bluuebunny/crossref_metadata_2025 huggingface dataset:
Annual Volume and Distribution of Physical Training in Norwegian Female Cross-Country Skiers and Biathletes: A Comparison Between Sports, Competition Levels, and Age Categories—The FENDURA Project
Authors: Osborne John O. , Solli Guro S. , Engseth Tina P. , Welde Boye , Morseth Bente , Noordhof Dionne A. , Sandbakk Øyvind , Andersson Erik P.
Published: October 2023

Purpose: To describe and compare the annual physical training characteristics between Norwegian female cross-country (XC) skiers and biathletes across competition levels and age categories. Methods: Daily training sessions for 1 year were recorded for 45 XC skiers and 26 biathletes, comprising international/national team (inter[national]) and nonnational/regional team members (nonnational) of both junior and senior age. Endurance, strength, flexibility, speed, and power training sessions were recorded. Data included exercise modality, intensity, and duration. Data were analyzed using linear mixed-effects models. Results: The total annual physical training volume consisted of ∼90% endurance training for both groups, although XC skiers had significantly higher total volumes (∼10%; P = .003; d = 0.78) than biathletes. Senior XC skiers performed more training hours of skiing and/or roller skiing compared with biathletes over the season. However, biathletes compensated for this lower volume by more skating and a higher proportion of endurance training as skiing (81% [17%]) compared with XC skiers (68% [16%]; P < .001; d = 0.94). Overall, (inter)national-level athletes completed a higher annual training volume than non-national-level athletes (740 [90] h vs 649 [95] h; P = .004;d = 0.81). Although juniors reported less endurance volume than seniors, they maintained a relatively stable level of endurance training across the preparatory and competition period, unlike senior athletes. Conclusions: The higher annual physical training volume by XC skiers compared with biathletes is likely caused by the different demands of the 2 sports; XC skiing necessitates training for 2 skiing styles, while biathlon requires additional shooting practice. However, biathletes compensate with a higher proportion of ski training, particularly in the skating technique.
Impact of an Online Rater Training on Scoring Accuracy of Two Skills on the Test of Gross Motor Development-3 Among Children With Developmental Disabilities: A Pilot Study
Authors: Maeng Hyokju , Shapiro Deborah R. , Webster Elizabeth Kipling , Kwon Hyunjin
Published: October 2023

Rater training is necessary to accurately evaluate fundamental motor skills among children with developmental disabilities (DD). The purpose of this pilot study was to examine the impact of an online rater training program for novice raters on scoring accuracy of the run and two-hand strike skills on the Test of Gross Motor Development—Third Edition (TGMD-3) among children with DD. A training program was created consisting of two units/modules. The first module introduced the TGMD-3 and the second outlined behavior, movement characteristics, and correct scoring for the run and two-hand strike for children with DD. Forty-one novice raters completed three rounds of training and scoring. Data analysis compared the change of rating accuracy of novice raters on the run and two-hand strike skills of the TGMD-3 across three different occasions. There was a statistically significant positive impact on the scoring accuracy of novice raters for the run, two-hand strike skills, and the total skill score (p < .001). The TGMD-3 online rater training program for novices in the present study could provide a model training program to improve the accuracy of scoring fundamental motor skills among children with DD.
An atomic-scale multi-qubit platform
Authors: Wang Yu , Chen Yi , Bui Hong T. , Wolf Christoph , Haze Masahiro , Mier Cristina , Kim Jinkyung , Choi Deung-Jang , Lutz Christopher P. , Bae Yujeong , Phark Soo-hyon , Heinrich Andreas J.
Published: October 2023

Individual electron spins in solids are promising candidates for quantum science and technology, where bottom-up assembly of a quantum device with atomically precise couplings has long been envisioned. Here, we realized atom-by-atom construction, coherent operations, and readout of coupled electron-spin qubits using a scanning tunneling microscope. To enable the coherent control of “remote” qubits that are outside of the tunnel junction, we complemented each electron spin with a local magnetic field gradient from a nearby single-atom magnet. Readout was achieved by using a sensor qubit in the tunnel junction and implementing pulsed double electron spin resonance. Fast single-, two-, and three-qubit operations were thereby demonstrated in an all-electrical fashion. Our angstrom-scale qubit platform may enable quantum functionalities using electron spin arrays built atom by atom on a surface.
Y chromosome proteins in female tissues
Authors: Gelfand Bradley D. , Ambati Jayakrishna
Published: October 2023

Unreliable protein-based tools are impeding sex-based research
Hormone-mediated neural remodeling orchestrates parenting onset during pregnancy
Authors: Ammari Rachida , Monaca Francesco , Cao Mingran , Nassar Estelle , Wai Patty , Del Grosso Nicholas A. , Lee Matthew , Borak Neven , Schneider-Luftman Deborah , Kohl Johannes
Published: October 2023

