bluuebunny/crossref_metadata_2025 huggingface dataset:
Strain-specific differences in the interactions of the cucumber mosaic virus 2b protein with the viral 1a and host Argonaute 1 proteins
Authors: Crawshaw Sam , Watt Lewis G. , Murphy Alex M. , Carr John P.
Published: August 2024

The cucumber mosaic virus (CMV) 2b protein is a potent counter-defense factor and symptom determinant that inhibits antiviral silencing by titrating short double-stranded RNAs. Expression of the CMV subgroup IA strain Fny-CMV 2b protein in transgenic
Arabidopsis thaliana
plants disrupts microRNA-mediated cleavage of host mRNAs by binding Argonaute 1 (AGO1), leading to symptom-like phenotypes. This also triggers AGO2-mediated antiviral resistance and resistance to CMV’s aphid vectors. However, in authentic viral infections, the Fny-CMV 1a protein modulates 2b-AGO1 interactions, inhibiting induction of AGO2-mediated virus resistance and aphid resistance. Contrastingly, 2b proteins encoded by the subgroup II strain LS-CMV and the recently discovered subgroup IA strain Ho-CMV induce no symptoms. Confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation showed that Fny-CMV and Ho-CMV 2b proteins interact with Fny-CMV and LS-CMV 1a proteins, while the CMV-LS 2b protein cannot. However, Fny-CMV, Ho-CMV, and LS-CMV 2b proteins, all interacted with AGO1, but while AGO1-Fny2b complexes occurred in the nucleus and cytoplasm, corresponding AGO1-2b complexes for LS-CMV and Ho-CMV accumulated almost exclusively in nuclei.
AGO2
transcript accumulation was used to assess the inhibition of AGO1-mediated mRNA degradation. Fny-CMV 2b induced a fivefold increase in
AGO2
accumulation, but LS-CMV and Ho-CMV 2b proteins induced only twofold increases. Thus, these 2b proteins bind AGO1 but are less effective at inhibiting AGO1 activity. We conclude that the intracellular localization of 2b-AGO1 complexes influences the degree to which a 2b protein inhibits microRNA-mediated host mRNA degradation and that cytoplasmic AGO1 has the strongest influence on miRNA-mediated cellular mRNA turnover.

IMPORTANCE

The cucumber mosaic virus (CMV) 2b protein was among the first discovered viral suppressors of RNA silencing. It has additional pro-viral functions through effects on plant defensive signaling pathways mediated by salicylic acid and jasmonic acid, the abscisic acid pathway and virus-induced drought resistance, and on host plant interactions with insect vectors. Many of these effects occur due to interaction with the important host RNA silencing component Argonaute 1 (AGO1). It was thought that only 2b proteins of “severe” CMV strains interacted with AGO1 and inhibited its microRNA-mediated “slicing” of cellular mRNAs and that the lack of interaction with AGO1 explained the moderate symptoms typically seen in plants infected with mild CMV strains. Our work overthrows this paradigm by showing that mild strain CMV 2b proteins can interact with AGO1, but their
in vivo
localization prevents them from interacting with AGO1 molecules present in the infected cell cytoplasm.
Zebrafish
phd1
enhances mavs-mediated antiviral responses in a hydroxylation-independent manner
Authors: Yu Guangqing , Yuan Le , Li Xiong , Zuo Mingzhong , Wang Rui , Chen Mengjuan , Liu Yuqing , Liu Xing , Xiao Wuhan
Published: August 2024

PHD1 is a member of the prolyl hydroxylase domain protein (PHD1–4) family, which plays a prominent role in the post-translational modification of its target proteins by hydroxylating proline residues. The best-characterized targets of PHD1 are hypoxia-inducible factor α (HIF-1α and HIF-2α), two master regulators of the hypoxia signaling pathway. In this study, we show that zebrafish phd1 positively regulates mavs-mediated antiviral innate immunity. Overexpression of
phd1
enhances the cellular antiviral response. Consistently, zebrafish lacking
phd1
are more susceptible to spring viremia of carp virus infection. Further assays indicate that phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, zebrafish phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. However, the enzymatic activity of phd1 is not required for phd1 to activate mavs. In conclusion, this study reveals a novel function of phd1 in the regulation of antiviral innate immunity.

IMPORTANCE

PHD1 is a key regulator of the hypoxia signaling pathway, but its role in antiviral innate immunity is largely unknown. In this study, we found that zebrafish
phd1
enhances cellular antiviral responses in a hydroxylation-independent manner. Phd1 interacts with mavs through the C-terminal transmembrane domain of mavs and promotes mavs aggregation. In addition, phd1 attenuates K48-linked polyubiquitination of mavs, leading to stabilization of mavs. Zebrafish lacking
phd1
are more susceptible to spring viremia of carp virus infection. These findings reveal a novel role for phd1 in the regulation of mavs-mediated antiviral innate immunity.
Prevotella timonensis
degrades the vaginal epithelial glycocalyx through high fucosidase and sialidase activities
Authors: Segui-Perez Celia , de Jongh Rivka , Jonkergouw Robin L. W. , Pelayo Paula , Balskus Emily P. , Zomer Aldert , Strijbis Karin
Published: August 2024

