Capsular Polysaccharide Production Research Articles

Environmental magnesium ion affects global gene expression, motility, biofilm formation and virulence of Vibrio parahaemolyticus

Vibrio parahaemolyticus is widely distributed in marine ecosystems. Magnesium ion (Mg2+) is the second most abundant metal cation in seawater, and plays important roles in the growth and gene expression of V. parahaemolyticus, but lacks the detailed mechanisms. In this study, the RNA sequencing data demonstrated that a total of 1494 genes was significantly regulated by Mg2+. The majority of the genes associated with lateral flagella, exopolysaccharide, type III secretion system 2, type VI secretion system (T6SS) 1, T6SS2, and thermostable direct hemolysin were downregulated. A total of 18 genes that may be involved in c-di-GMP metabolism and more than 80 genes encoding putative regulators were also significantly and differentially expressed in response to Mg2+, indicating that the adaptation process to Mg2+ stress may be strictly regulated by complex regulatory networks. In addition, Mg2+ promoted the proliferative speed, swimming motility and cell adhesion of V. parahaemolyticus, but inhibited the swarming motility, biofilm formation, and c-di-GMP production. However, Mg2+ had no effect on the production of capsular polysaccharide and cytoxicity against HeLa cells. Therefore, Mg2+ had a comprehensive impact on the physiology and gene expression of V. parahaemolyticus.

Host immunity involvement in the outcome of phage therapy against hypervirulent Klebsiella pneumoniae infections.

Highly encapsulated hypervirulent Klebsiella pneumoniae (hvKp) causes severe infections. Bacteriophage therapy, an antibiotic alternative, effectively treats bacterial infections. Phage φFK1979 encoding polysaccharide depolymerases can target and disarm the capsule of hvKp FK1979, showing promise against FK1979 infection. Resistant strains induced by φFK1979 are possibly eliminated by host immunity and new phage phiR3 targeting them. We constructed varied immunocompromised FK1979 infection mouse models to assess the therapy efficacy of φFK1979 alone or in combination with phiR3. Survival rates, bacterial loads, histopathology, inflammation, and immune cell distribution of mice were studied. Prompt and adequate administration of φFK1979, rather than phiR3, significantly improved survival rates in mice with different immune statuses. However, immunocompromised mice showed lower efficacy due to reduced tolerance to low-virulence φFK1979-resistant bacteria compared to immunocompetent mice. Adding phiR3 sequentially greatly enhanced therapy efficacy for them, leading to increased survival rates and notable improvements in pathology and inflammation. Immunocompetent mice exhibited the most favorable response to φFK1979 monotherapy, as their immune system cleared φFK1979-resistant bacteria while avoiding a robust response to phiR3 combating φFK1979-resistant bacteria. This study revealed host immunity involvement in the outcome of phage therapy against infections and introduced, for the first time, personalized phage therapy strategies for hvKp-infected mice with varying immune statuses.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp), with high capsular polysaccharide production, can cause severe invasive infections. Capsule-targeting phage poses the potential to fight against hvKp. We previously elucidated that the capsule-targeting phage induces resistance in hvKp, while phage-resistant strains exhibit sensitivity to host innate immunity and new phages targeting them. This indicated that phage-resistant strains can be eliminated by the immune system in immunocompetent patients, whereas they may require treatment with phages targeting resistant bacteria in immunocompromised patients. HvKp can infect individuals with varying immune statuses, including both immunocompetent and immunocompromised/deficient patients. This study, for the first time, developed personalized phage therapy strategies for hvKp-infected mice with different immune statuses, optimizing phage therapy against hvKp infections. This research is expected to provide a theoretical foundation and novel insights for clinical phage therapy against hvKp infections, offering significant societal benefits and clinical value.

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.