During pregnancy, physiological adaptations prepare the female body for the challenges of motherhood. Becoming a parent also requires behavioral adaptations. Such adaptations can occur as early as during pregnancy, but how pregnancy hormones remodel parenting circuits to instruct preparatory behavioral changes remains unknown. We found that action of estradiol and progesterone on galanin (Gal)–expressing neurons in the mouse medial preoptic area (MPOA) is critical for pregnancy-induced parental behavior. Whereas estradiol silences MPOA
Gal
neurons and paradoxically increases their excitability, progesterone permanently rewires this circuit node by promoting dendritic spine formation and recruitment of excitatory synaptic inputs. This MPOA
Gal
-specific neural remodeling sparsens population activity in vivo and results in persistently stronger, more selective responses to pup stimuli. Pregnancy hormones thus remodel parenting circuits in anticipation of future behavioral need.
Donor-derived regulatory dendritic cell infusion modulates effector CD8+T cell and NK cell responses after liver transplantation
Authors: Tran Lillian M. , Macedo Camila , Zahorchak Alan F. , Gu Xinyan , Elinoff Beth , Singhi Aatur D. , Isett Brian , Zeevi Adriana , Sykes Megan , Breen Kevin , Srivastava Avantika , Ables Erin M. , Landsittel Douglas , Styn Mindi A. , Humar Abhinav , Lakkis Fadi G. , Metes Diana M. , Thomson Angus W.
Published: October 2023

Immune cell–based therapies are promising strategies to facilitate immunosuppression withdrawal after organ transplantation. Regulatory dendritic cells (DCreg) are innate immune cells that down-regulate alloimmune responses in preclinical models. Here, we performed clinical monitoring and comprehensive assessment of peripheral and allograft tissue immune cell populations in DCreg-infused live-donor liver transplant (LDLT) recipients up to 12 months (M) after transplant. Thirteen patients were given a single infusion of donor-derived DCreg 1 week before transplant (STUDY) and were compared with 40 propensity-matched standard-of-care (SOC) patients. Donor-derived DCreg infusion was well tolerated in all STUDY patients. There were no differences in postoperative complications or biopsy-confirmed acute rejection compared with SOC patients up to 12M. DCreg administration was associated with lower frequencies of effector T-bet+Eomes+CD8+T cells and CD16brightnatural killer (NK) cells and an increase in putative tolerogenic CD141+CD163+DCs compared with SOC at 12M. Antidonor proliferative capacity of interferon-γ+(IFN-γ+) CD4+and CD8+T cells was lower compared with antithird party responses in STUDY participants, but not in SOC patients, at 12M. In addition, lower circulating concentrations of interleukin-12p40 (IL-12p40), IFN-γ, and CXCL10 were detected in STUDY participants compared with SOC patients at 12M. Analysis of 12M allograft biopsies revealed lower frequencies of graft-infiltrating CD8+T cells, as well as attenuation of cytolytic TH1 effector genes and pathways among intragraft CD8+T cells and NK cells, in DCreg-infused patients. These reductions may be conducive to reduced dependence on immunosuppressive drug therapy or immunosuppression withdrawal.
Antibacterial efficacy of an ultra-short palmitoylated random peptide mixture in mouse models of infection by carbapenem-resistant
Klebsiella pneumoniae
Authors: Lau Jonathan Z. , Kuo Shanny Hsuan , Belo Yael , Malach Einav , Maron Bar , Caraway Hannah E. , Oh Myung Whan , Zhang Yi , Ismail Nahed , Lau Gee W. , Hayouka Zvi
Published: October 2023

Indiscriminate use of antibiotics has imposed a selective pressure for the rapid rise in bacterial resistance, creating an urgent need for novel therapeutics for managing bacterial infectious diseases while counteracting bacterial resistance. Carbapenem-resistant
Klebsiella pneumoniae
strains have become a major challenge in modern medicine due to their ability to cause an array of severe infections. Recently, we have shown that the 20-mer random peptide mixtures are effective therapeutics against three ESKAPEE pathogens. Here, we evaluated the toxicity, biodistribution, bioavailability, and efficacy of the ultra-short palmitoylated 5-mer phenylalanine:lysine (FK5P) random peptide mixtures against multiple clinical isolates of carbapenem-resistant
K. pneumoniae
and
K. oxytoca
. We demonstrate the FK5P rapidly and effectively killed various strains of
K. pneumoniae
, inhibited the formation of biofilms, and disrupted mature biofilms. FK5P displayed strong toxicity profiles both
in vitro
and in mice, with prolonged favorable biodistribution and a long half-life. Significantly, FK5P reduced the bacterial burden in mouse models of acute pneumonia and bacteremia and increased the survival rate in a mouse model of bacteremia. Our results demonstrate that FK5P is a safe and promising therapy against
Klebsiella
species as well as other ESKAPEE pathogens.
Identification of potential inhibitors of casein kinase 2 alpha of
Plasmodium falciparum
with potent
in vitro
activity
Authors: Tomaz Kaira C. P. , Tavella Tatyana A. , Borba Joyce V. B. , Salazar-Alvarez Luis C. , Levandoski João E. , Mottin Melina , Sousa Bruna K. P. , Moreira-Filho José T. , Almeida Vitor M. , Clementino Leandro C. , Bourgard Catarina , Massirer Katlin B. , Couñago Rafael M. , Andrade Carolina H. , Sunnerhagen Per , Bilsland Elizabeth , Cassiano Gustavo C. , Costa Fabio T. M.
Published: October 2023

Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In
Plasmodium
, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative
Pf
CK2α inhibitors, we utilized an
in silico
chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (
542
), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that
542
inhibits yeast Cka1, which has a hinge region with high similarity to
Pf
CK2α. This finding is in agreement with our
in silico
results suggesting that
542
inhibits
Pf
CK2α via hinge region interaction.
Effect of malate on the activity of ciprofloxacin against
Pseudomonas aeruginosa
in different
in vivo
and
in vivo
-like infection models
Authors: Bao Xuerui , Goeteyn Ellen , Crabbé Aurélie , Coenye Tom
Published: October 2023

The clinical significance of
Pseudomonas aeruginosa
infections and the tolerance of this opportunistic pathogen to antibiotic therapy makes the development of novel antimicrobial strategies an urgent need. We previously found that D,L-malic acid potentiates the activity of ciprofloxacin against
P. aeruginosa
biofilms grown in a synthetic cystic fibrosis sputum medium by increasing metabolic activity and tricarboxylic acid cycle activity. This suggested a potential new strategy to improve antibiotic therapy in
P. aeruginosa
infections. Considering the importance of the microenvironment on microbial antibiotic susceptibility, the present study aims to further investigate the effect of D,L-malate on ciprofloxacin activity against
P. aeruginosa
in physiologically relevant infection models, aiming to mimic the infection environment more closely. We used
Caenorhabditis elegans
nematodes,
Galleria mellonella
larvae, and a 3-D lung epithelial cell model to assess the effect of D,L-malate on ciprofloxacin activity against
P. aeruginosa
. D,L-malate was able to significantly enhance ciprofloxacin activity against
P. aeruginosa
in both
G. mellonella
larvae and the 3-D lung epithelial cell model. In addition, ciprofloxacin combined with D,L-malate significantly improved the survival of infected 3-D cells compared to ciprofloxacin alone. No significant effect of D,L-malate on ciprofloxacin activity against
P. aeruginosa
in
C. elegans
nematodes was observed. Overall, these data indicate that the outcome of the experiment is influenced by the model system used which emphasizes the importance of using models that reflect the
in vivo
environment as closely as possible. Nevertheless, this study confirms the potential of D,L-malate to enhance ciprofloxacin activity against
P. aeru
ginosa-associated infections.
Competitive interactions facilitate resistance development against antimicrobials
Authors: Svet Luka , Parijs Ilse , Isphording Simon , Lories Bram , Marchal Kathleen , Steenackers Hans P.
Published: October 2023

While the evolution of antimicrobial resistance is well studied in free-living bacteria, information on resistance development in dense and diverse biofilm communities is largely lacking. Therefore, we explored how the social interactions in a duo-species biofilm composed of the brewery isolates
Pseudomonas rhodesiae
and
Raoultella terrigena
influence the adaptation to the broad-spectrum antimicrobial sulfathiazole. Previously, we showed that the competition between these brewery isolates enhances the antimicrobial tolerance of
P. rhodesiae
. Here, we found that this enhanced tolerance in duo-species biofilms is associated with a strongly increased antimicrobial resistance development in
P. rhodesiae
. Whereas
P. rhodesiae
was not able to evolve resistance against sulfathiazole in monospecies conditions, it rapidly evolved resistance in the majority of the duo-species communities. Although the initial presence of
R. terrigena
was thus required for
P. rhodesiae
to acquire resistance, the resistance mechanisms did not depend on the presence of
R. terrigena
. Whole genome sequencing of resistant
P. rhodesiae
clones showed no clear mutational hot spots. This indicates that the acquired resistance phenotype depends on complex interactions between low-frequency mutations in the genetic background of the strains. We hypothesize that the increased tolerance in duo-species conditions promotes resistance by enhancing the selection of partially resistant mutants and opening up novel evolutionary trajectories that enable such genetic interactions. This hypothesis is reinforced by experimentally excluding potential effects of increased initial population size, enhanced mutation rate, and horizontal gene transfer. Altogether, our observations suggest that the community mode of life and the social interactions therein strongly affect the accessible evolutionary pathways toward antimicrobial resistance.

IMPORTANCE

Antimicrobial resistance is one of the most studied bacterial properties due to its enormous clinical and industrial relevance; however, most research focuses on resistance development of a single species in isolation. In the present study, we showed that resistance evolution of brewery isolates can differ greatly between single- and mixed-species conditions. Specifically, we observed that the development of antimicrobial resistance in certain species can be significantly enhanced in co-culture as compared to the single-species conditions. Overall, the current study emphasizes the need of considering the within bacterial interactions in microbial communities when evaluating antimicrobial treatments and resistance evolution.
Rapid phenotypic antimicrobial susceptibility testing of Gram-negative rods directly from positive blood cultures using the novel Q-linea ASTar system
Authors: Esse Jan , Träger Johannes , Valenza Giuseppe , Bogdan Christian , Held Jürgen
Published: October 2023

