Opportunistic Human Pathogen Research Articles

The Ugd, a capsular polysaccharide synthesis protein, regulates the bacterial motility in Vibrio alginolyticus

Vibrio alginolyticus is one of the most common opportunistic pathogens in marine animals and humans. In this study, A transposon mutation library of the V. alginolyticus E110 was used to identify motility-related genes, and we found three flagellar and one capsular polysaccharide (CPS) synthesis-related genes were linked to swarming motility. Then, gene deletion and complementation further confirmed that CPS synthesis-related gene ugd is involved in the swarming motility of V. alginolyticus. Phenotype assays showed that the Δugd mutant reduced CPS production, decreased biofilm formation, impaired swimming ability, and increased cytotoxicity compared to the wild-type strain. Transcriptome analysis showed that 655 genes (15%) were upregulated and 914 genes (21%) were downregulated in the Δugd strain. KEGG pathway and heatmap analysis revealed that genes involved in two-component systems (TCSs), chemotaxis, and flagella assembly pathways were downregulated in the Δugd mutant. On the other hand, genes involved in pathways of human diseases, biosynthesis ABC transporters, and metabolism were upregulated in the Δugd mutant. The RT-qPCR further validated that ugd-regulated genes are associated with motility, biofilm formation, virulence, and TCSs. These findings imply that ugd may be an important player in the control of some physiological processes in V. alginolyticus, highlighting its potential as a target for future research and potential therapeutic interventions.

Preliminary Purification and Partial Characterization of a Functional Bacteriocin of Lacticaseibacillus paracasei Zhang and Mining for its Gene Cluster.

Bacteriocins produced by lactic acid bacteria (LAB) have good potential for use as food biopreservatives. Lacticaseibacillus paracasei Zhang (L. paracasei Zhang) is both a food use and a probiotic bacterium. This study aimed to purify and preliminary characterize the active antibacterial metabolite of L. paracasei Zhang. The cell-free supernatant of L. paracasei Zhang was collected and purified by ultrafiltration and gel filtration chromatography. The 1-3kDa active fraction could inhibit the growth of Staphylococcus aureus but not Escherichia coli. Further antibacterial activity assays revealed its capacity to suppress various foodborne and human opportunistic pathogens (including Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Listeria monocytogenes, and Bacillus cereus), but not fungi. The antibacterial activity showed good tolerance to heat (40 to 100°C), acid-base (pH 2-3 and pH 6-10), and digestions by a number of industrial and animal/human enzymes (such as trypsin, pepsin, α-amylase, and protease K, except papain); these desired properties make it a suitable biopreservative to be used in harsh and complex industrial production processes. The high papain sensitivity suggested a proteinaceous/peptide nature of the bioactivity. Moreover, our genomic data mining for bacteriocin through BAGEL4 revealed an area of interest encoding a complete set of putative genes required for bacteriocin production. In conclusion, our study showed that L. paracasei Zhang can produce extracellular functional antibacterial metabolite, likely a class II bacteriocin. Our preliminary extraction and characterization of the active metabolite demonstrated that it has good potential to be used as a biopreservative or an agent for suppressing gastrointestinal infections.

Marine-Derived Cytosine Arabinoside (Ara-C) Inhibits Biofilm Formation by Inhibiting PEL Operon Proteins (Pel A and Pel B) of Pseudomonas aeruginosa: An In Silico Approach.

Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative biofilm-forming opportunistic human pathogen whose vital mechanism is biofilm formation for better survival. PelA and PelB proteins of the PEL operon are essential for bacterial-synthesized pellicle polysaccharide (PEL), which is a vital structural component of the biofilm. It helps in adherence of biofilm on the surface and maintenance of cell-to-cell interactions and with other matrix components. Here, in-silico molecular docking and simulation studies were performed against PelA and PelB using ten natural bioactive compounds, individually [podocarpic acids, ferruginol, scopadulcic acid B, pisiferic acid, metachromin A, Cytarabine (cytosine arabinoside; Ara-C), ursolic acid, oleanolic acid, maslinic acid, and betulinic acid], those have already been established as anti-infectious compounds. The results obtained from AutoDock and Glide-Schordinger stated that a marine-derived cytosine arabinoside(Ara-C) among the ten compounds binds active sites of PelA and PelB, exhibiting strong binding affinity [Trp224 (hydrogen), Ser219 (polar), Val234 (hydrophobic) for PelA; Leu365 and Glu389 (hydrogen), Gln366 (polar) for PelB] with high negative binding energy -5.518kcal/mol and -6.056kcal/mol, respectively. The molecular dynamic and simulation studies for 100ns showed the MMGBSA binding energy scores are -16.4kcal/mol (Ara-C with PelA), and -22.25kcal/mol (Ara-C with PelB). Further, ADME/T studies indicate the IC50 values of AraC are 6.10mM for PelA and 18.78mM for PelB, which is a comparatively very low dose. The zero violation of Lipinski's Rule of Five further established that Ara-C is a good candidate for drug development. Thus, Ara-C could be considered a potent anti-biofilm compound against PEL operon-dependent biofilm formation of P. aeruginosa.

Assessing microplastics-antibiotics coexistence induced ciprofloxacin-resistant Pseudomonas aeruginosa at a water region scale

Microplastics (MPs) waste is widespread globally in water systems. The opportunistic human pathogen Pseudomonas aeruginosa can cause serious acute and chronic infections that are notoriously difficult to treat. Ciprofloxacin (CIP) is broadly applied as an anti-P. aeruginosa drug. A growing evidence reveals that antibiotic-resistance genes-carrying Pseudomonas aeruginosa were detected on MPs forming plastisphere due to their adsorbability along with high occurrence of CIP in water environments. The MPs-niched CIP-resistant P. aeruginosa has been likely to emerge as an unignorable public health issue. Here, we offered a novel approach to assess the development of CIP-resistant P. aeruginosa under MPs-antibiotic coexistence at a water region scale. By combing the adsorption isotherm models used to estimate CIP condensation around MPs and a pharmacokinetic/pharmacodynamic-based microbial population dynamic model, we predicted the P. aeruginosa development on CIP-adsorbed MPs in waters. Our assessment revealed a high antibiotic resistance in the P. aeruginosa populations (∼50 %) with a wider range of waterborne total cell counts (∼10−2–104 cfu mL−1) among water regions in that the resistance proportion was primarily determined by CIP pollution level and relative abundance of various polymer type of MPs. We implicate that water region-specific MPs were highly likely to provide media for P. aeruginosa propagation. Our results highlight the importance of antibiotic-resistant pathogen colonization−emerging environmental medium interactions when addressing global threat from MPs pollution, in the context of MPs-antibiotics co-contamination assessment and for the continued provision of water system management.

Epidemiology and genetic diversity of linezolid-resistant Enterococcus clinical isolates in Belgium from 2013 to 2021

ObjectivesLinezolid-resistant opportunistic human pathogens Enterococcus faecalis and Enterococcus faecium are emerging health threats as limited therapeutic options remain. The aim of this study was to investigate the epidemiology, resistance mechanisms, and genetic diversity of linezolid-resistant enterococci (LRE) isolated between 2013 and 2021 and received at the Belgian National Reference Centre (NRC) for Enterococci. MethodsLinezolid susceptibility testing was performed upon request on 2458 submitted enterococci strains. Whole-genome sequencing was performed on all LRE strains. ResultsSeventy-eight LRE human isolates, of which 63 (81%) E. faecalis and 15 (19%) E. faecium strains, were submitted to the Belgian NRC for Enterococci. Of the linezolid-resistant E. faecalis strains, 97% harboured the optrA gene (56% wild-type pE349) and 3% the poxtA gene. Of the linezolid-resistant E. faecium strains, 54% harboured the G2576T point mutation in the V domain of the 23S rRNA genes, 23% the poxtA, and 23% the optrA gene. Furthermore, two E. faecium strains were identified with a combination of two resistance mechanisms ([i] optrA and poxtA, and [ii] cfr(B) and G2576T point mutation, respectively). Vancomycin resistance was observed in 15% (n = 12) of the LRE. ST480 (n = 42/63 typed strains, 67%) was the most frequently detected sequence type (ST) in linezolid-resistant E. faecalis strains, while ST203 (n = 5/15 typed strains, 33%) was the most frequently detected ST in linezolid-resistant E. faecium strains. ConclusionsE. faecalis isolates harbouring optrA were the predominant LRE in Belgium, with ST480 as the most prominent multilocus sequence typing. Linezolid resistance in E. faecium could be attributed to either chromosomal mutations or transferable resistance determinants.