Streptococcus agalactiae serotype Ia capsular polysaccharide production from a Brazilian strain

The pathogen S. agalactiae, categorized as Lancefield group B streptococci (GBS), is a Gram-positive bacterium, encapsulated, anaerobic facultative bacillus, which occurs in pairs or short chains. It was responsible for more than 319,000 cases of infections in neonates. Early and late streptococci, in the course of an illness or medical condition, are infections that have high mortality and morbidity rates, with an incidence of 2.0 to 5.0 per 1000 live births in North America, Europe, and Australia. The infection of newborns usually happens through transmission from infected mothers during and after childbirth, with GBS being among the critical bacterial infections in the Brazilian epidemiological picture. S. agalactiae strains isolated from different regions of Brazil, indicate a great diversity among the isolated serotypes but with a higher incidence of serotype Ia. Publications report that, in pregnant women in Brazil, the prevalence of serotype Ia has been constant between 2002 and 2018, followed by serotypes II and IV. Serotypes Ib, III, and V also appear, but with some fluctuations in different regions of the country. The pathogen which causes invasive disease are usually surrounded by a polysaccharide capsule and is its major virulence factor, being that the key antigen in protective response triggered by polysaccharides and, more recently, protein-polysaccharide conjugate vaccines. The development of antibiotic resistance by these microorganisms to different antibiotics has been the guideline in leading therapeutic options against these infections including the use of prophylactic vaccines in pregnant women, immunosuppressed or older people. Such prophylactic alternatives can be constructed from capsular polysaccharides.Based on the experience in the production and purification of Neisseria meningitidis polysaccharides for vaccines, Bio-Manguinhos developed a process for the production, purification and characterization of S. agalactiae Ia polysaccharides. In the present study, the capsular polysaccharide from the strain isolated in clinical material of a public hospital, in Rio de Janeiro – Brazil, was obtained from simple batch cultures, with purification process achieving about 20 mg g−1 in yield for cell dry weight (CDW) and ca. of 100 % of purity. Hence, to prove that the CPS of GBS Ia can be consistently produced, purified, and characterized by NMR, HRGC-MS, and SEC-MALS – from such strain – to be used in vaccine productions against S. agalactiae.

Glaesserella parasuis QseBC two-component system senses epinephrine and regulates capD expression.

Glaesserella parasuis, a common inhabitant of the upper respiratory tract of pigs, is the etiological agent of Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis. Decreased production of capsular polysaccharide (CPS) promotes bacterial adhesion and uptake, whereas maximal expression of CPS is essential to evade the host immune response. For bacterial survival under highly variable host conditions, a coordinated regulation of CPS expression is necessary. In the present study, we investigated how epinephrine (Epi) affects the CPS production, biofilm formation, and adhesion ability of G. parasuis, as well as the specific mechanism of G. parasuis changes in response to Epi. The results demonstrated that Epi stimulation dramatically inhibited CPS expression, and both the action of Epi and the deletion of CPS biosynthesis gene capD had detrimental impacts on biofilm formation, and had the enhanced ability for cell adhesion. QseBC two-component system is a typical adrenaline-sensing system in bacteria. In the present study, by quantitative real-time PCR, Western blot, and electrophoretic mobility shift assay, we discovered that in the presence of Epi, G. parasuis negatively regulated capD expression via the interaction of QseB with the promoter of capD, thereby affecting the synthesis of CPS. In conclusion, we explored the impact of Epi on CPS synthesis and defined its regulatory mechanism in G. parasuis for the first time, which provides an important theoretical basis for understanding the pathogenic mechanism of G. parasuis. IMPORTANCE The key bacterial pathogen Glaesserella parasuis, which can cause Glässer's disease, has caused significant financial losses to the swine industry worldwide. Capsular polysaccharide (CPS) is an important virulence factor for bacteria, providing the ability to avoid recognition and killing by the host immune system. Exploring the alteration of CPS synthesis in G. parasuis in response to epinephrine stimulation can lay the groundwork for revealing the pathogenic mechanism of G. parasuis as well as providing ideas for Glässer's disease control.

L-arabinose affects the growth, biofilm formation, motility, c-di-GMP metabolism, and global gene expression of Vibrio parahaemolyticus.