Adequate and timely antibiotic therapy is crucial for the treatment of sepsis. Innovative systems, like the Q-linea ASTar, have been developed to perform rapid antimicrobial susceptibility testing (AST) directly from positive blood cultures (BCs). We conducted a prospective study to evaluate ASTar under real-life conditions with a focus on time-to-result and impact on antimicrobial therapy. Over 2 months, all positive BCs that showed Gram-negative rods upon microscopy were tested with the ASTar and our standard procedure (VITEK 2 from short-term culture). Additionally, we included multidrug-resistant Gram-negative bacteria from our archive. Both methods were compared to broth microdilution. In total, 78 bacterial strains (51 prospective and 27 archived) were tested. ASTar covered 94% of the species encountered. The categorical and essential agreement was 95.6% and 90.7%, respectively. ASTar caused 2.4% minor, 2.0% major, and 2.4% very major errors. The categorical agreement was similar to standard procedure. The average time between BC sampling and the availability of the antibiogram for the attending physician was 28 h 49 min for ASTar and 44 h 18 min for standard procedure. ASTar correctly identified all patients who required an escalation of antimicrobial therapy and 75% of those who were eligible for de-escalation. In conclusion, ASTar provided reliable AST results and significantly shortened the time to obtain an antibiogram. However, the percentage of patients that will profit from ASTar in a low-resistance setting is limited, and it is currently unclear if a change of therapy 29 h after BC sampling will have a significant impact on the patient’s prognosis.
A CRISPR-based system to investigate HBV cccDNA biology
Authors: Seeger Christoph
Published: October 2023

Chronic hepatitis B virus (HBV) infections affect over 250 million people worldwide, and over 800,000 are expected to die yearly from complications including cirrhosis and primary hepatocellular carcinoma. While clinically available nucleoside analog-based antiviral therapies inhibit virus production, they cannot cure infections due to the persistence of covalently closed circular (cccDNA) in the nuclei of infected hepatocytes. Hence, the goals of future curative antiviral therapies for chronic hepatitis B are to either eliminate or permanently silence transcription from cccDNA. Novel antiviral approaches building on a better understanding of the mechanisms underlying transcriptional regulation and the stability of HBV cccDNA will be necessary to accomplish such goals. To advance our understanding of cccDNA biology, we have employed a CRISPR/Cas9-based system to produce extrachromosomal circular (ec) DNA from plasmid DNA integrated into the host genome originally developed by Moller et al. [Nucleic Acid Research 46:(22) e131, 2018]. We modified the enhanced green fluorescent protein (EGFP) reporter system to include portions of the HBV genome encoding wild-type and inactive hepatitis B virus X (HBx) proteins on ecDNA to mimic cccDNA. We demonstrated that expression of EGFP is inhibited by the SMC5/6 complex, as is cccDNA, and that HBx expression can reverse transcriptional silencing of ecDNA. Moreover, we demonstrated that ecDNA is lost during cell division. The system described in this report permits investigations on ecDNA and enables large-scale screening of compound libraries for small molecules targeting host factors involved in ecDNA and, by inference, cccDNA transcription, maintenance, and loss during cell division.

IMPORTANCE

Hepatitis B virus cccDNA is the key target for the necessary development of antiviral therapies aimed at curing chronic hepatitis B. The CRISPR-based system to produce covalently closed circular (cccDNA)-like extrachromosomal DNAs described in this report enables large-scale screens of chemical libraries to identify drug candidates with the potential to permanently inactivate cccDNA. Moreover, this approach permits investigations on unresolved problems as described in this report concerning cccDNA biology including mechanisms of SMC5/6-dependent transcriptional silencing and the contributions of the SMC5/6 complex to cccDNA stability in resting and dividing hepatocytes.
The neuraminidase activity of influenza A virus determines the strain-specific sensitivity to neutralization by respiratory mucus
Authors: Iseli Alena N. , Pohl Marie O. , Glas Irina , Gaggioli Elisabeth , Martínez-Barragán Patricia , David Shannon C. , Schaub Aline , Luo Beiping , Klein Liviana K. , Bluvshtein Nir , Violaki Kalliopi , Motos Ghislain , Hugentobler Walter , Nenes Athanasios , Krieger Ulrich K. , Peter Thomas , Kohn Tamar , Stertz Silke
Published: October 2023

Respiratory mucus lining the airway epithelium constitutes an important first line of defense against infection with influenza A virus (IAV). Previous studies suggested that the inhibitory potential of mucus relies not only on the presence of decoy receptors but also on the dense meshwork blocking virus access to the epithelium independent of sialic acid. Here, we report for a panel of human and avian IAV isolates that susceptibility to neutralization by respiratory mucus varies in a strain-dependent manner. Our data reveal that viral neuraminidase (NA) activity inversely correlates with the mucus sensitivity of a given strain. As NA cleaves sialic acid from glycan chains and thus releases IAV from decoy receptor binding, our results imply that virus inhibition by mucus is mainly driven by decoy receptor binding of IAV. Other inhibitory properties of mucus, such as size exclusion, non-covalent interactions with mucus components, or the presence of immunomodulatory molecules, likely play minor roles in neutralizing IAV. Inhibition of NA activity using oseltamivir was sufficient to increase the sensitivity of a seasonal IAV strain to mucus during infection in a cell-based neutralization assay and in differentiated primary human airway epithelial cultures. Overall, our data highlight the importance of NA for the initiation of virus infection besides its major function in release, especially in overcoming the host defense mechanism of mucociliary clearance in the airway epithelium. Furthermore, strain-specific differences in the ability to penetrate respiratory mucus may be the result of host adaptation and can have consequences for the transmissibility and infectivity of circulating viruses.