Bacterial vaginosis (BV) is a polymicrobial infection of the female reproductive tract. BV is characterized by replacement of health-associated
Lactobacillus
species by diverse anerobic bacteria, including the well-known
Gardnerella vaginalis. Prevotella timonensis,
and
Prevotella bivia
are anerobes that are found in a significant number of BV patients, but their contributions to the disease process remain to be determined. Defining characteristics of anerobic overgrowth in BV are adherence to the mucosal surface and the increased activity of mucin-degrading enzymes such as sialidases in vaginal secretions. We demonstrate that
P. timonensis,
but not
P. bivia,
strongly adheres to vaginal and endocervical cells to a similar level as
G. vaginalis
but did not elicit a comparable proinflammatory epithelial response. The
P. timonensis
genome uniquely encodes a large set of mucus-degrading enzymes, including four putative fucosidases and two putative sialidases, PtNanH1 and PtNanH2. Enzyme assays demonstrated that fucosidase and sialidase activities in
P. timonensis
cell-bound and secreted fractions were significantly higher than for other vaginal anerobes. In infection assays,
P. timonensis
efficiently removed fucose and α2,3- and α2,6-linked sialic acid moieties from the epithelial glycocalyx. Recombinantly expressed
P. timonensis
NanH1 and NanH2 cleaved α2,3 and α2,6-linked sialic acids from the epithelial surface, and sialic acid removal by
P. timonensis
could be blocked using inhibitors. This study demonstrates that
P. timonensis
has distinct virulence-related properties that include initial adhesion and a high capacity for mucin degradation at the vaginal epithelial mucosal surface. Our results underline the importance of understanding the role of different anerobic bacteria in BV.

IMPORTANCE

Bacterial vaginosis (BV) is a common vaginal infection that affects a significant proportion of women and is associated with reduced fertility and increased risk of secondary infections.
Gardnerella vaginalis
is the most well-known BV-associated bacterium, but
Prevotella
species including
P. timonensis
and
P. bivia
may also play an important role. We showed that, similar to
G. vaginalis
,
P. timonensis
adhered well to the vaginal epithelium, suggesting that both bacteria could be important in the first stage of infection. Compared to the other bacteria,
P. timonensis
was unique in efficiently removing the protective mucin sugars that cover the vaginal epithelium. These results underscore that vaginal bacteria play different roles in the initiation and development of BV.
Transcriptional regulators
SP110
and
SP140
modulate inflammatory response genes in
Mycobacterium tuberculosis
-infected human macrophages
Authors: Nakamura Hajime , Hikichi Haruka , Seto Shintaro , Hijikata Minako , Keicho Naoto
Published: August 2024

Understanding the functions of human transcriptional regulatory genes
SP110
and
SP140
during
Mycobacterium tuberculosis
infection is crucial; in a mouse model, homologous genes
Sp110
and
Sp140
have been shown to negatively regulate inflammatory response genes, including the type I interferon (IFN) response. The reduction of these genes in mice is associated with susceptibility to
M. tuberculosis
infection and the development of necrotizing granulomatous lesions. To investigate the involvement of
SP110
and
SP140
in human inflammatory response, we analyzed their regulatory manner in THP-1 macrophages infected with
M. tuberculosis
. Genome-wide transcriptional profiling revealed that the depletion of
SP110
and/or
SP140
impaired the induction of gene expression associated with inflammatory responses, including IFN response genes, although it had little effect on the intracellular proliferation of
M. tuberculosis
. By contrast, genes related to phosphorylation were upregulated in infected macrophages with
SP110
and/or
SP140
knockdown, but downregulated in infected control macrophages without their knockdown. Reverse transcription-quantitative PCR and ELISA further confirmed the impairment of the induction of IFN response genes by the depletion of
SP110
and/or
SP140
in
M. tuberculosis
-infected macrophages. These findings suggest that human
SP110
and
SP140
act as positive regulators for genes associated with inflammatory responses in
M. tuberculosis
-infected macrophages.

IMPORTANCE

Tuberculosis (TB) is one of the most serious infectious diseases, with high morbidity and mortality worldwide. C3HeB/FeJ mice are widely utilized for evaluating anti-TB drugs because their drug sensitivity and pathology during
M. tuberculosis
infection resemble those of human TB, including the development of necrotizing granulomas. Downregulation of the transcriptional regulatory genes
Sp110
and
Sp140
in C3HeB/FeJ mice has been demonstrated to activate gene expression associated with inflammatory responses during
M. tuberculosis
infection, resulting in susceptibility to the infection. Here, we examined the regulatory manner of
SP110
and
SP140
using transcriptomic analysis in
M. tuberculosis
-infected human macrophages. Depletion of
SP110
and/or
SP140
in
M. tuberculosis
-infected THP-1 macrophages impaired the induction of gene expression associated with inflammatory responses, including interferon response genes, compared with that in control macrophages. These results suggest that human
SP110
and
SP140
act as positive regulators for genes associated with inflammatory responses upon
M. tuberculosis
infection.
Diversified humoral immunity and impacts of booster vaccines: SARS-CoV-2 antibody profile and Omicron BA.2 neutralization before and after first or second boosters
Authors: Zhang Xiaochun Susan , Windau Anne , Meyers Jamie , Yang Xiaohua , Dong Feng
Published: August 2024