The Putative Cytochrome b5 Domain-Containing Protein CaDap1 Homologue Is Involved in Antifungal Drug Tolerance, Cell Wall Chitin Maintenance, and Virulence in Candida albicans.

Candida albicans (Ca), a prominent opportunistic fungal pathogen in humans, has garnered considerable attention due to its infectious properties. Herein, we have identified and characterized CaCDAP1 (Ca orf19.1034), a homolog of ScDAP1 found in Saccharomyces cerevisiae. CaCDAP1 encodes a 183-amino acid protein with a conserved cytochrome b5-like heme-binding domain. The deletion of CaDAP1 renders Ca cells susceptible to caspofungin and terbinafine. CaDAP1 deletion confers resistance to Congo Red and Calcofluor White, and sensitivity to sodium dodecyl sulfate. The deletion of CaDAP1 results in a 50% reduction in chitin content within the cell wall, the downregulation of phosphorylation levels in CaMkc1, and the upregulation of phosphorylation levels in CaCek1. Notably, CaDAP1 deletion results in the abnormal hyphal development of Ca cells and diminishes virulence in a mouse systemic infection model. Thus, CaDAP1 emerges as a critical regulator governing cellular responses to antifungal drugs, the synthesis of cell wall chitin, and virulence in Ca.

Comparative genomic analysis provides insights into the genetic diversity and pathogenicity of the genus Brucella.

Some Brucella spp. are important pathogens. According to the latest prokaryotic taxonomy, the Brucella genus consists of facultative intracellular parasitic Brucella species and extracellular opportunistic or environmental Brucella species. Intracellular Brucella species include classical and nonclassical types, with different species generally exhibiting host preferences. Some classical intracellular Brucella species can cause zoonotic brucellosis, including B. melitensis, B. abortus, B. suis, and B. canis. Extracellular Brucella species comprise opportunistic or environmental species which belonged formerly to the genus Ochrobactrum and thus nowadays renamed as for example Brucella intermedia or Brucella anthropi, which are the most frequent opportunistic human pathogens within the recently expanded genus Brucella. The cause of the diverse phenotypic characteristics of different Brucella species is still unclear. To further investigate the genetic evolutionary characteristics of the Brucella genus and elucidate the relationship between its genomic composition and prediction of phenotypic traits, we collected the genomic data of Brucella from the NCBI Genome database and conducted a comparative genomics study. We found that classical and nonclassical intracellular Brucella species and extracellular Brucella species exhibited differences in phylogenetic relationships, horizontal gene transfer and distribution patterns of mobile genetic elements, virulence factor genes, and antibiotic resistance genes, showing the close relationship between the genetic variations and prediction of phenotypic traits of different Brucella species. Furthermore, we found significant differences in horizontal gene transfer and the distribution patterns of mobile genetic elements, virulence factor genes, and antibiotic resistance genes between the two chromosomes of Brucella, indicating that the two chromosomes had distinct dynamics and plasticity and played different roles in the survival and evolution of Brucella. These findings provide new directions for exploring the genetic evolutionary characteristics of the Brucella genus and could offer new clues to elucidate the factors influencing the phenotypic diversity of the Brucella genus.

Bacterial Metabarcoding of Raw Palm Sap Samples from Bangladesh with Nanopore Sequencing.

