Intracellular Bacteria Research Articles

“Nano knife” for efficient piezocatalytic inactivation of amoeba spores and their intracellular bacteria: Synergetic effect between physical damage and chemical oxidation

Microbial interactions between infectious agents severely interfere with the disinfection process, and current disinfection methods are unable to effectively inactivate intracellular pathogens, posing a new threat to drinking water safety. In this study, we first reported the high efficiency of piezocatalysis in inactivating amoebae and their intracellular bacteria. Results showed that the inactivation rates of the MoS2/rGO piezocatalytic system for amoebic spores and their intracellular bacteria were 4.18 and 5.02-log, respectively, within 180min. Based on scavenger studies and ESR tests, the efficient inactivation of pathogens can be attributed to the generation of reactive oxygen species (ROS), and different pathogens exhibit varying tolerances to distinct ROS. Moreover, TEM analysis revealed that the sharp edge of MoS2/rGO was conducive to the physical cutting of amoeba's cell wall and membrane, promoting the attack of ROS and ensuring a more thorough deactivation. Additionally, the intracellular ROS produced by amoebae is not only conducive to the inactivation of amoebae but also the main reason for the inactivation of bacteria in spores. This study provides a new solution for the inactivation of amoeba spores and their intracellular bacteria and emphasizes the high efficiency of the synergistic effect of physical damage and chemical oxidation.

Detection of Rickettsia amblyommatis in Amblyomma mixtum and Rhipicephalus microplus Ticks Collected from Cattle in Tamaulipas, Mexico.

Background: Rickettsia spp. are obligate intracellular bacteria of utmost importance for public health and the economy since their presence can generate significant losses in livestock production, affecting the health of animals, the quality of the meat, milk, and other products derived from affected animals. Therefore, prevention of the transmission of these pathogens, their early identification and timely treatment are essential to mitigate their impact on public health and animal production. Materials and Methods: In the present work, hard ticks were collected from infested Bos indicus cattle in Aldama, Tamaulipas, Mexico. They were identified by morphology using dichotomous keys and by sequencing and analyzing of a fragment of the mitochondrial 16S. PCR was performed using specific primers targeting Rickettsia sp. gltA. Phylogenetic analyses were performed, aligning the amino acid sequences with Muscle, and a phylogenetic tree was generated using PhyML. Results and Conclusions: Amblyomma mixtum and Rhipicephalus microplus ticks infesting the cattle were identified. Using molecular techniques, the presence of Rickettsia amblyommatis was identified in the cohort of analyzed ticks, suggesting a circulation of this pathogen in livestock in this region and granting further research in this area.

Pangenome-Wide Association Study in the Chlamydiaceae Family Reveals Key Evolutionary Aspects of Their Relationship with Their Hosts

The Chlamydiaceae are a family of obligate intracellular bacteria known for their unique biphasic developmental cycle. Chlamydial are associated with various host organisms, including humans, and have been proposed as emerging pathogens. Genomic studies have significantly enhanced our understanding of chlamydial biology, host adaptation, and evolutionary processes. In this study, we conducted a complete pangenome association analysis (pan-GWAS) using 101 genomes from the Chlamydiaceae family to identify differentially represented genes in Chlamydia and Chlamydophila, revealing their distinct evolutionary strategies for interacting with eukaryotic hosts. Our analysis identified 289 genes with differential abundance between the two clades: 129 showed a strong association with Chlamydia and 160 with Chlamydophila. Most genes in Chlamydia were related to the type III secretion system, while Chlamydophila genes corresponded to various functional categories, including translation, replication, transport, and metabolism. These findings suggest that Chlamydia has developed a high dependence on mammalian cells for replication, facilitated by a complex T3SS for intracellular manipulation. In contrast, the metabolic and functional diversity in Chlamydophila allows it to colonize a broad range of hosts, such as birds, reptiles, amphibians, and mammals, making it a less specialized clade.