This study aims to investigate humoral immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and assess the impact of booster vaccines. We recruited individuals scheduled to receive either the first (original formula) or the second (bivalent) booster following the initial two-dose SARS-CoV-2 vaccination. We tested for IgG antibodies targeting the spike protein receptor-binding domain (RBD), S1, S2, and nucleocapsid protein, as well as for neutralizing antibodies against Omicron BA.2, before and 14–28 days after receiving the boosters. One year after receiving the initial series of vaccinations, all participants maintained anti-RBD/S1 antibodies. However, levels were lower in individuals who were vaccinated only compared to those who had both vaccination and prior infection (hybrid immunity). Participants with hybrid immunity also showed higher retention of neutralizing antibodies (93% compared to 24% in vaccine-only individuals). Even before receiving any booster shots, participants with hybrid immunity had antibody levels similar to those of vaccine-only individuals after their first booster. After receiving booster shots, antibody levels at 14–28 days were similar regardless of the number of boosters or the type of immunity. About 1 year after the first booster, all participants maintained neutralizing antibodies, and vaccine-only individuals retained about 10 times higher levels of binding antibodies than those without a booster. Humoral immunity varies widely among individuals, and vaccination planning should consider both vaccination and infection history. Boosters are beneficial for increasing antibody levels to ensure sufficient protection against infection and helping bridge the immunity gap between vaccine-only and hybrid immunity.

IMPORTANCE

As we move into the era of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine boosters and shifting from pandemic to endemic, the landscape has changed for both the circulating SARS-CoV-2 variants and population immunity. Even though recent waves of infection have been clinically milder than earlier variants due to the high levels of population immunity and the properties of the Omicron subvariants, vaccination remains crucial for managing COVID-19 in the post-pandemic era. Our study unveils significant variations in the retention of anti-SARS-CoV-2 binding antibody profiles and neutralizing antibody levels 1 year after the primary and the first booster mRNA vaccination. It adds new information regarding how boosters change antibody levels and durability in individuals with hybrid (vaccination plus infection) or vaccine-only (never-infected) immunity. The findings can shed light on future vaccination planning.
Unveiling the multifaceted potential of
Pseudomonas khavaziana
strain SR9: a promising biocontrol agent for wheat crown rot
Authors: Guo Shengzhi , Liu Yuqi , Yin Yanling , Chen Yating , Jia Siyu , Wu Tong , Liao Jun , Jiang Xinyan , Kareem Hafiz Abdul , Li Xuejun , Pan Junfeng , Wang Yao , Shen Xihui
Published: August 2024

Fusarium pseudograminearum
, a soil-borne fungus, is the cause of the devastating wheat disease known as wheat crown rot (WCR). The persistence of this pathogen in the soil and crop residues contributes to the increased occurrence and severity of WCR. Therefore, developing effective strategies to prevent and manage WCR is of great importance. In this study, we isolated a bacterial strain, designated as SR9, from the stem of wheat, that exhibited potent antagonistic effects against
F. pseudograminearum
, as well as the biocontrol efficacy of SR9 on WCR was quantified at 83.99% ± 0.11%. We identified SR9 as
Pseudomonas khavaziana
and demonstrated its potential as a plant probiotic. SR9 displayed broad-spectrum antagonism against other fungal pathogens, including
Neurospora dictyophora
,
Botrytis californica
, and
Botryosphaeria dothidea
. Whole-genome sequencing analysis revealed that SR9 harbored genes encoding various cell wall-degrading enzymes, cellulases, and lipases, along with antifungal metabolites, which are responsible for its antagonistic activity. Gene knockout and quantitative PCR analyses reveal that phenazine is the essential factor for antagonism. SR9 possessed genes related to auxin synthesis, flagellar biosynthesis, biofilm adhesion, and the chemotaxis system, which play pivotal roles in plant colonization and growth promotion; we also evaluated the effects of SR9 on plant growth in wheat and
Arabidopsis
. Our findings strongly suggest that SR9 holds great promise as a biocontrol agent for WCR in sustainable agriculture.

IMPORTANCE

The escalating prevalence of wheat crown rot, primarily attributed to
Fusarium pseudograminearum
, necessitates the development of cost-effective and eco-friendly biocontrol strategies. While plant endophytes are recognized for their biocontrol potential, reports on effective strains targeting wheat crown rot are sparse. This study introduces the
Pseudomonas khavaziana
SR9 strain as an efficacious antagonist to the wheat crown rot pathogen
Fusarium pseudograminearum
. Demonstrating a significant reduction in wheat crown rot incidence and notable plant growth promotion, SR9 emerges as a key contributor to plant health and agricultural sustainability. Our study outlines a biological approach to tackle wheat crown rot, establishing a groundwork for innovative sustainable agricultural practices.
Distinct biogeographic patterns for bacteria and fungi in association with
Bursaphelenchus xylophilus
nematodes and infested pinewood
Authors: Cao Yuyu , Yang Nan , Gu Jianfeng , Zhang Xingyao , Ye Jianren , Chen Jianping , Li Hongjie
Published: August 2024

