in vivo phenotypes of a virus which may best reflect the sequence of Sindbis virus AR339 at the time of its isolation.