Overexpressing the ClpC AAA+ unfoldase accelerates developmental cycle progression in Chlamydia trachomatis.

Chlamydia is an obligate intracellular bacterium that undergoes a complex biphasic developmental cycle, alternating between the smaller, infectious, non-dividing elementary body (EB) and the larger, non-infectious but dividing reticulate body. Due to the differences between these functionally and morphologically distinct forms, we hypothesize protein degradation is essential to chlamydial differentiation. The bacterial Clp system, consisting of an ATPase unfoldase (e.g., ClpX or ClpC) and a proteolytic component (e.g., ClpP), is critical for the physiology of bacteria through its recognition, and usually degradation, of specific substrates. We observed by transmission electron microscopy that overexpression of wild-type ClpC, but not an ATPase mutant isoform, in Chlamydia increased glycogen accumulation within the vacuolar niche of the bacteria earlier in the developmental cycle than typically observed. This suggested ClpC activity may increase the expression of EB-associated genes. Consistent with this, targeted RT-qPCR analyses demonstrated a significant increase in several EB-associated gene transcripts earlier in development. These effects were not observed with overexpression of the ATPase mutant of ClpC, providing strong evidence that the activity of ClpC drives secondary differentiation. By analyzing the global transcriptional response to ClpC overexpression using RNA sequencing, we observed a shift to earlier expression of canonical late developmental cycle genes and other EB-associated genes. Finally, we directly linked overexpression of ClpC with earlier production of infectious chlamydiae. Conversely, disrupting normal ClpC function with an ATPase mutant caused a delay in developmental cycle progression. Overall, these findings provide the first mechanistic insight for initiation of secondary differentiation in Chlamydia.IMPORTANCEChlamydia species are obligate intracellular bacteria that require a host cell in which to complete their unique developmental cycle. Chlamydia differentiates between an infectious but non-replicating form, the elementary body, and a non-infectious but replicating form, the reticulate body. The signals that drive differentiation events are not characterized. We hypothesize that proteases are essential for mediating differentiation by allowing remodeling of the proteome as the organism transitions from one functional form to another. We previously reported that the Caseinolytic protease (Clp) system is essential for chlamydial growth. Here, we reveal a surprising function for ClpC, an unfoldase, in driving production of infectious chlamydiae during the chlamydial developmental cycle.

Molecular and Serological Findings in Sheep During Two Coxiella burnetii Outbreaks in Sicily (Southern Italy).

Q fever is a widespread zoonotic disease caused by the obligate intracellular bacterium Coxiella burnetii, primarily transmitted through the inhalation of contaminated aerosols. This study aimed to detect C. burnetii in two Sicilian sheep flocks, with no better defined reproductive disorders reported by the farmers. Blood, individual and bulk milk, ticks, and conjunctival swabs were collected from both flocks (A and B). Real-time and traditional PCRs were carried out to detect C. burnetii DNA and anti-C. burnetii antibodies were searched using an ELISA. In terms of Farm A, C. burnetii DNA was detected in 7.1% of blood samples, 20% of individual milk samples, bulk milk, 66.6% of conjunctival swabs, and in all the examined tick pools. Anti-C. burnetii antibodies were found in 77.0% of sera, 92.5% of individual milk samples, and bulk milk. In terms of Farm B, C. burnetii DNA was detected in 3.8% of blood samples, 39.4% of individual milk samples, bulk milk, 100% of conjunctival swabs, and in all tick pools; anti-C. burnetii antibodies were present in 53.6% of sera, 73.2% of milk samples, and in bulk milk. Our results highlight the high diffusion of C. burnetii in the two outbreaks, with widespread pathogen circulation, significant shedding in dairy products, and high environmental contamination, highlighting the need for enhanced surveillance and control measures in dairy sheep farms.

PH-Responsive Protein-Polycation Nanocarriers for Efficient Eradication of Bacterial Biofilms and Intracellular Bacteria.