Pinewood nematodes (PWN,
Bursaphelenchus xylophilus
) are destructive plant parasitic nematodes that cause pine wilt disease (PWD) by attacking the vascular systems of pine trees, resulting in widespread tree mortality. Research has shown that there are connections between nematode-associated microbes and PWD. Yet the variations in microbial communities across different geographic regions are not well-understood. In this study, we examined the bacterial and fungal communities associated with nematodes and infested wood collected from 34 sites across three vegetation zones in China, as well as samples from the United States, using 16S rRNA and internal transcribed spacer (ITS) gene amplicon sequencing. The predominant genera
Pseudomonas
and
Rhodococcus
were found in nematodes, and
Acinetobacter
was present in the wood of PWD-infected pine trees across China. Network analysis revealed that core bacterial taxa belonged to the Pseudomonadota and Actinomycetota phyla for the nematodes, whereas the Pseudomonadota and Bacteroidota phyla were dominant in the infested wood. Identification of enriched key microbial taxa in nematodes and infested wood across vegetation zones indicates distinct biogeographic microbial community structures and key bacterial species. Although the nematode-associated bacterial community showed consistency across geographic distances, the similarity of the PWD pine trees’ bacterial community decreased with distance, suggesting a spatial correlation with environmental variables. Our findings enhance our understanding of the microbiota associated with pinewood nematode (PWN) and offer valuable insights into PWD management.

IMPORTANCE

Our research uncovered specific bacteria and fungi linked to pinewood nematode (PWN) and infested wood in three different vegetation zones in China, as well as samples from the United States. This sheds light on the critical roles of certain microbial groups, such as
Pseudomonas, Acinetobacter
, and
Stenotrophomonas
, in influencing PWN fitness. Understanding these patterns provides valuable insights into the dynamics of PWN-associated microbiomes, offering potential strategies for managing pine wilt disease (PWD). We found significant correlations between geographic distance and similarity in bacterial communities in the infested wood, indicating a spatial influence on wood-associated microbial communities due to limited dispersal and localized environmental conditions. Further investigations of these spatial patterns and driving forces are crucial for understanding the ecological processes that shape microbial communities in complex ecosystems and, ultimately, for mitigating the transmission of PWN in forests.
A comparison of wild boar and domestic pig microbiota does not reveal a loss of microbial species but an increase in alpha diversity and opportunistic genera in domestic pigs
Authors: Rahman Rajibur , Fouhse Janelle M. , Ju Tingting , Fan Yi , S. Marcolla Camila , Pieper Robert , Brook Ryan K. , Willing Benjamin P.
Published: August 2024

The microbiome of wild animals is believed to be co-evolved with host species, which may play an important role in host physiology. It has been hypothesized that the rigorous hygienic practices in combination with antibiotics and diets with simplified formulas used in the modern swine industry may negatively affect the establishment and development of the gut microbiome. In this study, we evaluated the fecal microbiome of 90 domestic pigs sampled from nine farms in Canada and 39 wild pigs sampled from three different locations on two continents (North America and Europe) using 16S rRNA gene amplicon sequencing. Surprisingly, the gut microbiome in domestic pigs exhibited higher alpha-diversity indices than wild pigs (
P
< 0.0001). The wild pig microbiome showed a lower Firmicutes-to-Bacteroidetes ratio and a higher presence of bacterial phyla Elusimicrobiota, Verrucomicrobiota, Cyanobacteria, and Fibrobacterota when compared to their domestic counterparts. At the genus level, the wild pig microbiome had enriched genera that were known for fiber degradation and short-chain fatty acid production. Interestingly, the phylum Fusobacteriota was only observed in domestic pigs. We identified 31 ASVs that were commonly found in the pig gut microbiome, regardless of host sources, which could be recognized as members of the core gut microbiome. Interestingly, we found five ASVs missing in domestic pigs that were prevalent in wild ones, whereas domestic pigs harbored 59 ASVs that were completely absent in wild pigs. The present study sheds light on the impact of domestication on the pig gut microbiome, including the gain of new genera, which might provide the basis to identify novel targets to manipulate the pig gut microbiome for improved health.

IMPORTANCE

The microbiome of pigs plays a crucial role in shaping host physiology and health. This study sought to identify if domestication and current rearing practices have resulted in a loss of co-evolved bacterial species by comparing the microbiome of wild boar and conventionally raised pigs. It provides a comparison of domestic and wild pigs with the largest sample sizes and is the first to examine wild boars from multiple sites and continents. We were able to identify core microbiome members that were shared between wild and domestic populations, and on the contrary to expectation, few microbes were identified to be lost from wild boar. Nevertheless, the microbiome of wild boars had a lower abundance of important pathogenic genera and was distinct from domestic pigs. The differences in the microbial composition may identify an opportunity to shift the microbial community of domestic pigs towards that of wild boar with the intent to reduce pathogen load.
Mining metagenomic data to gain a new insight into the gut microbial biosynthetic potential in placental mammals
Authors: Hu Dini , Zhang Tongzuo , He Shunfu , Pu Tianchun , Yin Yanqiang , Hu Yibo
Published: August 2024

Mammals host a remarkable diversity and abundance of gut microbes. Biosynthetic gene clusters (BGCs) in microbial genomes encode biologically active chemical products and play an important role in microbe–host interactions. Traditionally, the exploration of gut microbial metabolic functions has relied on the pure culture method. However, given the limited amounts of microbes being cultivated, insights into the metabolism of gut microbes in mammals continued to be very limited. In this study, we adopted a computational pipeline for mining the metagenomic data (named taxonomy-guided identification of biosynthetic gene clusters, TaxiBGC) to identify experimentally verified BGCs in 373 metagenomes across 53 mammalian species in an unbiased manner. We demonstrated that polyketides (PKs) and nonribosomal peptides (NRPs) are representative of mammals, and the products derived from them were associated with cell–cell communication and resistance to inflammation. Large carnivores had the highest number of BGCs, followed by large herbivores and small mammals. We also observed that the large mammals had more common BGCs that aid in the biosynthesis of a variety of natural products. However, small mammals not only had fewer BGCs but were also unique to each species. Our results provide novel insights into the mining of metagenomic data sets to identify active BGCs and their products across mammals.