The traditional practice of harvesting and processing raw date palm sap is not only culturally significant but also provides an essential nutritional source in South Asia. However, the potential for bacterial or viral contamination from animals and environmental sources during its collection remains a serious and insufficiently studied risk. Implementing improved food safety measures and collection techniques could mitigate the risk of these infections. Additionally, the adoption of advanced food analytical methods offers the potential to identify pathogens and uncover the natural bacterial diversity of these products. The advancement of next-generation sequencing (NGS) technologies, particularly nanopore sequencing, offers a rapid and highly mobile solution. In this study, we employed nanopore sequencing for the bacterial metabarcoding of a set of raw date palm sap samples collected without protective coverage against animals in Bangladesh in 2021. We identified several bacterial species with importance in the natural fermentation of the product and demonstrated the feasibility of this NGS method in the surveillance of raw palm sap products. We revealed two fermentation directions dominated by either Leuconostoc species or Lactococcus species in these products at the first 6 h from harvest, along with opportunistic human pathogens in the background, represented with lower abundance. Plant pathogens, bacteria with the potential for opportunistic human infection and the sequences of the Exiguobacterium genus are also described, and their potential role is discussed. In this study, we demonstrate the potential of mobile laboratory solutions for food safety purposes in low-resource areas.

The emergence of highly resistant and hypervirulent Klebsiella pneumoniae CC14 clone in a tertiary hospital over 8 years

BackgroundKlebsiella pneumoniae is a major bacterial and opportunistic human pathogen, increasingly recognized as a healthcare burden globally. The convergence of resistance and virulence in K. pneumoniae strains has led to the formation of hypervirulent and multidrug-resistant strains with dual risk, limiting treatment options. K. pneumoniae clones are known to emerge locally and spread globally. Therefore, an understanding of the dynamics and evolution of the emerging strains in hospitals is warranted to prevent future outbreaks.MethodsIn this study, we conducted an in-depth genomic analysis on a large-scale collection of 328 multidrug-resistant (MDR) K. pneumoniae strains recovered from 239 patients from a single major hospital in the western coastal city of Jeddah in Saudi Arabia from 2014 through 2022. We employed a broad range of phylogenetic and phylodynamic methods to understand the evolution of the predominant clones on epidemiological time scales, virulence and resistance determinants, and their dynamics. We also integrated the genomic data with detailed electronic health record (EHR) data for the patients to understand the clinical implications of the resistance and virulence of different strains.ResultsWe discovered a diverse population underlying the infections, with most strains belonging to Clonal Complex 14 (CC14) exhibiting dominance. Specifically, we observed the emergence and continuous expansion of strains belonging to the dominant ST2096 in the CC14 clade across hospital wards in recent years. These strains acquired resistance mutations against colistin and extended spectrum β-lactamase (ESBL) and carbapenemase genes, namely blaOXA-48 and blaOXA-232, located on three distinct plasmids, on epidemiological time scales. Strains of ST2096 exhibited a high virulence level with the presence of the siderophore aerobactin (iuc) locus situated on the same mosaic plasmid as the ESBL gene. Integration of ST2096 with EHR data confirmed the significant link between colonization by ST2096 and the diagnosis of sepsis and elevated in-hospital mortality (p-value < 0.05).ConclusionsOverall, these results demonstrate the clinical significance of ST2096 clones and illustrate the rapid evolution of an emerging hypervirulent and MDR K. pneumoniae in a clinical setting.

Acinetobacter junii: an emerging One Health pathogen.

Acinetobacter baumannii is the most well-known species from the genus Acinetobacter. However, other much less studied Acinetobacter species could be important opportunistic pathogens of animals, plants and humans. Here, we conducted the largest genomic epidemiological study of A. junii, which has been described as a source not only of human but also of animal infections. Our analyses show that this bacterium has spread globally and that, in some instances, human and non-human isolates are not well differentiated. Remarkably, some non-human isolates have important antibiotic resistance genes against important antibiotics used in human medicine. Based on our results, we propose that this pathogen must be considered an issue not only for humans but also for veterinary medicine.