Bacterial biofilms and intracellular pathogens pose significant challenges in eradication, often leading to persistent infections that are difficult to treat. To address this issue, the hydrophobic biofilm dispersant D-tyrosine is encapsulated within protein-polycation nanoparticles, designed using a mannose-terminated cationic polymer and concanavalin through electrostatic interactions. Thermodynamic studies reveal that free mannosyl groups on the nanoparticle surface promote spontaneous binding to receptor molecules mimicking those on bacterial biofilms and host cells. Under mildly acidic conditions, the nanoparticles reduce in size from 550 to ≈48 nm within 2 h, releasing 76% of encapsulated D-tyrosine. The combination of mannose targeting, particle size reduction, and controlled D-tyrosine release enable the nanoparticles to eliminate 70%-80% of the Pseudomonas aeruginosa and Staphylococcus aureus biofilm biomass at minimum bactericidal concentration (MBC) and 2MBC while eradicating 8 log of bacteria embedded within the biofilm. In an intracellular Pseudomonas aeruginosa infection model using RAW 264.7 macrophages, the nanoparticles at 2MBC eliminate over 95% of the intracellular bacteria without inducing an increase in the inflammatory cytokine interleukin-6. These protein-polycation nanoparticles, which activate their antimicrobial properties under acidic conditions, efficiently penetrate bacterial biofilms and host cell barriers via their mannose-rich surface, offering a promising strategy for the treatment of persistent infections.

Antibacterial Mechanisms and Clinical Impact of Sitafloxacin.

Sitafloxacin is a 4th generation fluoroquinolone antibiotic with broad activity against a wide range of Gram-negative and Gram-positive bacteria. It is approved in Japan and used to treat pneumonia and urinary tract infections (UTIs) as well as other upper and lower respiratory infections, genitourinary infections, oral infections and otitis media. Compared to other fluoroquinolones, sitafloxacin displays a low minimal inhibitory concentration (MIC) for many bacterial species but also activity against anaerobes, intracellular bacteria, and persisters. Furthermore, it has also shown strong activity against biofilms of P. aeruginosa and S. aureus in vitro, which was recently validated in vivo with murine models of S. aureus implant-associated bone infection. Although limited in scale at present, the published literature supports the further evaluation of sitafloxacin in implant-related infections and other biofilm-related infections. The aim of this review is to summarize the chemical-positioning-based mechanisms, activity, resistance profile, and future clinical potential of sitafloxacin.

Role of Oral Yeast in Replenishing Gastric Mucosa with Yeast and Helicobacter pylori.

The relationship between oral and gastric yeasts and their role in the colonization of Helicobacter pylori in the stomach was studied. Four groups of 221, 7, 44, and 10 patients were used for the isolation of H. pylori and oral and gastric yeasts. In Group 1, gastric biopsies were used for the isolation of H. pylori and yeast, rapid urease test (RUT), staining with Gram's and hematoxylin & eosin (H&E), and immunohistochemistry (IHC) methods. In the other three groups, DNAs extracted from H. pylori and yeasts were used for the amplification of H. pylori-specific genes. Wet mounts of yeasts in Group 2 were examined to observe intracellular bacteria and released EVs. Among 221 patients, 65 (29.3%) had oral yeast, 35 (15.8%) H. pylori, and 31 (14%) gastric yeast. Culture of oral yeasts showed a significant correlation with the detection of H. pylori by IHC (10.3%), Gram stain (9%), RUT (6.3%), H&E (4.9%), and culture (4%) (p < 0.05). Gram-stained biopsies showed the occurrence of yeast and H. pylori, and the release of EVs from yeast. Detection of similar H. pylori genes in oral and gastric yeasts from patients in Group 2 showed their common source. Oral yeasts in Groups 3 and 4 also carried H. pylori genes. Wet mount preparations of yeasts showed intracellular bacteria inside the yeast vacuole and the release of EVs that could carry H. pylori. Oral yeast protects its intracellular H. pylori and releases it inside EVs to safely reach gastric mucosa. Yeast, as the environmental reservoir of H. pylori, plays a crucial role in bacterial reinfection after successful eradication.