IMPORTANCE

The gut microbes host numerous biosynthetic gene clusters (BGCs) that biosynthesize natural products and impact the host’s physiology. Historically, our understanding of BGCs in mammalian gut microbes was largely based on studies on cultured isolates; however, only a small fraction of mammal-associated microbes have been investigated. The biochemical diversity of the mammalian gut microbiota is poorly understood. Metagenomic sequencing contains data from a vast number of organisms and provides information on the total gene content of communities. Unfortunately, the existing BGC prediction tools are designed for individual microbial genomes. Recently, a BGC prediction tool called the taxonomy-guided identification of biosynthetic gene clusters (TaxiBGC) that directly mine the metagenome was developed. To gain new insights into the microbial metabolism, we used TaxiBGC to predict BGCs from 373 metagenomes across 53 mammalian species representing seven orders. Our findings elucidate the functional activities of complex microbial communities in the gut.
Plumbagin enhances antimicrobial and anti-biofilm capacities of chlorhexidine against clinical
Klebsiella pneumoniae
while reducing resistance mutations
Authors: Liu Haifeng , Chen Huanchang , Ma Zhexiao , Zhang Ying , Zhang Shihang , Zhao Deyi , Yao Zhuocheng , Zhou Tieli , Wang Zhongyong
Published: August 2024

With the widespread misuse of disinfectants, the clinical susceptibility of
Klebsiella pneumoniae
(
K. pneumoniae
) to chlorhexidine (CHX) has gradually diminished, posing significant challenges to clinical disinfection and infection control.
K. pneumoniae
employs overexpression of efflux pumps and the formation of thick biofilms to evade the lethal effects of CHX. Plumbagin (PLU) is a natural plant extract that enhances membrane permeability and reduces proton motive force. In this study, we elucidated the synergistic antimicrobial activity of PLU in combination with CHX, effectively reducing the MIC of CHX against
K. pneumoniae
to 1 µg/mL and below. Crucially, through crystal violet staining and confocal laser scanning microscopy live/dead staining, we discovered that PLU significantly enhances the anti-biofilm capability of CHX. Mechanistically, experiments involving membrane permeability, alkaline phosphatase leakage, reactive oxygen species, and RT-qPCR suggest that the combination of PLU and CHX improves the permeability of bacterial inner and outer membranes, promotes bacterial oxidative stress, and inhibits oqxA/B efflux pump expression. Furthermore, we conducted surface disinfection experiments on medical instruments to simulate clinical environments, demonstrating that the combination effectively reduces bacterial loads by more than 3 log
10
CFU/mL. Additionally, results from resistance mutation frequency experiments indicate that combined treatment reduces the generation of resistant mutants within the bacterial population. In summary, PLU can serve as an adjuvant, enhancing the anti-biofilm capability of CHX and reducing the occurrence of resistance mutations, thereby extending the lifespan of CHX.

IMPORTANCE

As disinfectants are extensively and excessively utilized worldwide, clinical pathogens are progressively acquiring resistance against these substances. However, high concentrations of disinfectants can lead to cross-resistance to antibiotics, and concurrent use of different disinfectants can promote bacterial resistance mutations and facilitate the horizontal transfer of resistance genes, which poses significant challenges for clinical treatment. Compared with the lengthy process of developing new disinfectants, enhancing the effectiveness of existing disinfectants with natural plant extracts is important and meaningful. CHX is particularly common and widely used compared with other disinfectants. Meanwhile,
Klebsiella pneumoniae
, as a clinically significant pathogen, exhibits high rates of resistance and pathogenicity. Previous studies and our data indicate a significant decrease in the sensitivity of clinical
K. pneumoniae
to CHX, highlighting the urgent need for novel strategies to address this issue. In light of this, our research is meaningful.
Impact of carbon dioxide concentrations on laboratory sensitivity of
Mycoplasma
species isolated from dairy cows
Authors: Biesheuvel Marit M. , Kalbfleisch Kristen , De Buck Jeroen , van Engelen Erik , van Schaik Gerdien , Moroni Paolo , Gioia Gloria , Nobrega Diego , Samera Gennerick J. , Adams Paul J. , Walcott Jermaine R. , Barkema Herman W.
Published: August 2024