The L-arabinose inducible pBAD vectors are commonly used to turn on and off the expression of specific genes in bacteria. The utilization of certain carbohydrates can influence bacterial growth, virulence factor production, and biofilm formation. Vibrio parahaemolyticus, the causative agent of seafood-associated gastroenteritis, can grow in media with L-arabinose as the sole carbon source. However, the effects of L-arabinose on V. parahaemolyticus physiology have not been investigated. In this study, we show that the growth rate, biofilm formation capacity, capsular polysaccharide production, motility, and c-di-GMP production of V. parahaemolyticus are negatively affected by L-arabinose. RNA-seq data revealed significant changes in the expression levels of 752 genes, accounting for approximately 15.6% of V. parahaemolyticus genes in the presence of L-arabinose. The affected genes included those associated with L-arabinose utilization, major virulence genes, known key biofilm-related genes, and numerous regulatory genes. In the majority of type III secretion system, two genes were upregulated in the presence of L-arabinose, whereas in those of type VI secretion system, two genes were downregulated. Ten putative c-di-GMP metabolism-associated genes were also significantly differentially expressed, which may account for the reduced c-di-GMP levels in the presence of L-arabinose. Most importantly, almost 40 putative regulators were significantly differentially expressed due to the induction by L-arabinose, indicating that the utilization of L-arabinose is strictly regulated by regulatory networks in V. parahaemolyticus. The findings increase the understanding of how L-arabinose affects the physiology of V. parahaemolyticus. Researchers should use caution when considering the use of L-arabinose inducible pBAD vectors in V. parahaemolyticus. IMPORTANCE The data in this study show that L-arabinose negatively affects the growth rate, biofilm formation, capsular polysaccharide production, motility, and c-di-GMP production of V. parahaemolyticus. The data also clarify the gene expression profiles of the bacterium in the presence of L-arabinose. Significantly differentially expressed genes in response to L-arabinose were involved in multiple cellular pathways, including L-arabinose utilization, virulence factor production, biofilm formation, motility, adaptation, and regulation. The collective findings indicate the significant impact of L-arabinose on the physiology of V. parahaemolyticus. There may be similar effects on other species of bacteria. Necessary controls should be established when pBAD vectors must be used for ectopic gene expression.

Enhancement of capsular hypermucoviscosity in Klebsiella pneumoniae by Acanthamoeba.

Acanthamoeba and Klebsiella pneumoniae are both environmental commensals. Recently, clinical harm caused by hypermucoviscous K. pneumoniae has been observed. However, the interaction between these microbes and the origin of hypermucoviscous K. pneumoniae have not been reported. Here, we report that the bacterial capsule is enlarged when co-cultured with Acanthamoeba using India ink staining, and this effect depends on the number of parasites present. This interaction results in an enhancement of capsular polysaccharide production in the subsequent generations of K. pneumoniae, even without co-culturing with Acanthamoeba. The hypermucoviscosity of the capsule was examined using the sedimentation assay and string test. We also screened other K. pneumoniae serotypes, including K1, K2, K5, and K20, for interaction with Acanthamoeba using India ink staining, and found the same interaction effect. These findings suggest that the interaction between Acanthamoeba and K. pneumoniae could lead to harmful consequences in public health and nosocomial disease control, particularly hypermucoviscous K. pneumoniae infections.

Extracellular Polysaccharide Receptor and Receptor-Binding Proteins of the Rhodobacter capsulatus Bacteriophage-like Gene Transfer Agent RcGTA.

A variety of prokaryotes produce a bacteriophage-like gene transfer agent (GTA), and the alphaproteobacterial Rhodobacter capsulatus RcGTA is a model GTA. Some environmental isolates of R. capsulatus lack the ability to acquire genes transferred by the RcGTA (recipient capability). In this work, we investigated the reason why R. capsulatus strain 37b4 lacks recipient capability. The RcGTA head spike fiber and tail fiber proteins have been proposed to bind extracellular oligosaccharide receptors, and strain 37b4 lacks a capsular polysaccharide (CPS). The reason why strain 37b4 lacks a CPS was unknown, as was whether the provision of a CPS to 37b4 would result in recipient capability. To address these questions, we sequenced and annotated the strain 37b4 genome and used BLAST interrogations of this genome sequence to search for homologs of genes known to be needed for R. capsulatus recipient capability. We also created a cosmid-borne genome library from a wild-type strain, mobilized the library into 37b4, and used the cosmid-complemented strain 37b4 to identify genes needed for a gain of function, allowing for the acquisition of RcGTA-borne genes. The relative presence of CPS around a wild-type strain, 37b4, and cosmid-complemented 37b4 cells was visualized using light microscopy of stained cells. Fluorescently tagged head spike fiber and tail fiber proteins of the RcGTA particle were created and used to measure the relative binding to wild-type and 37b4 cells. We found that strain 37b4 lacks recipient capability because of an inability to bind RcGTA; the reason it is incapable of binding is that it lacks CPS, and the absence of CPS is due to the absence of genes previously shown to be needed for CPS production in another strain. In addition to the head spike fiber, we found that the tail fiber protein also binds to the CPS.