InSeq analysis of defined Legionella pneumophila libraries identifies a transporter-encoding gene cluster important for intracellular replication in mammalian hosts.

Legionella pneumophila is an intracellular bacterial pathogen that replicates inside human alveolar macrophages to cause a severe pneumonia known as Legionnaires' disease. L. pneumophila requires the Dot/Icm Type IV secretion system to deliver hundreds of bacterial proteins to the host cytosol that manipulate cellular processes to establish a protected compartment for bacterial replication known as the Legionella-containing vacuole. To better understand mechanisms apart from the Dot/Icm system that support survival and replication in this vacuole, we used transposon insertion sequencing in combination with defined mutant sublibraries to identify L. pneumophila fitness determinants in primary mouse macrophages and the mouse lung. This approach validated that many previously identified genes important for intracellular replication were critical for infection of a mammalian host. Further, the screens uncovered additional genes contributing to L. pneumophila replication in mammalian infection models. This included a cluster of seven genes in which insertion mutations resulted in L. pneumophila fitness defects in mammalian hosts. Generation of isogenic deletion mutants and genetic complementation studies verified the importance of genes within this locus for infection of mammalian cells. Genes in this cluster are predicted to encode nucleotide-modifying enzymes, a protein of unknown function, and an atypical ATP-binding cassette (ABC) transporter with significant homology to multidrug efflux pumps that has been named Lit, for Legionella infectivity transporter. Overall, these data provide a comprehensive overview of the bacterial processes that support L. pneumophila replication in a mammalian host and offer insight into the unique challenges posed by the intravacuolar environment.IMPORTANCEIntracellular bacteria employ diverse mechanisms to survive and replicate inside the inhospitable environment of host cells. Legionella pneumophila is an opportunistic human pathogen and a model system for studying intracellular host-pathogen interactions. Transposon sequencing is an invaluable tool for identifying bacterial genes contributing to infection, but current animal models for L. pneumophila are suboptimal for conventional screens using saturated mutant libraries. This study employed a series of defined transposon mutant libraries to identify determinants of L. pneumophila fitness in mammalian hosts, which include a newly identified bacterial transporter called Lit. Understanding the requirements for survival and replication inside host cells informs us about the environment bacteria encounter during infection and the mechanisms they employ to make this environment habitable. Such knowledge will be key to addressing future challenges in treating infections caused by intracellular bacteria.

Targeted Sonodynamic Therapy Platform for Holistic Integrative Helicobacter pylori Therapy.

Helicobacter pylori (H. pylori) is a primary pathogen associated with gastrointestinal diseases, including gastric cancer. The increase in resistance to antibiotics, along with the adverse effects caused by complicated medication protocols, has made the eradication of H. pylori a more formidable challenge, necessitating alternative therapeutics. Herein, a targeted nanoplatform is reported based on sonodynamic therapy, the chitosan-conjugated fucose loaded with indocyanine green (ICG@FCS). It penetrates the gastric mucosa and homes in on H. pylori through dual targeting mechanisms: molecular via fucose and physical via ultrasound. Upon ultrasound activation, it generates singlet oxygen, effectively attacking planktonic bacteria, disrupting biofilms, and facilitating the clearance of intracellular bacteria by promoting autophagy, including multidrug-resistant strains. The ICG@FCS nanoplatform minimally affects the gut microbiota and aids in gastric mucosa repair. a holistic integrative H. pylori therapy strategy is proposed that targets eradication while preserving gastrointestinal health. This strategy emphasizes the importance of maintaining patient health while eradicating the pathogen. This advancement is set to refine the comprehensive antibacterial approach, offering a promising horizon in the ongoing battle against antibiotic resistance and more effective gastric cancer prevention strategies.