Conventional
Mycoplasma
spp. diagnostics involve culture, often considered the gold standard in diagnostic test evaluation. However, culture protocols lack empirical derivation and primarily adhere to National Mastitis Council recommendations, tracing back to initial cultivation of
Mycoplasma bovis
. Despite a wide range of carbon dioxide (CO
2
) supplementation reported in literature, specific impacts of CO
2
on
Mycoplasma
spp. growth remain unexplored. Our objective was to assess the effect of CO
2
concentration on growth detection rates of 24
Mycoplasma
spp. isolates from dairy cows. These isolates, mainly
M. bovis
, were incubated at 37°C in triplicate and three dilution ranges under three CO
2
conditions: ambient air or 5% CO
2
or 10% CO
2
. Bacterial growth was evaluated on incubation days 3, 5, 7, and 10. When cultured using ambient air, log
10
cfu/mL was lower on days 3, 5, and 7 of incubation compared with isolates incubated in the recommended 5% or 10% CO
2
, with less variation observed in ambient air compared with 5% or 10% CO
2
. However, by 10 days of incubation, no differences in the detection of observable growth were noted among isolates incubated in ambient air, 5% CO
2
, or 10% CO
2
. Consequently,
Mycoplasma
spp. isolated from dairy cattle demonstrated growth after the recommended 7–10 days of culture, even in the absence of supplemental CO
2
. Given the expected concentration of
M. bovis
in (sub)clinical samples had similar concentrations to those used in our study, with the majority of isolates being
M. bovis
, we recommend expanding CO
2
concentration ranges in
M. bovis
culture from 10% CO
2
to ambient air when incubating for 10 days. However, the turnaround time could be shortened when incubating with supplemental CO
2
.

IMPORTANCE

Current
Mycoplasma
spp. culture protocols lack empirical derivation concerning carbon dioxide (CO
2
) supplementation and are primarily based on the initial cultivation of
Mycoplasma bovis
. This study indicates that the suitable range for CO
2
supplementation is broader than what is currently recommended by the National Mastitis Council for culturing within the specified 7–10 days. No differences in bacterial growth detection rates were observed among ambient air, 5% CO
2
, or 10% CO
2
supplementation during the 7- and 10-day incubation intervals. These new insights provide evidence supporting the possibility of culturing
Mycoplasma
spp. under ambient air conditions in a laboratory setting.
Immune gene expression changes more during a malaria transmission season than between consecutive seasons
Authors: Tebben Kieran , Yirampo Salif , Coulibaly Drissa , Koné Abdoulaye K. , Laurens Matthew B. , Stucke Emily M. , Dembélé Ahmadou , Tolo Youssouf , Traoré Karim , Niangaly Amadou , Berry Andrea A. , Kouriba Bourèma , Plowe Christopher V. , Doumbo Ogobara K. , Lyke Kirsten E. , Takala-Harrison Shannon , Thera Mahamadou A. , Travassos Mark A. , Serre David
Published: August 2024

Plasmodium
parasites, the causative organism of malaria, caused over 600,000 deaths in 2022. In Mali,
Plasmodium falciparum
causes the majority of malaria cases and deaths and is transmitted seasonally. Anti-malarial immunity develops slowly over repeated exposures to
P. falciparum
and some aspects of this immunity (e.g., antibody titers) wane during the non-transmission, dry season. Here, we sequenced RNA from 33 pediatric blood samples collected during
P. falciparum
infections at the beginning or end of a transmission season, and characterized the host and parasite gene expression profiles for paired, consecutive infections. We found that human gene expression changes more over the course of one transmission season than between seasons, with signatures of partial development of an adaptive immune response during one transmission season and stability in gene expression during the dry season. Additionally, we found that
P. falciparum
gene expression did not vary with timing during the season and remained stable both across and between seasons, despite varying human immune pressures. Our results provide insights into the dynamics of anti-malarial immune response development over short time frames that could be exploited by future vaccine and prevention efforts.

IMPORTANCE

Our work seeks to understand how the immune response to
Plasmodium falciparum
malaria changes between infections that occur during low and high malaria transmission seasons, and highlights that immune gene expression changes more during the high transmission season. This provides important insight into the dynamics of the anti-malarial immune response that are important to characterize over these short time frames to better understand how to exploit this immune response with future vaccine efforts.
Bile acids activate the antibacterial T6SS1 in the gut pathogen
Vibrio parahaemolyticus
Authors: Schiffmann Sarah , Mass Shir , Salomon Dor
Published: August 2024

The marine bacterium
Vibrio parahaemolyticus
is a major cause of seafood-borne gastroenteritis in humans and of acute hepatopancreatic necrosis disease in shrimp. Bile acids, produced by the host and modified into secondary bile acids by commensal bacteria in the gastrointestinal tract, induce the virulence factors leading to disease in humans and shrimp. Here, we show that secondary bile acids also activate this pathogen’s type VI secretion system 1, a toxin delivery apparatus mediating interbacterial competition. This finding implies that
Vibrio parahaemolyticus
exploits secondary bile acids to activate its virulence factors and identify the presence of commensal bacteria that it needs to outcompete in order to colonize the host.

IMPORTANCE

Bacterial pathogens often manipulate their host and cause disease by secreting toxic proteins. However, to successfully colonize a host, they must also remove commensal bacteria that reside in it and may compete with them over resources. Here, we find that the same host-derived molecules that activate the secreted virulence toxins in a gut bacterial pathogen,
Vibrio parahaemolyticus
, also activate an antibacterial toxin delivery system that targets such commensal bacteria. These findings suggest that a pathogen can use one cue to launch a coordinated,
trans
-kingdom attack that enables it to colonize a host.
Efficient and rapid one-step method to generate gene deletions in
Streptococcus pyogenes
Authors: Schiavolin Lionel , Lakhloufi Dalila , Botquin Gwenaelle , Deneubourg Geoffrey , Bruyns Corentin , Steinmetz Jenny , Henrot Charlotte , Delforge Valérie , Smeesters Pierre R. , Botteaux Anne
Published: August 2024

Streptococcus pyogenes
or Group A
Streptococcus
(GAS) remains a significant infectious problem around the world, particularly in low- and middle-income settings. Moreover, a recent invasive GAS infection (iGAS) upsurge has been observed in high-income settings. However, to date, no vaccine is available. Finding a good vaccine antigen and understanding the role of virulence factors in GAS infections have been hampered, in part, by technical difficulties to transform the many different strains and generate knockout mutants. Using colE1-type plasmid as a suicide vector, we have set up a method allowing the generation of non-polar mutants of GAS in 3 days.

