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Q fever is a disease caused by Coxiella burnetii, which can cause serious illness in humans and abortions in goats. A Q fever outbreak among an unvaccinated goat herd led to a 65% loss of the kid crop in spring 2018. To assess the impact of the outbreak on the herd and environment, longitudinal surveillance of the ranch was conducted across three samplings in September 2018, April 2019, and May 2022. Antibodies against C. burnetii were monitored by an indirect immunofluorescence assay. Shedding was monitored through analysis of vaginal/fecal swabs and milk. Environmental swabs and bulk soil were collected from various locations around the ranch. Animal and environmental samples were analyzed for C. burnetii DNA by PCR. Herd-level seroprevalence decreased from 89% in 2018 to 84.3% in 2019, and 64.5% in 2022. Overall herd shedding was 14.4% in 2018, 7.4% in 2019, and 6.7% in 2022. The percentage of C. burnetii-positive environmental samples was 83.7% in 2018, 51.7% in 2019, and 28.6% in 2022. Serological evidence suggests that new infections were occurring in the herd 4 years post-abortion storm. This study demonstrates the presence of C. burnetii shedding and environmental contamination in a goat operation at least four kidding seasons after an outbreak. A better understanding of management practices that can improve outcomes for infected herds, particularly in areas without access to vaccines against C. burnetii, is needed to better protect operators and the public. [ABSTRACT FROM AUTHOR]

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease (COVID-19), originally relied mainly on nasopharyngeal swab (NPS) samples collected by healthcare workers (HCWs). However, [...]

The iron overload disorder hereditary hemochromatosis (HH) predisposes humans to serious disseminated infection with pathogenic Yersinia as well as several other pathogens. Recently, we showed that the iron-sulfur cluster coordinating transcription factor IscR is required for type III secretion in Y. pseudotuberculosis by direct control of the T3SS master regulator LcrF. In E. coli and Yersinia, IscR levels are predicted to be regulated by iron bioavailability, oxygen tension, and oxidative stress, such that iron depletion should lead to increased IscR levels. To investigate how host iron overload influences Y. pseudotuberculosis virulence and the requirement for the Ysc type III secretion system (T3SS), we utilized two distinct murine models of HH: hemojuvelin knockout mice that mimic severe, early-onset HH as well as mice with the Hfe (C282Y∕C282Y) mutation carried by 10% of people of Northern European descent, associated with adult-onset HH. Hjv (-∕-) and Hfe (C282Y∕C282Y) transgenic mice displayed enhanced colonization of deep tissues by Y. pseudotuberculosis following oral inoculation, recapitulating enhanced susceptibility of humans with HH to disseminated infection with enteropathogenic Yersinia. Importantly, HH mice orally infected with Y. pseudotuberculosis lacking the T3SS-encoding virulence plasmid, pYV, displayed increased deep tissue colonization relative to wildtype mice. Consistent with previous reports using monocytes from HH vs. healthy donors, macrophages isolated from Hfe (C282Y∕C282Y) mice were defective in Yersinia uptake compared to wildtype macrophages, indicating that the anti-phagocytic property of the Yersinia T3SS plays a less important role in HH animals. These data suggest that Yersinia may rely on distinct virulence factors to cause disease in healthy vs. HH hosts.

Iron-sulfur clusters act as important cofactors for a number of transcriptional regulators in bacteria, including many mammalian pathogens. The sensitivity of iron-sulfur clusters to iron availability, oxygen tension, and reactive oxygen and nitrogen species enables bacteria to use such regulators to adapt their gene expression profiles rapidly in response to changing environmental conditions. In this review, we discuss how the [4Fe-4S] or [2Fe-2S] cluster-containing regulators FNR, Wbl, aconitase, IscR, NsrR, SoxR, and AirSR contribute to bacterial pathogenesis through control of both metabolism and classical virulence factors. In addition, we briefly review mammalian iron homeostasis as well as oxidative/nitrosative stress to provide context for understanding the function of bacterial iron-sulfur cluster sensors in different niches within the host.

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.