Orientia tsutsugamushi infection reduces host gluconeogenic but not glycolytic substrates.

Orientia tsutsugamushi a causal agent of scrub typhus, is an obligate intracellular bacterium that, akin to other rickettsiae, is dependent on host cell-derived nutrients for survival and thus pathogenesis. Based on limited experimental evidence and genome-based in silico predictions, O. tsutsugamushi is hypothesized to parasitize host central carbon metabolism (CCM). Here, we (re-)evaluated O. tsutsugamushi dependency on host cell CCM as initiated by glucose and glutamine. Orientia infection had no effect on host glucose and glutamine consumption or lactate accumulation, indicating no change in overall flux through CCM. However, host cell mitochondrial activity and ATP levels were reduced during infection and correspond with lower intracellular glutamine and glutamate pools. To further probe the essentiality of host CCM in O. tsutsugamushi proliferation, we developed a minimal medium for host cell cultivation and paired it with chemical inhibitors to restrict the intermediates and processes related to glucose and glutamine metabolism. These conditions failed to negatively impact O. tsutsugamushi intracellular growth, suggesting the bacterium is adept at scavenging from host CCM. Accordingly, untargeted metabolomics was utilized to evaluate minor changes in host CCM metabolic intermediates across O. tsutsugamushi infection and revealed that pathogen proliferation corresponds with reductions in critical CCM building blocks, including amino acids and TCA cycle intermediates, as well as increases in lipid catabolism. This study directly correlates O. tsutsugamushi proliferation to alterations in host CCM and identifies metabolic intermediates that are likely critical for pathogen fitness.IMPORTANCEObligate intracellular bacterial pathogens have evolved strategies to reside and proliferate within the eukaryotic intracellular environment. At the crux of this parasitism is the balance between host and pathogen metabolic requirements. The physiological basis driving O. tsutsugamushi dependency on its mammalian host remains undefined. By evaluating alterations in host metabolism during O. tsutsugamushi proliferation, we discovered that bacterial growth is independent of the host's nutritional environment but appears dependent on host gluconeogenic substrates, including amino acids. Given that O. tsutsugamushi replication is essential for its virulence, this study provides experimental evidence for the first time in the post-genomic era of metabolic intermediates potentially parasitized by a scrub typhus agent.

Race for the surface between THP-1 macrophages and Staphylococcus aureus on various titanium implants with well-defined topography and wettability.

Gristina et al. (1987) suggested the fate of a biomaterial is decided in a “race for the surface” between pathogens and the host. To gain deeper insight into the mechanisms behind this concept, we investigated the “race for the surface” across three co-culture scenarios with THP-1 macrophages and Staphylococcus aureus (1:1 ratio), varying the order of addition: (i) simultaneous, (ii) macrophages first, and (iii) S. aureus first, on six Ti6Al4V-ELI surfaces modified with specific topographies and wettability. The outcome of the race for the surface was not influenced by these biomaterials but by the chronological introduction of macrophages and S. aureus. When macrophages and S. aureus arrived simultaneously, macrophages won the race, leading to the lowest number of viable S. aureus through rapid phagocytosis and killing. When macrophages arrived and established first, macrophages still prevailed but under greater challenge resulting from the lower bacterial killing efficiency of adherent macrophages and numerous viable intracellular bacteria, supporting the concept of the so-called immunocompromised zone around implants (upregulation of TLR-2 receptor and pro-inflammatory IL-1β). When S. aureus arrived first establishing a biofilm, bacteria won the race, leading to macrophage dysfunction and cell death (upregulation of FcγR and TLR-2 receptors, NF-κB signaling, NOX2 mediated reactive oxygen species), contributing to a persistent biofilm phenotype (upregulation of clfA, icaA, sarA, downregulation of agrA, hld, lukAB) and intracellular survival of S. aureus (lipA upregulation). The clinical implications are bacterial colonization of the implant and persistence of intracellular bacteria in periprosthetic tissues, which can lead to infection chronicity. Statement of SignificanceGristina et al. (1987) suggested the fate of a biomaterial is decided in a “race for the surface” between bacterial pathogens and host cells. There is a lack of in vitro co-culture models and knowledge on macrophage-S. aureus interactions on biomaterial surfaces, and none has evaluated the expression of virulence factors in S. aureus biofilms.We have successfully developed co-culture models and molecular panels and elucidated important cellular and molecular interactions between macrophages and S. aureus on a broad range of titanium biomaterials with well-defined surface topography and wettability. Our findings highlight the critical role of biofilm formation and the chronological order of bacteria or macrophage arrival in determining the fate of the surface.

