Longer-term effectiveness of a heterologous coronavirus disease 2019 (COVID-19) vaccine booster in healthcare workers in Brazil.

Objective: To compare the long-term vaccine effectiveness between those receiving viral vector [Oxford-AstraZeneca (ChAdOx1)] or inactivated viral (CoronaVac) primary series (2 doses) and those who received an mRNA booster (Pfizer/BioNTech) (the third dose) among healthcare workers (HCWs). Methods: We conducted a retrospective cohort study among HCWs (aged ≥18 years) in Brazil from January 2021 to July 2022. To assess the variation in the effectiveness of booster dose over time, we estimated the effectiveness rate by taking the log risk ratio as a function of time. Results: Of 14,532 HCWs, coronavirus disease 2019 (COVID-19) was confirmed in 56.3% of HCWs receiving 2 doses of CoronaVac vaccine versus 23.2% of HCWs receiving 2 doses of CoronaVac vaccine with mRNA booster (P < .001), and 37.1% of HCWs receiving 2 doses of ChAdOx1 vaccine versus 22.7% among HCWs receiving 2 doses of ChAdOx1 vaccine with mRNA booster (P < .001). The highest vaccine effectiveness with mRNA booster was observed 30 days after vaccination: 91% for the CoronaVac vaccine group and 97% for the ChAdOx1 vaccine group. Vacine effectiveness declined to 55% and 67%, respectively, at 180 days. Of 430 samples screened for mutations, 49.5% were SARS-CoV-2 delta variants and 34.2% were SARS-CoV-2 omicron variants. Conclusions: Heterologous COVID-19 vaccines were effective for up to 180 days in preventing COVID-19 in the SARS-CoV-2 delta and omicron variant eras, which suggests the need for a second booster.

Risk factors for long coronavirus disease 2019 (long COVID) among healthcare personnel, Brazil, 2020-2022.

OBJECTIVE: To determine risk factors for the development of long coronavirus disease 2019 (COVID-19) in healthcare personnel (HCP). METHODS: We conducted a case-control study among HCP who had confirmed symptomatic COVID-19 working in a Brazilian healthcare system between March 1, 2020, and July 15, 2022. Cases were defined as those having long COVID according to the Centers for Disease Control and Prevention definition. Controls were defined as HCP who had documented COVID-19 but did not develop long COVID. Multiple logistic regression was used to assess the association between exposure variables and long COVID during 180 days of follow-up. RESULTS: Of 7,051 HCP diagnosed with COVID-19, 1,933 (27.4%) who developed long COVID were compared to 5,118 (72.6%) who did not. The majority of those with long COVID (51.8%) had 3 or more symptoms. Factors associated with the development of long COVID were female sex (OR, 1.21; 95% CI, 1.05-1.39), age (OR, 1.01; 95% CI, 1.00-1.02), and 2 or more SARS-CoV-2 infections (OR, 1.27; 95% CI, 1.07-1.50). Those infected with the SARS-CoV-2 δ (delta) variant (OR, 0.30; 95% CI, 0.17-0.50) or the SARS-CoV-2 o (omicron) variant (OR, 0.49; 95% CI, 0.30-0.78), and those receiving 4 COVID-19 vaccine doses prior to infection (OR, 0.05; 95% CI, 0.01-0.19) were significantly less likely to develop long COVID. CONCLUSIONS: Long COVID can be prevalent among HCP. Acquiring >1 SARS-CoV-2 infection was a major risk factor for long COVID, while maintenance of immunity via vaccination was highly protective.

Effectiveness of two coronavirus disease 2019 (COVID-19) vaccines (viral vector and inactivated viral vaccine) against severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection in a cohort of healthcare workers.