Background: We previously identified an ECF sigma factor, σσS, that is important in the stress and virulence response of Staphylococcus aureus. Transcriptional profiling of sigS revealed that it is differentially expressed in many laboratory and clinical isolates, suggesting the existence of regulatory networks that modulates its expression. Results: To identify regulators of sigS, we performed a pull down assay using S. aureus lysates and the sigS promoter. Through this we identified CymR as a negative effector of sigS expression. Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription. To more globally explore genetic regulation of sigS, a Tn551 transposon screen was performed, and identified insertions in genes that are involved in amino acid biosynthesis, DNA replication, recombination and repair pathways, and transcriptional regulators. In efforts to identify gain of function mutations, methyl nitro-nitrosoguanidine mutagenesis was performed on a sigS-lacZ reporter fusion strain. From this a number of clones displaying sigS upregulation were subject to whole genome sequencing, leading to the identification of the lactose phosphotransferase repressor, lacR, and the membrane histidine kinase, kdpD, as central regulators of sigS expression. Again using EMSAs we determined that LacR is an indirect regulator of sigS expression, while the response regulator, KdpE, directly binds to the promoter region of sigS. Conclusions: Collectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS. [ABSTRACT FROM AUTHOR]

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF. [ABSTRACT FROM AUTHOR]

S. aureus is a highly successful pathogen that is speculated to be the most common cause of human disease. The progression of disease in S. aureus is subject to multi-factorial regulation, in response to the environments encountered during growth. This adaptive nature is thought to be central to pathogenesis, and is the result of multiple regulatory mechanisms employed in gene regulation. In this work we describe the existence of a novel S. aureus regulator, an as yet uncharacterized ECF-sigma factor (σS), that appears to be an important component of the stress and pathogenic responses of this organism. Using biochemical approaches we have shown that sS is able to associates with core-RNAP, and initiate transcription from its own coding region. Using a mutant strain we determined that σS is important for S. aureus survival during starvation, extended exposure to elevated growth temperatures, and Triton X-100 induced lysis. Coculture studies reveal that a sS mutant is significantly outcompeted by its parental strain, which is only exacerbated during prolonged growth (7 days), or in the presence of stressor compounds. Interestingly, transcriptional analysis determined that under standard conditions, S. aureus SH1000 does not initiate expression of sigS. Assays performed hourly for 72h revealed expression in typically background ranges. Analysis of a potential anti-sigma factor, encoded downstream of sigS, revealed it to have no obvious role in the upregulation of sigS expression. Using a murine model of septic arthritis, sigS-mutant infected animals lost significantly less weight, developed septic arthritis at significantly lower levels, and had increased survival rates. Studies of mounted immune responses reveal that sigS-mutant infected animals had significantly lower levels of IL-6, indicating only a weak immunological response. Finally, strains of S. aureus lacking sigS were far less able to undergo systemic dissemination, as determined by bacterial loads in the kidneys of infected animals. These results establish that sS is an important component in S. aureus fitness, and in its adaptation to stress. Additionally it appears to have a significant role in its pathogenic nature, and likely represents a key component in the S. aureus regulatory network. [ABSTRACT FROM AUTHOR]

Since 1999, doxycycline and hydroxychloroquine have been the recommended treatment for chronic Q fever, a life-threatening disease caused by the bacterial pathogen, Coxiella burnetii . Despite the duration of its use, the treatment is not ideal due to the lengthy treatment time, high mortality rate, resistant strains, and the potential for contraindicated usage. A literature search was conducted to identify studies that screened large panels of drugs against C. burnetii to identify novel targets with potential efficacy against C. burnetii . Twelve candidate antimicrobials approved for use in humans by the US Food and Drug Administration were selected and minimum inhibitory concentrations (MICs) were determined against the low virulence strain Nine Mile phase II. Rifabutin and rifaximin were the best performing antibiotics tested with MICs of ≤0.01 µg mL -1 . Further screening of these top candidates was conducted alongside two drugs from the same class, rifampin, well-characterized , and rifapentine, not previously reported against C. burnetii . These were screened against virulent strains of C. burnetii representing three clinically relevant genotypes. Rifapentine was the most effective in the human monocytic leukemia cell line, THP-1, with a MIC ≤0.01 µg mL -1 . In the human kidney epithelial cell line, A-498, efficacy of rifapentine, rifampin, and rifabutin varied across C. burnetii strains with MICs between ≤0.001 and 0.01 µg mL -1 . Rifampin, rifabutin, and rifapentine were all bactericidal against C. burnetii ; however, rifabutin and rifapentine demonstrated impressive bactericidal activity as low as 0.1 µg mL -1 and should be further explored as alternative Q fever treatments given their efficacy in vitro ., Importance: This work will help inform investigators and physicians about potential alternative antimicrobial therapies targeting the causative agent of Q fever, Coxiella burnetii . Chronic Q fever is difficult to treat, and alternative antimicrobials are needed. This manuscript explores the efficacy of rifamycin antibiotics against virulent strains of C. burnetii representing three clinically relevant genotypes in vitro . Importantly, this study determines the susceptibility of C. burnetii to rifapentine, which has not been previously reported. Evaluation of the bactericidal activity of the rifamycins reveals that rifabutin and rifapentine are bactericidal at low concentrations, which is unusual for antibiotics against C. burnetii ., Competing Interests: The authors declare no conflict of interest.