Bartonella Species in Small Mammals in Turkey: Bartonella bilalgolemii sp. nov. Isolated from a Ural Field Mouse (Apodemus uralensis).

Background: The genus Bartonella is composed of Gram-negative, fastidious, facultative intracellular bacteria that can cause bacteremia in mammals and various disorders in humans. Rodents have been reported as reservoirs of more than 30 Bartonella species, seven of which cause zoonotic infections. Materials and Methods: In the present study, the isolation of Bartonella sp. was attempted from 150 spleen samples from 13 rodent species (mostly Apodemus species) from three geographically different regions in Turkey. Results: Bartonella sp. was successfully isolated from 65 of these 150 samples (43%). The prevalences of Bartonella sp. in tested rodents in the regions of Giresun, Yozgat, and Burdur were 68%, 44%, and 16%, respectively. Using polymerase chain reaction/sequence analysis of the citrate synthase-coding gene (gltA), Bartonellaisolates were classified seven species including B. taylorii, B. grahamii, B. birtlesii, B. mastomydis, and three putatively new Bartonella species. We performed further identification techniques for one of the three Bartonella species that were different from the validated Bartonella species according to the gltA sequence analysis. Conclusion: Here, we report the genomic and phenotypic characterization of Bartonella sp. strain G70 that was isolated from the splenic tissue of an Apodemus uralensis (Pallas 1881), the Ural field mouse, captured in the Giresun region of northeastern Turkey. Bartonella sp. strainG70 (RSKK 22001) was characterized by whole genome and partial gene (gltA, 16S ribosomal RNA) sequencing and comparison, scanning electron microscopy, biochemical tests, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This novel Bartonella is a Gram-negative, rod-shaped bacterium and has neither flagella nor pili. The genome from strain G70 was 1,606,969-bp-long with a G + C content of 35.7%. Bartonella rochalimae was found to be the closest phylogenetic relative of strain G70 (OrthoANI = 90.5%, digital DNA-DNA hybridization = 41.4%). We therefore propose that this new species be named Bartonella bilalgolemii sp. nov. with strain G70T as the type strain.

Antimicrobial resistance: Biofilms, small colony variants, and intracellular bacteria

Soft tissue and bone infections continue to be a serious complication in orthopedic and trauma surgery. Both can lead to a high burden for the patients and the healthcare system. Musculoskeletal infections can be induced by intraoperative contamination, bacterial contamination of open wounds or hematogenous bacterial spread. During the recent decades, advances were achieved in the understanding of pathogenesis and antibiotic resistance. Despite some progress in the diagnosis and advancing of therapeutic concepts, groundbreaking successful improvement of treatment concepts is still missing. Current therapy concepts are based on the two pillars consisting of surgical debridement with joint or bone reconstruction as well as prolonged antibiotic therapy. An improved understanding of both host and pathogen-related factors leading to treatment failure is essential in musculoskeletal infections. Therefore, this review aims to give an overview of pathogen-related pathophysiology in musculoskeletal infections. It describes defense strategies of pathogens such as (1) biofilm, its development, characteristics, and treatment options. In addition, (2) characteristics of small colony variants and (3) intracellular bacteria are highlighted. Lastly (4) an outlook for potential and promising future therapeutic strategies is provided.