OBJECTIVE: We investigated real-world vaccine effectiveness for Oxford-AstraZeneca (ChAdOx1) and CoronaVac against laboratory-confirmed severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among healthcare workers (HCWs). METHODS: We conducted a retrospective cohort study among HCWs (aged ≥18 years) working in a private healthcare system in Brazil between January 1, 2021 and August 3, 2021, to assess vaccine effectiveness. We calculated vaccine effectiveness as 1 - rate ratio (RR), with RR determined by adjusting Poisson models with the occurrence of SARS-CoV-2 infection as the outcome and the vaccination status as the main variable. We used the logarithmic link function and simple models adjusting for sex, age, and job types. RESULTS: In total, 13,813 HCWs met the inclusion criteria for this analysis. Among them, 6,385 (46.2%) received the CoronaVac vaccine, 5,916 (42.8%) received the ChAdOx1 vaccine, and 1,512 (11.0%) were not vaccinated. Overall, COVID-19 occurred in 6% of unvaccinated HCWs, 3% of HCWs who received 2 doses of CoronaVac vaccine, and 0.7% of HCWs who received 2 doses of ChAdOx1 vaccine (P < .001). In the adjusted analyses, the estimated vaccine effectiveness rates were 51.3% for CoronaVac, and 88.1% for ChAdOx1 vaccine. Both vaccines reduced the number of hospitalizations, the length of hospital stay, and the need for mechanical ventilation. In addition, 19 SARS-CoV-2 samples from 19 HCWs were screened for mutations of interest. Of 19 samples, 18 were the γ (gamma) variant. CONCLUSIONS: Although both COVID-19 vaccines (viral vector and inactivated virus) can significantly prevent COVID-19 among HCWs, CoronaVac was much less effective. The COVID-19 vaccines were also effective against the dominant γ variant.

Effectiveness of Heterologous Coronavirus Disease 2019 (COVID-19) Vaccine Booster Dosing in Brazilian Healthcare Workers, 2021.

BACKGROUND: Little is currently known about vaccine effectiveness (VE) for either 2 doses of Oxford-AstraZeneca (ChAdOx1) viral vector vaccine or CoronaVac (Instituto Butantan) inactivated viral vaccine followed by a third dose of mRNA vaccine (Pfizer/BioNTech) among healthcare workers (HCWs). METHODS: We conducted a retrospective cohort study among HCWs (aged ≥18 years) working in a private healthcare system in Brazil from January to December 2021. VE was defined as 1 - incidence rate ratio (IRR), with IRR determined using Poisson models with the occurrence of laboratory-confirmed coronavirus disease 2019 (COVID-19) infection as the outcome, adjusting for age, sex, and job type. We compared those receiving viral vector or inactivated viral primary series (2 doses) with those who received an mRNA booster. RESULTS: A total of 11 427 HCWs met the inclusion criteria. COVID-19 was confirmed in 31.5% of HCWs receiving 2 doses of CoronaVac vaccine versus 0.9% of HCWs receiving 2 doses of CoronaVac vaccine with mRNA booster (P < .001) and 9.8% of HCWs receiving 2 doses of ChAdOx1 vaccine versus 1% among HCWs receiving 2 doses of ChAdOx1 vaccine with mRNA booster (P < .001). In the adjusted analyses, the estimated VE was 92.0% for 2 CoronaVac vaccines plus mRNA booster and 60.2% for 2 ChAdOx1 vaccines plus mRNA booster, when compared with those with no mRNA booster. Of 246 samples screened for mutations, 191 (77.6%) were Delta variants. CONCLUSIONS: While 2 doses of ChAdOx1 or CoronaVac vaccines prevent COVID-19, the addition of a Pfizer/BioNTech booster provided significantly more protection.

The AraC-type transcription factor TagK is a new player in the signaling cascade that induces the anti-eukaryotic T6SS of Xanthomonas citri.