IMPORTANCE

The respiratory tract of humans is constantly exposed to potentially harmful agents, such as small particles or pathogens, and thus requires protective measures. Respiratory mucus that lines the airway epithelia plays a major role in the prevention of viral infections by limiting the mobility of viruses, allowing subsequent mucociliary clearance. Understanding the interplay between respiratory mucus and viruses can help elucidate host and virus characteristics that enable the initiation of infection. Here, we tested a panel of primary influenza A viruses of avian or human origin for their sensitivity to mucus derived from primary human airway cultures and found that differences between virus strains can be mapped to viral neuraminidase activity. We also show that binding of influenza A viruses to decoy receptors on highly glycosylated mucus components constitutes the major inhibitory function of mucus against influenza A viruses.
Establishment of tongue microbiota by 18 months of age and determinants of its microbial profile
Authors: Kageyama Shinya , Ma Jiale , Furuta Michiko , Takeshita Toru , Asakawa Mikari , Okabe Yuka , Yamashita Yoshihisa
Published: October 2023

Acquisition of oral bacteria early in life is considered to have an important role in the establishment of oral microbiota. In this study, we examined tongue swab samples collected from 216 infants at the 18-month checkup, along with samples collected from them and from their mothers at the 4-month checkup. The bacterial composition of each sample was determined using PacBio single-molecule long-read sequencing of the full-length 16S rRNA gene and amplicon sequence variant (ASV) analysis. At 18 months of age, the tongue microbiota was dominated by
Streptococcus salivarius
,
Neisseria perflava
, and
Granulicatella adiacens
; and the overall bacterial composition was more similar to that of the mothers than at 4 months of age. The bacterial composition at 18 months of age was classified into three microbiota profiles:
S. salivarius
-dominant,
Neisseria
-dominant, and infant profile dominated by 4 months specific ASVs such as
S. salivarius
,
Streptococcus lactarius
, and
Streptococcus peroris
. Those with infant profiles exhibited significantly higher rates of current breastfeeding, dental caries or white spot lesions, and dental plaque accumulation compared to infants with the other profiles. Comparison between the
S. salivarius
- and
Neisseria
-dominant profiles showed that incomplete weaning, low intake of fruits, and frequent intake of sweetened beverages or sweet snacks were significantly associated with the
S. salivarius
-dominant profile. Our findings, based on a high-resolution profiling approach, demonstrate that the foundation of the adult tongue microbiota is established by 18 months of age, and the bacterial profile is possibly determined by dietary habits during this period.

IMPORTANCE

Understanding the development of oral microbiota early in life and the factors that influence it is important for preventing the establishment of dysbiotic oral microbiota later in life. This study demonstrates that the tongue microbiota undergoes early development from 4 to 18 months of age and converges into two types of microbiota showing indications of adult characteristics, with either
S. salivarius
or
Neisseria
-dominance. Interestingly, their divergence was strongly determined by their weaning status and the dietary frequencies of sweetened beverages, snacks, and fruits, suggesting that dietary habits during this period might influence the establishment of the oral microbiota. These findings may contribute to the development of novel preventive strategies against oral microbiota-related diseases.
Genome sequences of
bla
NDM-1
producing
Acinetobacter lactucae
isolated from immunocompromised patients in India
Authors: Amar Ashutosh Kumar , Sawant Ajit Ramesh , Manoharan Meerabai , Tomar Archana , Sistla Sujatha , Prashanth K
Published: October 2023

Among the species within
Acb-
complex,
Acinetobacter lactucae
has not been frequently isolated from clinical settings, unlike
Acinetobacter baumannii
, which is an important nosocomial pathogen. We report the genomic sequences of
A. lactucae
strains (PKAL1732 and 1828C) harboring multiple-resistance determinants including metallo-β-lactamase (
bla
NDM-1
) isolated from immunocompromised patients admitted to a referral hospital in India.
Stenotrophomonas maltophilia
affects the gene expression profiles of the major pathogens
Pseudomonas aeruginosa
and
Staphylococcus aureus
in an
in vitro
multispecies biofilm model
Authors: Alio Ifey , Moll Raphael , Hoffmann Tim , Mamat Uwe , Schaible Ulrich E. , Pappenfort Kai , Alawi Malik , Schie Marcel , Thünauer Roland , Stamm Johanna , Rohde Holger , Streit Wolfgang R.
Published: October 2023

Stenotrophomonas maltophilia
is an emerging lung pathogen. In its natural habitat, it lives together with
Pseudomonas aeruginosa
,
Staphylococcus aureus
, and other pathogens. Here, we provide the first evidence that
S. maltophilia
interferes with the metabolism and physiology of other species when co-cultivated in dual- and triple-species biofilms. CSLM analyses implied that
S. maltophilia
was in general the first to colonize the bottom layer in multispecies biofilms. Structural patterns and niche formation resulting in distinct layer formation within the biofilm were observed on a species- and strain-dependent level for
S. maltophilia
. Furthermore, gene expression profiles of
S. aureus
and
P. aeruginosa
were strongly affected by the presence of
S. maltophilia
. The
S. maltophilia
metabolism was mostly fermentative in multispecies biofilms with varying sets of cytochromes used for anaerobic respiration. One of the most striking observations was that
S. maltophilia
interfered with
P. aeruginosa
LasI-dependent expression of quorum sensing-regulated pathogenicity factors in multispecies biofilms.