Questionnaire data have linked contact with ruminants to the risk of esophageal squamous cell carcinoma (ESCC) in high-risk Asian populations. To better understand this observed association, we investigated exposure to two major zoonotic ruminant pathogens relative to ESCC risk. Using enzyme-linked immunosorbent assay, immunofluorescence assay, and Brucella microagglutination test assays, we measured immunoglobulin G anti- Coxiella burnetii and anti- Brucella spp. antibodies in patients with ESCC ( n  = 177) and population-based controls ( n  = 177) matched by age, gender, and residence area from the Golestan case-control study in Iran. We found a similarly high seroprevalence of C. burnetii in ESCC cases and controls (75% and 80%, respectively), and a similarly low seroprevalence of Brucella spp. (0% and 0.6%, respectively). While documenting a high exposure to one of two zoonotic ruminant infections, this exposure failed to explain the observed association of ruminant contact and ESCC risk in this high-risk population.

Staphylococcus aureus is an opportunistic pathogen that presents severe health care concerns due to the prevalence of multiple antibiotic-resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component by aureolysin (Aur). We demonstrate here that four major extracellular proteases of S. aureus are potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8 and the metalloproteinase Aur resulted in a drastic decrease in the hemolytic activity of serum, whereas two staphylococcal serine proteases D and E, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureus mutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureus appear to be important for pathogen-mediated evasion of the human complement system., (© 2013 S. Karger AG, Basel.)

Previously we identified a novel component of the Staphylococcus aureus regulatory network, an extracytoplasmic function σ-factor, σ(S), involved in stress response and disease causation. Here we present additional characterization of σ(S), demonstrating a role for it in protection against DNA damage, cell wall disruption, and interaction with components of the innate immune system. Promoter mapping reveals the existence of three unique sigS start sites, one of which appears to be subject to autoregulation. Transcriptional profiling revealed that sigS expression remains low in a number of S. aureus wild types but is upregulated in the highly mutated strain RN4220. Further analysis demonstrates that sigS expression is inducible upon exposure to a variety of chemical stressors that elicit DNA damage, including methyl methanesulfonate and ciprofloxacin, as well as those that disrupt cell wall stability, such as ampicillin and oxacillin. Significantly, expression of sigS is highly induced during growth in serum and upon phagocytosis by RAW 264.7 murine macrophage-like cells. Phenotypically, σ(S) mutants display sensitivity to a broad range of DNA-damaging agents and cell wall-targeting antibiotics. Furthermore, the survivability of σ(S) mutants is strongly impacted during challenge by components of the innate immune system. Collectively, our data suggest that σ(S) likely serves dual functions within the S. aureus cell, protecting against both cytoplasmic and extracytoplasmic stresses. This further argues for its important, and perhaps novel, role in the S. aureus stress and virulence responses.

S. aureus is a highly successful pathogen that is speculated to be the most common cause of human disease. The progression of disease in S. aureus is subject to multi-factorial regulation, in response to the environments encountered during growth. This adaptive nature is thought to be central to pathogenesis, and is the result of multiple regulatory mechanisms employed in gene regulation. In this work we describe the existence of a novel S. aureus regulator, an as yet uncharacterized ECF-sigma factor (sigma(S)), that appears to be an important component of the stress and pathogenic responses of this organism. Using biochemical approaches we have shown that sigma(S) is able to associates with core-RNAP, and initiate transcription from its own coding region. Using a mutant strain we determined that sigma(S) is important for S. aureus survival during starvation, extended exposure to elevated growth temperatures, and Triton X-100 induced lysis. Coculture studies reveal that a sigma(S) mutant is significantly outcompeted by its parental strain, which is only exacerbated during prolonged growth (7 days), or in the presence of stressor compounds. Interestingly, transcriptional analysis determined that under standard conditions, S. aureus SH1000 does not initiate expression of sigS. Assays performed hourly for 72 h revealed expression in typically background ranges. Analysis of a potential anti-sigma factor, encoded downstream of sigS, revealed it to have no obvious role in the upregulation of sigS expression. Using a murine model of septic arthritis, sigS-mutant infected animals lost significantly less weight, developed septic arthritis at significantly lower levels, and had increased survival rates. Studies of mounted immune responses reveal that sigS-mutant infected animals had significantly lower levels of IL-6, indicating only a weak immunological response. Finally, strains of S. aureus lacking sigS were far less able to undergo systemic dissemination, as determined by bacterial loads in the kidneys of infected animals. These results establish that sigma(S) is an important component in S. aureus fitness, and in its adaptation to stress. Additionally it appears to have a significant role in its pathogenic nature, and likely represents a key component in the S. aureus regulatory network.