Chlamydiosis in Animals.

The Chlamydiaceae family consists of Gram-negative, obligate intracellular bacteria that replicate within the cells of a diverse range of hosts. These hosts include domesticated animals such as cats, dogs, and livestock, as well as wildlife like koalas and birds, exotic species such as reptiles and amphibians, and humans. Chlamydial infection can result in various clinical signs, including respiratory diseases, reproductive failures, ocular pathologies, and enteritis, though the infected organism may remain asymptomatic. In recent years, chlamydial nomenclature has undergone several revisions due to the wide range of hosts, the frequent discovery of novel strains, and the reclassification of existing ones. Given this and the clinical significance of these infections, ranging from asymptomatic to fatal, an updated review is essential. This article outlines key characteristics of Chlamydia species and provides an updated overview of their nomenclature, offering a concise reference for future research on chlamydial diseases.

Understanding melioidosis: Review

Melioidosis is an infectious disease caused by the Gram-negative intracellular bacterium Burkholderia pseudomallei, first identified in 1911. While it is predominantly reported in Southeast Asia, cases have also emerged in other regions, including parts of the United States. The clinical presentation of melioidosis often mimics that of tuberculosis and influenza, leading to diagnostic confusion that can delay appropriate treatment. This delay is particularly concerning, as timely diagnosis and intervention are critical to prevent severe complications, including septicemia and potential death. Education about this disease and taking preventive measures play a crucial role in managing this disease. The possibility of using the causative bacterium as a biological weapon poses a serious threat to public health.

MMV 1804559 is a potential antistaphylococcal and antibiofilm agent targeting the clfA gene of Staphylococcus aureus.

Staphylococcus aureus, a high-priority pathogen proclaimed to cause infections ranging from mild to life-threatening, presents significant challenges in treatment. New therapies can be developed quicker using open drug discovery platforms offering a distinct approach to expedite the development of innovative antibacterial and anti-biofilm therapeutics. This study set out to address these issues by finding new uses for current medications to find compounds that are effective against S. aureus. In this study, we screened the global priority health box, launched by Medicines for Malaria Ventures containing 240 compounds, for their effectiveness against S. aureus. MMV1795508, MMV1542799, MMV027331, MMV1593278, and MMV1804559 showed potential antibacterial activity at 10µM concentration. These compounds underwent further evaluation for their ability to clear intracellular bacteria, disrupt biofilm formation and eradicate existing biofilms. MMV1804559 demonstrated strong efficacy across all tested parameters, achieving 94% inhibition of intracellular bacteria, 79.19% disruption of biofilm cells, and 66.18% inhibition of biofilm formation. Scanning electron microscopy revealed notable membrane perforations and blebbing in MMV1804559-treated cells, indicating its impact on bacterial membranes. Gene expression analysis of cells treated with MMV1804559 showed downregulation of clfA and clfB genes, critical for biofilm formation. Additionally, docking studies confirmed the binding affinity of MMV1804559 with clfA, supported by favorable docking scores, MM/GBSA binding energy, and increased hydrogen bond interactions in the binding pocket, suggesting clfA as a target for MMV1804559. MMV1804559 could serve as a potential therapy for S. aureus by targeting biofilm development and cell adhesion processes.