IMPORTANCE

In the past, studies have focused on bacterial pathogenicity in mono-species infections, in part ignoring the clinical relevance of diseases caused by more than one pathogen (i.e., polymicrobial infections). However, it is now common knowledge that multiple bacteria species are often involved in the course of an infection. For treatment of such infections, it is absolutely important to understand the dynamics of species interactions at possible infection sites and the molecular mechanisms behind these interactions. Here, we studied the impact of
Stenotrophomonas maltophilia
on its commensals
Pseudomonas aeruginosa
and
Staphylococcus aureus
in multispecies biofilms. We analyzed the 3D structural architectures of dual- and triple-species biofilms, niche formation within the biofilms, and the interspecies interactions on a molecular level. RNAseq data identified key genes involved in multispecies biofilm formation and interaction as potential drug targets for the clinical combat of multispecies infection with these major pathogens.
Association of intestinal bacteria with immune activation in a cohort of healthy adults
Authors: Riazati Niknaz , Kable Mary E. , Stephensen Charles B.
Published: October 2023

Interactions among intestinal bacteria and the immune system contribute to the maintenance of a functional intestinal barrier in healthy individuals, and possibly to systemic immune activity. We hypothesized that intestinal bacteria would be associated with systemic biomarkers of innate and adaptive immune responses in healthy adults. 79 immune function markers were subjected to factor analysis resulting in 17 Immune Factors (IFs), each composed of 2–10 immune variables. Bacterial taxa from stool samples were identified at the family and genus levels by 16S rRNA amplicon sequence analysis and their read counts and relative abundances were utilized in a multiple linear regression model to identify microbial taxa associated with the IFs. A total of 10 significant associations were identified between bacterial taxa and IFs. The family
Rikenellaceae
showed a positive association with innate IF5 (including 5 chemokines, 2 cytokines, 2 adhesion molecules, and the macrophage metabolite neopterin) and a negative association with adaptive IF4 (including T-cells with activation marker HLA-DR). Additionally,
Pseudomonadaceae
and its genus
Pseudomonas
showed a negative relationship with innate IF5, and adaptive IF13 (including T-cell cytokines IL-10, IL-17, and IFN-γ) was negatively associated with
Butyrivibrio
and positively associated with
Slackia
. These associations suggest ongoing interactions between gut bacteria and the systemic immune system in healthy adults. The association of these taxa with the IFs may result from specific microbial-immune system interactions that play a role in maintenance of a healthy barrier integrity in our cohort of healthy adults.

IMPORTANCE

Chronic inflammation may develop over time in healthy adults as a result of a variety of factors, such as poor diet directly affecting the composition of the intestinal microbiome, or by causing obesity, which may also affect the intestinal microbiome. These effects may trigger the activation of an immune response that could eventually lead to an inflammation-related disease, such as colon cancer. Before disease develops it may be possible to identify subclinical inflammation or immune activation attributable to specific intestinal bacteria normally found in the gut that could result in future adverse health impacts. In the present study, we examined a group of healthy men and women across a wide age range with and without obesity to determine which bacteria were associated with particular types of immune activation to identify potential preclinical markers of inflammatory disease risk. Several associations were found that may help develop dietary interventions to lower disease risk.
Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand
Authors: Noparatvarakorn Chawitar , Jakkul Wallop , Seng Rathanin , Tandhavanant Sarunporn , Ottiwet Orawan , Janon Rachan , Saikong Wadsamon , Chantratita Narisara
Published: October 2023

Melioidosis is a severe infectious disease caused by
Burkholderia pseudomallei
. Effective treatment of melioidosis requires rapid detection of
B. pseudomallei
infection. Previous studies demonstrated that real-time PCR for the diagnosis of melioidosis in blood samples was insensitive. We optimized the DNA extraction method and evaluated real-time PCRs with an internal control for the detection of
B. pseudomallei
in plasma samples and compared the performance of real-time PCRs based on six target genes. The lower limit of detection (LOD) of TTS1-
orf2
, BPSS0745, BPSS1187, and BPSS1498 real-time PCRs was ≤10 genome equivalent (GE)/reaction, while the LOD of BPSS0087 and BPSS1492 was 100 GE/reaction. In the initial phase, we evaluated four targets (TTS1-
orf2
, BPSS0745, BPSS1187, BPSS1498) for the diagnosis of melioidosis using 87 plasma samples from 38 bacteremic-melioidosis patients and 49 other infection patients at a hospital in Northeast Thailand. Among these, BPSS1187 exhibited the highest sensitivity of 76.3% and specificity of 100%. A second evaluation phase was conducted at two hospitals in Northeast Thailand to compare two real-time PCRs between BPSS1187 and widely used TTS1-
orf2
targets. A total of 705 clinical samples were collected from 421 suspected melioidosis cases; 77 of them were subsequently confirmed as melioidosis by culture using one or more specimens. The sensitivities of BPSS1187 and TTS1-
orf2
were 89.6% and 79.2%, respectively (
P
= 0.004), and the specificities were 96% and 99%, respectively. The sensitivities of these two assays in plasma samples were 84.6% and 67.7%, respectively (
P
= 0.007). Therefore, the BPSS1187 real-time PCR was sensitive and specific for early diagnosis of melioidosis.