Identification of Capsular Polysaccharide Synthesis Loci Determining Bacteriophage Susceptibility in Tetragenococcus halophilus.

Bacteriophages infecting Tetragenococcus halophilus, a halophilic lactic acid bacterium, have been a major industrial concern due to their detrimental effects on the quality of food products. Previously characterized tetragenococcal phages displayed narrow host ranges, but there is little information on these mechanisms. Here, we revealed the host's determinant factors for phage susceptibility using two virulent phages, phiYA5_2 and phiYG2_4, that infect T. halophilus YA5 and YG2, respectively. Phage-resistant derivatives were obtained from these host strains, and mutations were found at the capsular polysaccharide (CPS) synthesis (cps) loci. Quantification analysis verified that capsular polysaccharide production by the cps derivatives from YG2 was impaired. Transmission electron microscopy observation confirmed the presence of filamentous structures outside the cell walls of YG2 and their absence in the cps derivatives of YG2. Phage adsorption assays revealed that phiYG2_4 adsorbed to YG2 but not its cps derivatives, which suggests that the capsular polysaccharide of YG2 is the specific receptor for phiYG2_4. Interestingly, phiYA5_2 adsorbed and infected cps derivatives of YG2, although neither adsorption to nor infection of the parental strain YG2 by phiYA5_2 was observed. The plaque-surrounding halos formed by phiYA5_2 implied the presence of the virion-associated depolymerase that degrades the capsular polysaccharide of YA5. These results indicated that the capsular polysaccharide is a physical barrier rather than a binding receptor for phiYA5_2 and that phiYA5_2 specifically overcomes the capsular polysaccharide of YA5. Thus, it is suggested that tetragenococcal phages utilize CPSs as binding receptors and/or degrade CPSs to approach host cells. IMPORTANCE T. halophilus is a halophilic lactic acid bacterium that contributes to the fermentation processes for various salted foods. Bacteriophage infections of T. halophilus have been a major industrial problem causing fermentation failures. Here, we identified the cps loci in T. halophilus as genetic determinants of phage susceptibility. The structural diversity of the capsular polysaccharide is responsible for the narrow host ranges of tetragenococcal phages. The information provided here could facilitate future studies on tetragenococcal phages and the development of efficient methods to prevent bacteriophage infections.

A link between STK signalling and capsular polysaccharide synthesis in Streptococcus suis

Synthesis of capsular polysaccharide (CPS), an important virulence factor of pathogenic bacteria, is modulated by the CpsBCD phosphoregulatory system in Streptococcus. Serine/threonine kinases (STKs, e.g. Stk1) can also regulate CPS synthesis, but the underlying mechanisms are unclear. Here, we identify a protein (CcpS) that is phosphorylated by Stk1 and modulates the activity of phosphatase CpsB in Streptococcus suis, thus linking Stk1 to CPS synthesis. The crystal structure of CcpS shows an intrinsically disordered region at its N-terminus, including two threonine residues that are phosphorylated by Stk1. The activity of phosphatase CpsB is inhibited when bound to non-phosphorylated CcpS. Thus, CcpS modulates the activity of phosphatase CpsB thereby altering CpsD phosphorylation, which in turn modulates the expression of the Wzx-Wzy pathway and thus CPS production.

Functional Characterization of Plasmid-Borne rmpADC Homologues in Klebsiella pneumoniae.