Clinical manifestations, antimicrobial resistance and genomic feature analysis of multidrug-resistant Elizabethkingia strains

BackgroundElizabethkingia is emerging as an opportunistic pathogen in humans. The aim of this study was to investigate the clinical epidemiology, antimicrobial susceptibility, virulence factors, and genome features of Elizabethkingia spp.MethodsClinical data from 71 patients who were diagnosed with Elizabethkingia-induced pneumonia and bacteremia between August 2019 and September 2021 were analyzed. Whole-genome sequencing was performed on seven isolates, and the results were compared with a dataset of 83 available Elizabethkingia genomes. Genomic features, Kyoto Encyclopedia of Genes and Genomes (KEGG) results and clusters of orthologous groups (COGs) were analyzed.ResultsThe mean age of the patients was 56.9 ± 20.7 years, and the in-hospital mortality rate was 29.6% (21/71). Elizabethkingia strains were obtained mainly from intensive care units (36.6%, 26/71) and emergency departments (32.4%, 23/71). The majority of the strains were isolated from respiratory tract specimens (85.9%, 61/71). All patients had a history of broad-spectrum antimicrobial exposure. Hospitalization for invasive mechanical ventilation or catheter insertion was found to be a risk factor for infection. The isolates displayed a high rate of resistance to cephalosporins and carbapenems, but all were susceptible to minocycline and colistin. Genomic analysis identified five β-lactamase genes (blaGOB, blaBlaB, blaCME, blaOXA, and blaTEM) responsible for β-lactam resistance and virulence genes involved in stress adaptation (ureB/G, katA/B, and clpP), adherence (groEL, tufA, and htpB) and immune modulation (gmd, tviB, cps4J, wbtIL, cap8E/D/G, and rfbC). Functional analysis of the COGs revealed that “metabolism” constituted the largest category within the core genome, while “information storage and processing” was predominant in both the accessory and unique genomes. The unique genes in our 7 strains were mostly enriched in KEGG pathways related to microRNAs in cancer, drug resistance (β-lactam and vancomycin), ABC transporters, biological metabolism and biosynthesis, and nucleotide excision repair mechanisms.ConclusionThe Elizabethkingia genus exhibits multidrug resistance and carries carbapenemase genes. This study presents a comparative genomic analysis of Elizabethkingia, providing knowledge that facilitates a better understanding of this microorganism.

Spatial variation of airborne bacterial heterogeneity and potential opportunistic human pathogens: a comparative study of sites in Korea and Japan

Spatial variation of airborne bacterial heterogeneity and potential opportunistic human pathogens: a comparative study of sites in Korea and Japan

A highly conserved tRNA modification contributes to C. albicans filamentation and virulence.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections.

Emerging Issues on Antibiotic-Resistant Bacteria Colonizing Plastic Waste in Aquatic Ecosystems.

Antibiotic-resistant bacteria (ARB) adhesion onto plastic substrates is a potential threat to environmental and human health. This current research investigates the prevalence of two relevant human pathogens, Staphylococcus spp. and Klebsiella spp., and their sophisticated equipment of antibiotic-resistant genes (ARGs), retrieved from plastic substrates submerged into an inland water body. The results of microbiological analysis on selective and chromogenic media revealed the presence of colonies with distinctive phenotypes, which were identified using biochemical and molecular methods. 16S rDNA sequencing and BLAST analysis confirmed the presence of Klebsiella spp., while in the case of Staphylococcus spp., 63.6% of strains were found to be members of Lysinibacillus spp., and the remaining 36.3% were identified as Exiguobacterium acetylicum. The Kirby-Bauer disc diffusion assay was performed to test the susceptibility of the isolates to nine commercially available antibiotics, while the genotypic resistant profile was determined for two genes of class 1 integrons and eighteen ARGs belonging to different classes of antibiotics. All isolated bacteria displayed a high prevalence of resistance against all tested antibiotics. These findings provide insights into the emerging risks linked to colonization by potential human opportunistic pathogens on plastic waste commonly found in aquatic ecosystems.