Type 6 secretion systems (T6SSs) are specialized multiprotein complexes that inject protein effectors into prokaryotic and/or eukaryotic cells. We previously described the role of the T6SS of the phytopathogen Xanthomonas citri pv. citri as an anti-eukaryotic nanoweapon that confers resistance to predation by the amoeba Dictyostelium discoideum. Transcription of the X. citri T6SS genes is induced by a signaling cascade involving the Ser/Thr kinase PknS and the extracytoplasmic function sigma factor EcfK. Here, we used a strain overexpressing a phosphomimetic constitutively active version of EcfK (EcfKT51E ) to identify the EcfK regulon, which includes a previously uncharacterized transcription factor of the AraC-family (TagK), in addition to T6SS genes and genes encoding protein homeostasis factors. Functional studies demonstrated that TagK acts downstream of EcfK, binding directly to T6SS gene promoters and inducing T6SS expression in response to contact with amoeba cells. TagK controls a small regulon, consisting of the complete T6SS, its accessory genes and additional genes encoded within the T6SS cluster. We conclude that a singular regulatory circuit consisting of a transmembrane kinase (PknS), an alternative sigma factor (EcfK) and an AraC-type transcriptional regulator (TagK) promotes expression of the X. citri T6SS in response to a protozoan predator.

An Extracytoplasmic Function Sigma Factor Required for Full Virulence in Xanthomonas citri pv. citri.

The genus Xanthomonas includes more than 30 phytopathogenic species that infect a wide range of plants and cause severe diseases that greatly impact crop productivity. These bacteria are highly adapted to the soil and plant environment, being found in decaying material, as epiphytes, and colonizing the plant mesophyll. Signal transduction mechanisms involved in the responses of Xanthomonas to environmental changes are still poorly characterized. Xanthomonad genomes typically encode several representatives of the extracytoplasmic function σ (σECF) factors, whose physiological roles remain elusive. In this work, we functionally characterized the Xanthomonas citri pv. citri EcfL, a σECF factor homologous to members of the iron-responsive FecI-like group. We show that EcfL is not required or induced during iron starvation, despite presenting the common features of other FecI-like σECF factors. EcfL positively regulates one operon composed of three genes that encode a TonB-dependent receptor involved in cell surface signaling, an acid phosphatase, and a lectin-domain containing protein. Furthermore, we demonstrate that EcfL is required for full virulence in citrus, and its regulon is induced inside the plant mesophyll and in response to acid stress. Together, our study suggests a role for EcfL in the adaptation of X. citri to the plant environment, in this way contributing to its ability to cause citrus canker disease. IMPORTANCE The Xanthomonas genus comprises a large number of phytopathogenic species that infect a wide variety of economically important plants worldwide. Bacterial adaptation to the plant and soil environment relies on their repertoire of signal transduction pathways, including alternative sigma factors of the extracytoplasmic function family (σECF). Here, we describe a new σECF factor found in several Xanthomonas species, demonstrating its role in Xanthomonas citri virulence to citrus plants. We show that EcfL regulates a single operon containing three genes, which are also conserved in other Xanthomonas species. This study further expands our knowledge on the functions of the widespread family of σECF factors in phytopathogenic bacteria.

DegP protease is essential for tolerance to salt stress in the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5.

The use of plant growth-promoting bacteria represents an alternative to the massive use of mineral fertilizers in agriculture. However, some abiotic stresses commonly found in the environment, like salinity, can affect the efficiency of this approach. Here, we investigated the key mechanisms involved in the response of the plant growth-promoting bacterium Gluconacetobacter diazotrophicus to salt stress by using morphological and cell viability analyses, comparative proteomics, and reverse genetics. Our results revealed that the bacteria produce filamentous cells in response to salt at 100 mM and 150 mM NaCl. However, such a response was not observed at higher concentrations, where cell viability was severely affected. Proteomic analysis showed that salt stress modulates proteins involved in several pathways, including iron uptake, outer membrane efflux, osmotic adjustment, cell division and elongation, and protein transport and quality control. Proteomic data also revealed the repression of several extracytoplasmic proteins, especially those located at periplasm and outer membrane. The role of such pathways in the tolerance to salt stress was analyzed by the use of mutant defectives for Δtbdr (iron uptake), ΔmtlK and ΔotsA (compatible solutes synthesis), and ΔdegP (quality control of nascent extracytoplasmic proteins). ΔdegP presented the highest sensitivity to salt stress, Δtbdr, andΔmtlK also showed increased sensitivity, but ΔotsA was not affected. This is the first demonstration that DegP protein, a protease with minor chaperone activity, is essential for tolerance to salt stress in G. diazotrophicus. Our data contribute to a better understanding of the molecular bases that control the bacterial response/tolerance to salt stress, shedding light on quality control of nascent extracytoplasmic proteins.