IMPORTANCE

Melioidosis is a serious infectious disease caused by
Burkholderia pseudomallei
, an environmental Gram-negative bacterium. Early detection of
B. pseudomallei
infection is crucial for successful antibiotic treatment and reducing mortality rates associated with melioidosis. Bacteria culture is currently used to identify
B. pseudomallei
in clinical samples, but the method is slow. Therefore, there is a need for more accurate and sensitive molecular-based diagnostic methods that can detect
B. pseudomallei
in all sample types, including samples from blood. We developed an optimal DNA extraction method for
B. pseudomallei
from plasma samples and used an internal control for real-time PCR. We evaluated six PCR target genes and identified the most effective target for the early detection of
B. pseudomallei
infection in patients. To prevent delays in the treatment of melioidosis that can lead to fatal outcomes, we recommend implementing this new approach for routine early detection of
B. pseudomallei
in clinical settings.
Enhancing bacterial fitness and recombinant enzyme yield by engineering the quality control protease HtrA of
Bacillus subtilis
Authors: Öktem Ayşegül , Núñez-Nepomuceno David , Ferrero-Bordera Borja , Walgraeve Jonathan , Seefried Michael , Gesell Salazar Manuela , Steil Leif , Michalik Stephan , Maaß Sandra , Becher Dörte , Mäder Ulrike , Völker Uwe , van Dijl Jan Maarten
Published: October 2023

Bacillus subtilis
is a Gram-positive bacterium that is well-known for its high secretory capacity. Although secretion of homologous proteins is extremely efficient, secretion of heterologous proteins imposes what is known as “secretion stress” on the cells. This is not desirable in an industrial setting, because the bacterial secretion stress responses can adversely affect product yields. So far, the main approach to reduce such counterproductive secretion stress responses has been to remove components of the CssR-CssS two-component regulatory system, including the CssR-CssS-regulated quality control proteases HtrA and/or HtrB. However, there are several examples indicating that a complete loss of these proteases does not correlate with improved protein secretion or a reduced secretion stress response. In this study, an alternative approach to limit the effects of secretion stress by modulating the protease activity of HtrA rather than completely removing this protease was investigated. Indeed, expression of the proteolytically inactive HtrA led to a generally improved fitness of the bacteria when producing the heterologous α-amylase AmyQ. Moreover, compared to control strains, the strain expressing the mutant HtrA provided higher AmyQ yields, which correlated with superior α-amylase enzymatic activity in shake flasks and fermenter-scale cultivations. Proteome analysis of the engineered strains revealed critical differences in stress responses and metabolism. Altogether, our present findings show how proteolytically inactive HtrA can be employed to achieve improved bacterial fitness and higher enzyme yields.

IMPORTANCE

In the expanding market of recombinant proteins, microbial cell factories such as
Bacillus subtilis
are key players. Microbial cell factories experience secretion stress during high-level production of secreted proteins, which can negatively impact product yield and cell viability. The CssRS two-component system and CssRS-regulated quality control proteases HtrA and HtrB play critical roles in the secretion stress response. HtrA has a presumptive dual function in protein quality control by exerting both chaperone-like and protease activities. However, its potential role as a chaperone has not been explored in
B. subtilis
. Here, we describe for the first time the beneficial effects of proteolytically inactive HtrA on α-amylase yields and overall bacterial fitness.
Lasso peptide MccY alleviates non-typhoidal salmonellae-induced mouse gut inflammation via regulation of intestinal barrier function and gut microbiota
Authors: Li Yu , Li Wenjing , Zeng Zhiwei , Han Yu , Chen Qinxi , Dong Xinyi , Wang Zepeng , Feng Saixiang , Cao Weisheng
Published: October 2023

Non-typhoidal salmonellae-induced zoonotic intestinal bleeding infections are a crucial threat to public health worldwide, and the development of new medications is required to prevent and treat non-typhoid
Salmonella
(NTS) infections. MccY, a class of lasso peptide, is effective in biological sterilization against
Salmonella
and
Shigella
. In this study, the therapeutic effect of MccY on
S
. Typhimurium-infected BALB/c mice has been discussed. specific pathogen free (SPF) mice were infected with the ST53 strain with bacterial loads of 10
6
and 10
3
CFU/mL, respectively, and then treated with 9.92 mg/kg BW MccY. Results revealed that MccY significantly reduced mouse mortality by 20%–40% and effectively alleviated weight loss and diarrhea. The integrity of the intestinal structure was protected, and the expression of TNF-α, IL-6, IL-10, and IL-18 was inhibited. Particularly, MccY exerted multiple effects on gut microbiota ecology, including direct elimination of
Salmonella
, showing promise in limiting infection to the gut level and thereby halting the transmission of NTS. MccY indirectly inhibited other opportunistic pathogens and partially restored the microbiota, promoting an increase in
Bacteroidetes
,
Lactobacillus
,
Muribaculum
, and
Clostridium
. Consequently, this work provides an understanding of the link between host infection and therapeutic application of MccY.