Genetic diversity of Bartonella species in small mammals in the Qinghai Menyuan section of Qilian Mountain National Park, China

Bartonella are vector-borne gram-negative facultative intracellular bacteria that can infect a wide spectrum of mammals, and are recognized as emerging human pathogens. This study aimed to investigate the prevalence and molecular characteristics of Bartonella infections in small mammals within the Qinghai Menyuan section of Qilian Mountain National Park, China. Small mammals were captured, and the liver, spleen and kidney were collected for Bartonella detection and identification using a combination of real-time PCR targeting the transfer-mRNA (ssrA) gene and followed by sequencing of the citrate synthase (gltA) gene. A total of 52 rodents were captured, and 36 rodents were positive for Bartonella, with a positivity rate of 69.23% (36/52). Bartonella was detected in Cricetulus longicaudatus, Microtus oeconomus, Mus musculus, and Ochotona cansus. The positivity rate was significantly different in the different habitats. Two Bartonella species were observed, including Bartonella grahamii and Bartonella heixiaziensis, and B. grahamii was the dominant epidemic strain in this area. Phylogenetic analysis showed that B. grahamii mainly clustered into two clusters, which were closely related to the Apodemus isolates from China and Japan and the local plateau pika isolates, respectively. In addition, genetic polymorphism analysis showed that B. grahamii had high genetic diversity, and its haplotype had certain regional differences and host specificity. Overall, high prevalence of Bartonella in small mammals in the Qinghai Menyuan section of Qilian Mountain National Park. B. grahamii is the dominant strain with high genetic diversity and potential pathogenicity to humans, and corresponding control measures should be considered.

FtsK Initiates the Assembly of a Unique Divisome Complex in the FtsZ-less Chlamydia trachomatis.

Chlamydia trachomatis serovar L2 (Ct), an obligate intracellular bacterium that does not encode FtsZ, divides by a polarized budding process. In the absence of FtsZ, we show that divisome assembly in Ct is initiated by FtsK, a chromosomal translocase. Chlamydial FtsK forms discrete foci at the septum and at the base of the progenitor mother cell, and our data indicate that FtsK foci at the base of the mother cell mark the location of nascent divisome complexes that form at the site where a daughter cell will emerge in the next round of division. The divisome in Ct has a hybrid composition, containing elements of the divisome and elongasome from other bacteria, and FtsK is recruited to nascent divisomes prior to the other chlamydial divisome proteins assayed, including the PBP2 and PBP3 transpeptidases, and MreB and MreC. Knocking down FtsK prevents divisome assembly in Ct and inhibits cell division and septal peptidoglycan synthesis. We further show that MreB does not function like FtsZ and serve as a scaffold for the assembly of the Ct divisome. Rather, MreB is one of the last proteins recruited to the chlamydial divisome, and it is necessary for the formation of septal peptidoglycan rings. Our studies illustrate the critical function of chlamydial FtsK in coordinating divisome assembly and peptidoglycan synthesis in this obligate intracellular bacterial pathogen.

Genetic variation, structural analysis, and virulence implications of BimA and BimC in clinical isolates of Burkholderia pseudomallei in Thailand

Melioidosis is a life-threatening tropical disease caused by an intracellular gram-negative bacterium Burkholderia pseudomallei. B. pseudomallei polymerizes the host cell actin through autotransporters, BimA, and BimC, to facilitate intracellular motility. Two variations of BimA in B. pseudomallei have been reported previously: BimABp and BimA B. mallei-like (BimABm). However, little is known about genetic sequence variations within BimA and BimC, and their potential effect on the virulence of B. pseudomallei. This study analyzed 1,294 genomes from clinical isolates of patients admitted to nine hospitals in northeast Thailand between 2015 and 2018 and performed 3D structural analysis and plaque-forming efficiency assay. The genomic analysis identified 10 BimABp and 5 major BimC types, in the dominant and non-dominant lineages of the B. pseudomallei population structure. Our protein prediction analysis of all BimABp and major BimC variants revealed that their 3D structures were conserved compared to those of B. pseudomallei K96243. Sixteen representative strains of the most distant BimABp types were tested for plaque formation and the development of polar actin tails in A549 epithelial cells. We found that all isolates retained these functions. These findings enhance our understanding of the prevalence of BimABp and BimC variants and their implications for B. pseudomallei virulence.