c-di-GMP-related phenotypes are modulated by the interaction between a diguanylate cyclase and a polar hub protein.

c-di-GMP is a major player in the switch between biofilm and motile lifestyles. Several bacteria exhibit a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be demonstrated in Pseudomonas aeruginosa. We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a polar peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells but scattered in the cytoplasm of cells lacking FimV. Overexpression of dgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a ΔfimV background. Therefore, our findings suggest that DgcP activity is regulated by FimV. The polar localization of DgcP might contribute to a local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.

The Atypical Response Regulator AtvR Is a New Player in Pseudomonas aeruginosa Response to Hypoxia and Virulence.

Two-component systems are widespread in bacteria, allowing adaptation to environmental changes. The classical pathway is composed of a histidine kinase that phosphorylates an aspartate residue in the cognate response regulator (RR). RRs lacking the phosphorylatable aspartate also occur, but their function and contribution during host-pathogen interactions are poorly characterized. AtvR (PA14_26570) is the only atypical response regulator with a DNA-binding domain in the opportunistic pathogen Pseudomonas aeruginosa Macrophage infection with the atvR mutant strain resulted in higher levels of tumor necrosis factor alpha secretion as well as increased bacterial clearance compared to those for macrophages infected with the wild-type strain. In an acute pneumonia model, mice infected with the atvR mutant presented increased amounts of proinflammatory cytokines, increased neutrophil recruitment to the lungs, reductions in bacterial burdens, and higher survival rates in comparison with the findings for mice infected with the wild-type strain. Further, several genes involved in hypoxia/anoxia adaptation were upregulated upon atvR overexpression, as seen by high-throughput transcriptome sequencing (RNA-Seq) analysis. In addition, atvR was more expressed in hypoxia in the presence of nitrate and required for full expression of nitrate reductase genes, promoting bacterial growth under this condition. Thus, AtvR would be crucial for successful infection, aiding P. aeruginosa survival under conditions of low oxygen tension in the host. Taken together, our data demonstrate that the atypical response regulator AtvR is part of the repertoire of transcriptional regulators involved in the lifestyle switch from aerobic to anaerobic conditions. This finding increases the complexity of regulation of one of the central metabolic pathways that contributes to Pseudomonas ubiquity and versatility.

A steroidal molecule present in the egg wax of the tick Rhipicephalus (Boophilus) microplus inhibits bacterial biofilms.

The cattle tick Rhipicephalus (Boophilus) microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. Tick eggs have a protective wax coating that may be a source of nutrients for microorganisms. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the coating might have anti-biofilm properties. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. We have identified the anti-biofilm molecule as N-(3-sulfooxy-25-cholest-5-en-26-oyl)-L-isoleucine (boophiline), and we show that it inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in Pseudomonas aeruginosa. Boophiline is a novel biofilm inhibitor being also effective against Staphylococcus epidermidis biofilm. In our study we show evidences of the boophiline mode of action in the protection of arthropod eggs against biofilm colonization.

A novel role for an ECF sigma factor in fatty acid biosynthesis and membrane fluidity in Pseudomonas aeruginosa.