IMPORTANCE

Group A Streptococcus (GAS) is a major human pathogen, causing diseases ranging from mild and superficial infections of the skin and pharyngeal epithelium to severe systemic and invasive diseases. Since June 2022, several European countries, the US, and Australia are facing an upsurge of invasive life-threatening GAS infections. Finding a good vaccine antigen and understanding the role of virulence factors in GAS infections have been hampered, in part, by technical difficulties to transform the many different GAS strains and generate knockout mutants. Moreover, these tools must be adapted to a large range of different strains, since GAS are divided into more than 260 emm-types (M-type). We have set up a method allowing the generation of non-polar mutants of GAS in 3 days and in diverse backgrounds, which contrasts with previously published protocols.
An automated positive selection screen in yeast provides support for boron-containing compounds as inhibitors of SARS-CoV-2 main protease
Authors: Sigurdardóttir Sunniva , Silva Suélen Fernandes , Tiukova Ievgeniia , Alalam Hanna , King Ross D. , Grøtli Morten , Eriksson Leif A. , Sunnerhagen Per
Published: August 2024

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus continues to cause severe disease and deaths in many parts of the world, despite massive vaccination efforts. Antiviral drugs to curb an ongoing infection remain a priority. The virus-encoded 3C-like main protease (MPro; nsp5) is seen as a promising target. Here, with a positive selection genetic system engineered in
Saccharomyces cerevisiae
using cleavage and release of MazF toxin as an indicator, we screened in a robotized setup small molecule libraries comprising ~2,500 compounds for MPro inhibitors. We detected eight compounds as effective against MPro expressed in yeast, five of which are characterized proteasome inhibitors. Molecular docking indicates that most of these bind covalently to the MPro catalytically active cysteine. Compounds were confirmed as MPro inhibitors in an
in vitro
enzymatic assay. Among those were three previously only predicted
in silico
; the boron-containing proteasome inhibitors bortezomib, delanzomib, and ixazomib. Importantly, we establish reaction conditions
in vitro
preserving the MPro-inhibitory activity of the boron-containing drugs. These differ from the standard conditions, which may explain why boron compounds have gone undetected in screens based on enzymatic
in vitro
assays. Our screening system is robust and can find inhibitors of a specific protease that are biostable, able to penetrate a cell membrane, and are not generally toxic. As a cellular assay, it can detect inhibitors that fail in a screen based on an
in vitro
enzymatic assay using standardized conditions, and now give support for boron compounds as MPro inhibitors. This method can also be adapted for other viral proteases.

IMPORTANCE

The coronavirus disease 2019 (COVID-19) pandemic triggered the realization that we need flexible approaches to find treatments for emerging viral threats. We implemented a genetically engineered platform in yeast to detect inhibitors of the virus’s main protease (MPro), a promising target to curb severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Screening molecule libraries, we identified candidate inhibitors and verified them in a biochemical assay. Moreover, the system detected boron-containing molecules as MPro inhibitors. Those were previously predicted computationally but never shown effective in a biochemical assay. Here, we demonstrate that they require a non-standard reaction buffer to function as MPro inhibitors. Hence, our cell-based method detects protease inhibitors missed by other approaches and provides support for the boron-containing molecules. We have thus demonstrated that our platform can screen large numbers of chemicals to find potential inhibitors of a viral protease. Importantly, the platform can be modified to detect protease targets from other emerging viruses.
Comparing the activity of broad-spectrum beta-lactams in combination with aminoglycosides against VIM-producing
Enterobacteriaceae
Authors: Clark Justin A. , Burgess David S.
Published: August 2024

Metallo-beta-lactamase (MBL)-producing carbapenem-resistant
Enterobacteriaceae
(CRE) infections continue to pose a serious threat to healthcare. Due to their unique active site, MBLs evade the activity of many novel beta-lactam/beta-lactamase inhibitor combinations, which have been specifically targeted toward those carbapenemases with serine active sites. Furthermore, resistance to most, if not all, other clinically relevant antimicrobial classes leaves few reliable therapeutic options. Combination therapy has thus played a vital role in the treatment of MBL-producing CRE infections. In this study, we utilized the static time-kill assay to investigate clinically relevant concentrations of cefepime, piperacillin-tazobactam, and meropenem alone and in combination with either amikacin or the novel plazomicin to determine if combinations of routinely used beta-lactam therapy with an aminoglycoside would achieve bactericidal activity against eight clinically isolated Verona integron-encoded MBL (VIM)-producing CRE. Furthermore, we compared this activity to the combination of aztreonam/avibactam, which has shown potent activity against MBL-producing CRE. Both aztreonam/avibactam and meropenem with either aminoglycoside were rapidly bactericidal within 4 hours and remained bactericidal through 24 hours against all isolates with few exceptions. Combinations including cefepime and piperacillin-tazobactam were also rapidly bactericidal, but activity after 24 hours was inconsistent depending upon the partner aminoglycoside and isolate. Further investigation is warranted to elucidate optimal antibiotic exposures against MBL-producing CRE, including novel agents in the pipeline.