IMPORTANCE

Diseases caused by
Enterobacteriaceae
multidrug-resistant strains have become increasingly difficult to manage. It is necessary to verify the new antibacterial drug MccY effect on non-typhoid
Salmonella
infection in mice since it is regarded as a promising microcin. The results demonstrated that MccY has a potential therapeutic application value in the protection against
Salmonella
-induced intestinal damage and alleviating related intestinal dysbiosis and metabolic disorders. MccY could be a promising candidate as an antimicrobial or anti-inflammatory agent for treating infectious diseases.
Protective efficacy of a candidate live attenuated vaccine derived from the SD-R strain of lineage 1 porcine reproductive and respiratory syndrome virus against a lethal challenge with HP-PRRSV HuN4 in piglets
Authors: Zhang Hongliang , Li Chao , Xu Hu , Gong Bangjun , Li Wansheng , Guo Zhenyang , Xiang Lirun , Sun Qi , Zhao Jing , Peng Jinmei , Wang Qian , Zhou Guohui , Tang Yan-Dong , An Tongqing , Cai Xue-Hui , Tian Zhi-Jun
Published: October 2023

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen endangering the swine industry worldwide. Our previous study confirmed that the live attenuated vaccine SD-R strain could provide broad cross-protection against homologous and heterologous NADC30-like viruses. However, it is unclear whether SD-R can provide protection for piglets against HP-PRRSV. In the present study, 4-week-old PRRSV-free piglets were vaccinated intramuscularly with 10
6.2
TCID
50
of SD-R and then challenged intramuscularly (2 mL) and intranasally (2 mL) with HP-PRRSV HuN4 (1 × 10
5.0
TCID
50
/mL). The results showed that two of five piglets died in the HuN4 challenge group and five of five immunized piglets survived in the immune challenge group. Five piglets in the HuN4 challenge group had a fever for 6–12 days and showed obvious clinical symptoms, but 3 of 5 piglets had a fever for only 1 day at 5 days post inoculation, and only 1 of the 5 piglets in the immune challenge group had a cough. Compared to challenged control piglets, vaccinated piglets had significantly improved daily weight gain and obviously alleviated pathological lesions. Furthermore, piglets immunized with SD-R had reduced thymus atrophy caused by HP-PRRSV HuN4. These results demonstrate that the lineage 1 PRRSV attenuated vaccine SD-R strain provides protection against lethal HP-PRRSV HuN4 challenge. As such, it is a promising vaccine candidate for use against PRRSV infection.

IMPORTANCE

Both highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and NADC30-like PRRSV have caused tremendous economic losses to the Chinese pig industry. In this study, a good challenge model was established to evaluate the protection afforded by the candidate SD-R vaccine against infection with a representative HP-PRRSV strain (HuN4). The control piglets in the challenge experiment displayed obvious clinical symptoms of PRRSV infection, with a mortality rate up to 40%. In contrast, all the piglets in the vaccinated challenged group survived, and only some pigs had transient fever. The daily gain of SD-R immunized group piglets was significantly increased, and the pathological changes were significantly reduced. In addition, the viral replication levels in the serum of the immunized group were significantly lower than those of the challenged control group. The live attenuated vaccine SD-R strain can provide protection against HP-PRRSV challenge, indicating that the SD-R strain is a promising vaccine candidate for use in the swine industry.
Emergence and genomic insights of non-pandemic O1
Vibrio cholerae
in Zhejiang, China
Authors: Luo Yun , Payne Michael , Kaur Sandeep , Octavia Sophie , Jiang Jianmin , Lan Ruiting
Published: October 2023

Vibrio cholerae
O1 has caused cholera pandemics. Non-pandemic
V. cholerae
O1 strains, which are genetically distinctive from the pandemic clones, have been isolated from both human infections and the environment. We aimed to better understand the non-pandemic O1 strains and their pandemic potential. We sequenced 109 non-pandemic O1 isolates from Zhejiang, China (from 1963 to 1996) and compared them with 62 publicly available non-pandemic O1 genomes. The isolates from Zhejiang can be classified into three lineages (L1–L3). All grouped together with L3 sharing the most recent common ancestor with the pandemic clones. L2 and L3 emerged in the 1960s while L1 emerged in the 1970s. L1 and L2 disappeared after the 1990s, but L3 persisted until recently. All isolates contained the type VI secretion system. The
Vibrio
pathogenicity island was present in all L3 isolates, whereas the type III secretion system was present in all L1 isolates. L2 did not carry any unique virulence genes. An intact CTXφ was present in only two L3 isolates. An intact
Vibrio
seventh pandemic island 1 was present in only three L3 isolates. The

bla
CARB-7

gene was identified in 96.3% of L2 isolates. Each of the non-pandemic O1 lineages has unique properties contributing to their capacity to cause disease. Our findings offer new insight into the evolution of O1
V. cholerae
for cholera prevention and control.

IMPORTANCE

It is well recognized that only
Vibrio cholerae
O1 causes cholera pandemics. However, not all O1 strains cause pandemic-level disease. In this study, we analyzed non-pandemic O1
V. cholerae
isolates from the 1960s to the 1990s from China and found that they fell into three lineages, one of which shared the most recent common ancestor with pandemic O1 strains. Each of these non-pandemic O1 lineages has unique properties that contribute to their capacity to cause cholera. The findings of this study enhanced our understanding of the emergence and evolution of both pandemic and non-pandemic O1
V. cholerae
.