Extracytoplasmic function (ECF) sigma factors are members of cell-surface signaling systems, abundant in the opportunistic pathogen Pseudomonas aeruginosa. Twenty genes coding for ECF sigma factors are present in P. aeruginosa sequenced genomes, most of them being part of TonB systems related to iron uptake. In this work, poorly characterized sigma factors were overexpressed in strain PA14, in an attempt to understand their role in the bacterium's physiology. Cultures overexpressing SigX displayed a biphasic growth curve, reaching stationary phase earlier than the control strain, followed by subsequent growth resumption. During the first stationary phase, most cells swell and die, but the remaining cells return to the wild type morphology and proceed to a second exponential growth. This is not due to compensatory mutations, since cells recovered from late time points and diluted into fresh medium repeated this behavior. Swollen cells have a more fluid membrane and contain higher amounts of shorter chain fatty acids. A proteomic analysis was performed to identify differentially expressed proteins due to overexpression of sigX, revealing the induction of several fatty acid synthesis (FAS) enzymes. Using qRT-PCR, we showed that at least one isoform from each of the FAS pathway enzymes were upregulated at the mRNA level in the SigX overexpressing strain thus pointing to a role for this ECF sigma factor in the FAS regulation in P. aeruginosa.

Pseudomonas aeruginosa PA14 cupD transcription is activated by the RcsB response regulator, but repressed by its putative cognate sensor RcsC.

The opportunistic pathogen Pseudomonas aeruginosa PA14 possesses four fimbrial cup clusters, which may confer the ability to adapt to different environments. cupD lies in the pathogenicity island PAPI-1 next to genes coding for a putative phosphorelay system composed of the hybrid histidine kinase RcsC and the response regulator RcsB. The main focus of this work was the regulation of cupD at the mRNA level. It was found that the HN-S-like protein MvaT does not exert a strong influence on cupD transcript levels, as it does for cupA. cupD transcription is higher in cultures grown at 28 degrees C, which agrees with a cupD mutant presenting attenuated virulence only in a plant model, but not in a mouse model of infection. Whereas an rcsC in-frame deletion mutant presented higher levels of cupD mRNA, rcsB deletion had the opposite effect. Accordingly, overexpression of RcsB increased the levels of cupD transcription, and promoted biofilm formation and the appearance of fimbriae. A single transcription start site was determined for cupD and transcription from this site was induced by RcsB. A motif similar to the enterobacterial RcsB/RcsA-binding site was detected adjacent to the -35 region, suggesting that this could be the RcsB-binding site. Comparison of P. aeruginosa and Escherichia coli Rcs may provide insights into how similar systems can be used by different bacteria to control gene expression and to adapt to various environmental conditions.

The pathogenic properties of a novel and conserved gene product, KerV, in proteobacteria.

Identification of novel virulence factors is essential for understanding bacterial pathogenesis and designing antibacterial strategies. In this study, we uncover such a factor, termed KerV, in Proteobacteria. Experiments carried out in a variety of eukaryotic host infection models revealed that the virulence of a Pseudomonas aeruginosa kerV null mutant was compromised when it interacted with amoebae, plants, flies, and mice. Bioinformatics analyses indicated that KerV is a hypothetical methyltransferase and is well-conserved across numerous Proteobacteria, including both well-known and emerging pathogens (e.g., virulent Burkholderia, Escherichia, Shigella, Vibrio, Salmonella, Yersinia and Brucella species). Furthermore, among the 197 kerV orthologs analyzed in this study, about 89% reside in a defined genomic neighborhood, which also possesses essential DNA replication and repair genes and detoxification gene. Finally, infection of Drosophila melanogaster with null mutants demonstrated that KerV orthologs are also crucial in Vibrio cholerae and Yersinia pseudotuberculosis pathogenesis. Our findings suggested that KerV has a novel and broad significance as a virulence factor in pathogenic Proteobacteria and it might serve as a new target for antibiotic drug design.