Functional identification of bacterial spermine, thermospermine, norspermine, norspermidine, spermidine, and N1-aminopropylagmatine synthases

Spermine synthase is an aminopropyltransferase that adds an aminopropyl group to the essential polyamine spermidine to form tetraamine spermine, needed for normal human neural development, plant salt and drought resistance, and yeast CoA biosynthesis. We functionally identify for the first time bacterial spermine synthases, derived from phyla Bacillota, Rhodothermota, Thermodesulfobacteriota, Nitrospirota, Deinococcota and Pseudomonadota. We also identify bacterial aminopropyltransferases that synthesize the spermine same mass isomer thermospermine, from phyla Cyanobacteriota, Thermodesulfobacteriota, Nitrospirota, Dictyoglomota, Armatimonadota and Pseudomonadota, including the human opportunistic pathogen Pseudomonas aeruginosa. Most of these bacterial synthases were capable of synthesizing spermine or thermospermine from the diamine putrescine, and so possess also spermidine synthase activity. We found that most thermospermine synthases could synthesize tetraamine norspermine from triamine norspermidine, i.e., they are potential norspermine synthases. This finding could explain the enigmatic source of norspermine in bacteria. Some of the thermospermine synthases could synthesize norspermidine from diamine 1,3-diaminopropane, demonstrating that they are potential norspermidine synthases. Of 18 bacterial spermidine synthases identified, 17 were able to aminopropylate agmatine to form N1-aminopropylagmatine, including the spermidine synthase of Bacillus subtilis, a species known to be devoid of putrescine. This suggests that the N1-aminopropylagmatine pathway for spermidine biosynthesis, which bypasses putrescine, may be far more widespread than realized and may be the default pathway for spermidine biosynthesis in species encoding L-arginine decarboxylase for agmatine production. Some thermospermine synthases were able to aminopropylate N1-aminopropylagmatine to form N12-guanidinothermospermine. Our study reveals an unsuspected diversification of bacterial polyamine biosynthesis, and suggests a more prominent role for agmatine.

Herbal Bioactive Compounds for Skin Infections and Inflammatory Conditions

Skin microbiota is an integral part of the human immune system. Staphylococcus aureus is one of the essential components of the normal flora. Approximately 20–30% of healthy individuals are persistently colonized with S. aureus, whereas the remainders are considered low-level intermittent carriers. Despite these natural aspects of existence, S. aureus can be a major opportunistic human pathogen. This versatile microorganism can infect a variety of anatomical sites, causing a broad spectrum of pathologies ranging from superficial to invasive infections. It developed a variety of strategies to adopt to a changing microenvironment. This attributed to the emergence of resistance to antibiotics of different classes during the past six decades. Methicillin-resistant S. aureus (MRSA) was originally confined to health-care settings (health-care-associated MRSA). Later on, community-acquired MRSA was identified as another source of infections. Recent figures indicate that MRSA strains have been associated with approximately 75% of all S. aureus infections worldwide. Several guidelines have been published to establish an adequate treatment of skin and soft tissue infections (SSTIs) caused by MRSA strains. In the first part of this review, we focus on current treatment guidelines with a focus on medical drug therapy, but drug therapy has its own limitations. Recently, the interest in herbal remedies has greatly increased. There is growing evidence of antimicrobial activity of medicinal plants and their extracts. The second part of this review is dedicated to herbal compounds to circumvent antibiotic resistance. Herbal compounds may potentiate the action of antibiotics and restore the activities of antibacterial agents against which S. aureus has developed a drug resistance. Part 2 focuses on the role of S. aureus in pathology of the two major inflammatory skin diseases, i.e., atopic dermatitis (AD) and psoriasis. Finally, Part 3 provides an overview on natural compounds with antimicrobial activity against S. aureus and possible use in the treatment of SSTIs,

Removal of Pseudomonas type IV pili by a small RNA virus.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

Complete sequence of carbapenem-resistant Ralstonia mannitolilytica clinical isolate co-producing novel class D β-lactamase OXA-1176 and OXA-1177 in Japan.