Expression of the hypermucoviscosity (HMV) phenotype and capsular polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae were reported to be encoded by genes located in the chromosomal rmp locus. However, the functions of the rmp locus in the virulence plasmid remained unclear, and most of the rmp loci in clinical K. pneumoniae are plasmid carried. In this study, we investigated the functional characteristics of plasmid-borne rmp homologues in clinical hypervirulent K. pneumoniae (hvKP) strains by cloning and introducing such gene homologues into K. pneumoniae strains of different capsule types, followed by the evaluation of phenotypic changes in these strains. Acquisition of the plasmid-borne prmpADC and prmpA2D2 loci were found to result in an increase in mucoviscosity and CPS production in K1 and K2 K. pneumoniae, while only the prmpA2D2 locus contributed to phenotypic changes in the ST11/KL64 strain. Consistently, both rmpD and rmpD2 increased HMV in K1 and K2 K. pneumoniae, while only rmpD2 contributed to HMV in the ST11/KL64 strain; rmpC contributed to CPS overproduction in K1 and K2 strains but not in the ST11/KL64 strain. Furthermore, we proposed a logistic molecular basis of the HMV phenotype of K. pneumoniae on which prmpD2-mediated HMV is attributed to the increase of cell-free CPS production. Our data confirm that the rmp homologues carried by the virulence plasmid play a key role in virulence expression in K. pneumoniae, but the phenotype is highly dependent on the genetic background of the host strain and explained why most of the clinical ST11 strains carry only the prmpA2D2 locus. IMPORTANCE Klebsiella pneumoniae has become the most frequently isolated bacterial pathogen in hospital settings, with a very high mortality rate worldwide. Factors contributing to the virulence of K. pneumoniae are the overproduction of capsular polysaccharide (CPS) as well as the hypermucoviscosity (HMV) phenotype. These two phenotypes were reported to be regulated by rmpA/A2 homologues, which are often carried by virulence plasmids. Here, we determined the functional role of two plasmid-borne rmpA in mediating expression of the HMV phenotype and CPS production in K. pneumoniae. Different capsule types exhibited differences in the expression of HMV and CPS production although they harbored an identical plasmid-borne rmpA or rmpA2 locus, indicating that these virulence-related phenotypes are strongly related to the genetic background of the host strains. Our study provides a novel understanding of the regulation of virulence-related phenotypes and clinical management of K. pneumoniae infections.

Reduced virulence in tigecycline-resistant Klebsiella pneumoniae caused by overexpression of ompR and down-regulation of ompK35

BackgroundThe development of tigecycline resistance in hypervirulent Klebsiella pneumoniae strains has resulted in decreased virulence that is associated with reduced production of capsular polysaccharides (CPS). In this study, we investigated the mechanisms that link tigecycline susceptibility to decreased virulence.MethodsWe compared transcriptomes from tigecycline-susceptible wild-type strains and tigecycline-resistant mutants using mRNA sequencing. ompR-overexpressed and ompR-deleted mutants were constructed from wild-type strains and tigecycline-resistant mutants, respectively. Antibiotic susceptibility tests were performed, and string tests and precipitation assays were conducted to identify phenotypic changes related to tigecycline susceptibility and ompR expression. Bacterial virulence was assessed by serum resistance and Galleria mellonella infection assays.ResultsTranscriptomic analyses demonstrated a significant decrease in the expression of ompK35 in the tigecycline-resistant mutants. We observed that tigecycline-resistant mutants overexpressed ompR, and that the expression of ompK35 was regulated negatively by ompR. While tigecycline-resistant mutants and ompR-overexpressed mutants exhibited reduced hypermucoviscosity and virulence, deletion of ompR from tigecycline-resistant mutants restored their hypermucoviscosity and virulence.ConclusionsIn hypervirulent K. pneumoniae strains, ompR expression, which is regulated by exposure to tigecycline, may affect the production of CPS, leading to bacterial virulence.

The Space Environment Activates Capsular Polysaccharide Production in Lacticaseibacillus rhamnosus Probio-M9 by Mutating the wze (ywqD) Gene.