IMPORTANCE

Carbapenem-resistant
Enterobacterales
(CRE) are one of the most pressing antimicrobial-resistant threats at present. In addition to exhibiting resistance to many, if not all, commonly used antimicrobial agents, CRE achieves these resistant phenotypes through a variety of mechanisms, each of which can uniquely affect available treatment options. The present study is an
in vitro
investigation of several Verona integron-encoded metallo-beta-lactamase (VIM)-producing CRE isolated from patients at our academic medical center. Because metallo-beta-lactamases (MBLs) are inherently resistant to many of the novel treatments designed to treat CRE due to their different active site composition, we tested several antimicrobial combinations containing routinely utilized broad-spectrum beta-lactams and aminoglycosides. Our results further our understanding of combination therapy options against VIM-producing CRE, including with non-carbapenem-beta-lactams cefepime and piperacillin. By optimizing combinations of existing antimicrobial agents, we hope to expand the available armamentarium against these resistant pathogens.
Supplemental Material: Radiometric dating (40Ar/39Ar and 14C), compositions, and erupted volumes of volcanoes of the Valle de Santiago area (Michoacán-Guanajuato Volcanic Field, Mexico)
Authors: Rangel-Granados Elizabeth , al. et
Published: August 2024

Figures S1–S3 and Tables S1–S3.
Comparative treatment of homeless persons with an infectious disease in the US emergency department setting: a retrospective approach
Authors: Barnes Jessica , Segars Larry , Wasserman Jason Adam , Karabon Patrick , Taylor Tracey A H
Published: August 2024

Background

Research has long documented the increased emergency department usage by persons who are homeless compared with their housed counterparts, as well as an increased prevalence of infectious diseases. However, there is a gap in knowledge regarding the comparative treatment that persons who are homeless receive. This study seeks to describe this potential difference in treatment, including diagnostic services tested, procedures performed and medications prescribed.

Methods

This study used a retrospective, cohort study design to analyse data from the 2007–2010 United States National Hospital Ambulatory Medical Care Survey database, specifically looking at the emergency department subset. Complex sample logistic regression analysis was used to compare variables, including diagnostic services, procedures and medication classes prescribed between homeless and private residence individuals seeking emergency department treatment for infectious diseases. Findings were then adjusted for potential confounding variables.

Results

Compared with private residence individuals, persons who are homeless and presenting with an infectious disease were more likely (adjusted OR: 10.99, CI 1.08 to 111.40, p<0.05) to receive sutures or staples and less likely (adjusted OR: 0.29, CI 0.10 to 0.87, p<0.05) to be provided medications when presenting with an infectious disease in US emergency departments. Significant differences were also detected in prescribing habits of multiple anti-infective medication classes.

Conclusion

This study detected a significant difference in suturing/stapling and medication prescribing patterns for persons who are homeless with an infectious disease in US emergency departments. While some findings can likely be explained by the prevalence of specific infectious organisms in homeless populations, other findings would benefit from further research.
Sex differences in early-onset atrial fibrillation in Norwegian primary care: a retrospective national database analysis
Authors: Kalstø Silje Madeleine , Nygård Ståle , Ariansen Inger , Tveit Arnljot , Christophersen Ingrid Elisabeth
Published: August 2024

Background

Individual variation in the need for healthcare constitutes knowledge gaps for young atrial fibrillation (AF) patients. We aimed to estimate the prevalence and primary care burden of early-onset AF in Norway, emphasising sex differences, in a nationwide healthcare database.

Methods

We used data from the Norwegian Control and Payment of Health Reimbursement database to identify all Norwegian residents ≥18 years of age registered with a primary care physician (PCP) in 2019, with onset of AF at ≤50 years of age (early-onset AF) in the period 2006–2019. From the accumulated number of early-onset AF cases among current residents, we calculated the prevalence in 2019. The group-level primary care burden was calculated as the total number of annual AF consultations divided by the annual number of AF patients (2014–2018), and individual burden as the mean number of consultations per AF patient per year within the study period. We analysed the distribution of AF consultations between PCP and primary care emergency room (ER) services in total and by sex.

Results

We identified 10 925 Norwegian residents with early-onset AF in 2019 (26.3% women, mean age 48.4 years). The prevalence of early-onset AF was 0.34% (women: 0.19%, men: 0.50%). The early-onset AF population had on average one annual primary care consultation for AF. The individual burden of annual AF consultations varied widely; <1: 66% of women and 54% of men, (1–5]: 25% of women and 36% of men, (5–10]: 6% of women and 8% of men, ≥10: 2% of women and 2% of men. A higher proportion of men (71%) than women (38%) attended both PCP and ER services due to AF.

Conclusions

The study confirmed a low prevalence of early-onset AF, with substantial sex differences and individual variation in primary healthcare needs. Our results signal a need for a higher resolution with regard to age groups in future research on burden and sex differences in early-onset AF.