In 2020, the Ralstonia mannitolilytica strain JARB-RN-0044 was isolated from a midstream urine sample of an elderly hospitalized patient in Japan and was highly resistant to carbapenem (i.e., imipenem, meropenem, and doripenem). Whole-genome sequencing revealed that the complete genome consists of two replicons, a 3.5-Mb chromosome and a 1.5-Mb large non-chromosomal replicon which has not been reported in R. mannitolilytica, and referred to as the "megaplasmid" in this study based on Cluster of Orthologous Group of proteins functional analysis. The strain JARB-RN-0044 harbored two novel OXA-60 and OXA-22 family class D β-lactamase genes blaOXA-1176 and blaOXA-1177 on the megaplasmid. Cloning experiments indicated that Escherichia coli recombinant clone expressing blaOXA-1176 gene showed increased minimum inhibitory concentrations (MICs) of imipenem, meropenem, and doripenem, indicating that blaOXA-1176 gene encodes carbapenemase. In contrast, E. coli recombinant clone expressing blaOXA-1177 gene showed increased MICs of piperacillin and cefazolin, but not of carbapenem. Interestingly, the 44.6 kb putative prophage region containing genes encoding phage integrase, terminase, head and tail protein was identified in the downstream region of blaOXA-1176 gene, and comparative analysis with some previously reported R. mannitolilytica isolates revealed that the prophage region was unique to strain JARB-RN-0044. The existence of a highly carbapenem-resistant R. mannitolilytica isolate may raise human health concerns in Japan, where the population is rapidly aging.IMPORTANCERalstonia mannitolilytica is an aerobic non-fermenting Gram-negative rod commonly found in aquatic environments and soil. The bacteria can occasionally cause severe hospital-acquired bloodstream infections in immunocompromised patients and it has been recently recognized as an emerging opportunistic human pathogen. Furthermore, some R. mannitolilytica isolates are resistant to various antimicrobial agents, including β-lactams and aminoglycosides, making antimicrobial therapy challenging and clinically problematic. However, clinical awareness of this pathogen is limited. To our knowledge, in Japan, there has been only one report of a carbapenem-resistant R. mannitolilytica clinical isolate from urine by Suzuki et al. in 2015. In this study, whole-genome sequencing analysis revealed the presence and genetic context of novel blaOXA-1176 and blaOXA-1177 genes on the 1.5 Mb megaplasmid from highly carbapenem-resistant R. mannitolilytica isolate and characterized the overall distribution of functional genes in the chromosome and megaplasmid. Our findings highlight the importance of further attention to R. mannitolilytica isolate in clinical settings.

Estimating between-country migration in pneumococcal populations.

Streptococcus pneumoniae (the pneumococcus) is a globally distributed, human obligate opportunistic bacterial pathogen which, although often carried commensally, is also a significant cause of invasive disease. Apart from multi-drug resistant and virulent clones, the rate and direction of pneumococcal dissemination between different countries remains largely unknown. The ability for the pneumococcus to take a foothold in a country depends on existing population configuration, the extent of vaccine implementation, as well as human mobility since it is a human obligate bacterium. To shed light on its international movement, we used extensive genome data from the Global Pneumococcal Sequencing project and estimated migration parameters between multiple countries in Africa. Data on allele frequencies of polymorphisms at housekeeping-like loci for multiple different lineages circulating in the populations of South Africa, Malawi, Kenya, and The Gambia were used to calculate the fixation index (Fst) between countries. We then further used these summaries to fit migration coalescent models with the likelihood-free inference algorithms available in the ELFI software package. Synthetic datawere additionally used to validate the inference approach. Our results demonstrate country-pair specific migration patterns and heterogeneity in the extent of migration between different lineages. Our approach demonstrates that coalescent models can be effectively used for inferring migration rates for bacterial species and lineages provided sufficiently granular population genomics surveillance data. Further, it can demonstrate the connectivity of respiratory disease agents between countries to inform intervention policy in the longer term.

A quorum-sensing regulatory cascade for siderophore-mediated iron homeostasis in Chromobacterium violaceum.

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.