The study of microorganisms in outer space has focused mainly on investigating phenotypic changes in microbial pathogens induced by factors encountered in space. This study aimed to investigate the effect of space exposure on a probiotic bacterium, Lacticaseibacillus rhamnosus Probio-M9. Probio-M9 cells were exposed to space in a spaceflight. Interestingly, our results showed that a substantial proportion of space-exposed mutants (35/100) exhibited a ropy phenotype, characterized by their larger colony sizes and an acquired ability to produce capsular polysaccharide (CPS), compared with the original Probio-M9 or the ground control isolates without space exposure. Whole-genome sequencing analyses on both the Illumina and PacBio platforms revealed a skewed distribution of single nucleotide polymorphisms (12/89 [13.5%]) toward the CPS gene cluster, particularly in the wze (ywqD) gene. The wze gene encodes a putative tyrosine-protein kinase that regulates CPS expression through substrate phosphorylation. Transcriptomics analysis of two space-exposed ropy mutants revealed increased expression in the wze gene relative to a ground control isolate. Finally, we showed that the acquired ropy phenotype (CPS-producing ability) and space-induced genomic changes could be stably inherited. Our findings confirmed that the wze gene directly influences the capacity for CPS production in Probio-M9, and space mutagenesis is a potential strategy for inducing stable physiological changes in probiotics. IMPORTANCE This work investigated the effect of space exposure on a probiotic bacterium, Lacticaseibacillus rhamnosus Probio-M9. Interestingly, the space-exposed bacteria became capable of producing capsular polysaccharide (CPS). Some probiotic-derived CPSs have nutraceutical potential and bioactive properties. They also enhance the survival of probiotics through the gastrointestinal transit and ultimately strengthen the probiotic effects. Space mutagenesis seems to be a promising strategy for inducing stable changes in probiotics, and the obtained high-CPS-yielding mutants are valuable resources for future applications.

Staphylococcus aureus chronic intramammary infections in dairy cows: Pathogen-specific characteristics

Abstract Staphylococcus aureus is one of the main causes of mastitis in dairy cows worldwide. The most common form of the disease is characterized by chronic subclinical infection with sporadic clinical episodes that can persist throughout the life of the animal. Once infection evolves to chronicity, the bacteriological cure rate following classic antibiotic therapy is low and persistently infected cows become the main reservoir of the organism in a dairy herd. The establishment and persistence of S. aureus intramammary infection (IMI) has been associated with certain characteristics of the pathogen that allow it to evade the immune response or induce a deficient immune response that is unable to eliminate the pathogen. The main pathogen characteristics that have been suggested to be linked to S. aureus persistence in the bovine mammary gland (MG), include the capacity to invade cells and/or survive intracellularly and to form biofilms, the capsular polysaccharides (CP) production, the ability to form small colony variants (SCVs), the accessory gene regulator ( agr ) type of the strain and the capacity to acquire antibiotic resistance. This review summarizes current knowledge about the most relevant pathogen-specific characteristics that are associated with the establishment and chronicity of S. aureus IMI. A better understanding of these pathogen characteristics will allow for the development of intervention strategies aimed at improving different aspects of the control of this disease, including diagnostic methods, therapeutics and modulation of the host immune response.

Effects of chlorogenic acid on antimicrobial, antivirulence, and anti-quorum sensing of carbapenem-resistant Klebsiella pneumoniae.

The rise in infections caused by the hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is an emergent threat to public health. We assessed the effects of chlorogenic acid (CA), a natural phenolic compound, on antibacterial, antivirulence, and anti-quorum sensing (QS) of hv-CRKP. Five hv-CRKP were selected for antimicrobial susceptibility test and confirmed to carry virulence genes and carbapenem-resistant genes by polymerase chain reaction (PCR). Subsequently, a series of time-kill assay, determinations of protease activity and capsule content, biofilm-related experiment, scanning electron microscopy (SEM) and transmission electron microscope (TEM) observation, G. mellonella infection model, quantitative real-time PCR (qRT-PCR) of QS-related genes and biofilm formation genes, as well as AI-2 binding test were conduct to verify the effect of CA on hv-CRKP. Five CRKP strains showed varying degrees of resistance to antibacterial agents. All strains carried the bla KPC-2 gene, primarily carrying rmpA2, iucA, and peg-344. CA showed no effect on CRKP growth at the 1/2 minimum inhibitory concentration (MIC), 1/4 MIC, and 1/8 MIC, CA could reduce the production of extracellular protease and capsular polysaccharides, and improve the survival rate of larvae in Galleria mellonella (G. mellonella) infection model. By means of crystal violet staining and scanning electron microscopy experiments, we observed that CA can inhibit the formation of CRKP biofilm. On the quantitative real-time PCR analysis, the expression of the luxS, mrkA and wbbm genes in most CRKP strains appeared downregulated because of the CA treatment. Besides, CA significantly inhibited the effect of AI-2 activity of BB170. Our study suggests that CA can be an effective antimicrobial, antivirulent compound that can target QS in hv-CRKP infections, thus providing a new therapeutic direction for treating bacterial infections.

Within-Host Genotypic and Phenotypic Diversity of Contemporaneous Carbapenem-Resistant Klebsiella pneumoniae from Blood Cultures of Patients with Bacteremia.

It is unknown whether bacterial bloodstream infections (BSIs) are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole-genome sequences of 10 sequence type 258 (ST258) CRKP strains from blood cultures in each of 6 patients (Illumina HiSeq). Strains clustered by patient by core genome and pan-genome phylogeny. In 5 patients, there was within-host strain diversity by gene mutations, presence/absence of antibiotic resistance or virulence genes, and/or plasmid content. Accessory gene phylogeny revealed strain diversity in all 6 patients. Strains from 3 patients underwent long-read sequencing for genome completion (Oxford Nanopore) and phenotypic testing. Genetically distinct strains within individuals exhibited significant differences in carbapenem and other antibiotic responses, capsular polysaccharide (CPS) production, mucoviscosity, and/or serum killing. In 2 patients, strains differed significantly in virulence during mouse BSIs. Genetic or phenotypic diversity was not observed among strains recovered from blood culture bottles seeded with index strains from the 3 patients and incubated in vitro at 37°C. In conclusion, we identified genotypic and phenotypic variant ST258 CRKP strains from blood cultures of individual patients with BSIs, which were not detected by the clinical laboratory or in seeded blood cultures. The data suggest a new paradigm of CRKP population diversity during BSIs, at least in some patients. If validated for BSIs caused by other bacteria, within-host microbial diversity may have implications for medical, microbiology, and infection prevention practices and for understanding antibiotic resistance and pathogenesis. IMPORTANCE The long-standing paradigm for pathogenesis of bacteremia is that, in most cases, a single organism passes through a bottleneck and establishes itself in the bloodstream (single-organism hypothesis). In keeping with this paradigm, standard practice in processing positive microbiologic cultures is to test single bacterial strains from morphologically distinct colonies. This study is the first genome-wide analysis of within-host diversity of Klebsiella pneumoniae strains recovered from individual patients with bloodstream infections (BSIs). Our finding that positive blood cultures comprised genetically and phenotypically heterogeneous carbapenem-resistant K. pneumoniae strains challenges the single-organism hypothesis and suggests that at least some BSIs are caused by mixed bacterial populations that are unrecognized by the clinical laboratory. The data support a model of pathogenesis in which pressures in vivo select for strain variants with particular antibiotic resistance or virulence attributes and raise questions about laboratory protocols and treatment decisions directed against single strains.

Subinhibitory Concentrations of Antibiotics Alter the Response of Klebsiella pneumoniae to Components of Innate Host Defense.

Carbapenem-resistant Klebsiella pneumoniae isolates classified as multilocus sequence type 258 (ST258) are a problem in health care settings in many countries globally. ST258 isolates are resistant to multiple classes of antibiotics and can cause life-threatening infections, such as pneumonia and sepsis, in susceptible individuals. Treatment strategies for such infections are limited. Understanding the response of K. pneumoniae to host factors in the presence of antibiotics could reveal mechanisms employed by the pathogen to evade killing in the susceptible host, as well as inform treatment of infections. Here, we investigated the ability of antibiotics at subinhibitory concentrations to alter K. pneumoniae capsular polysaccharide (CPS) production and survival in normal human serum (NHS). Unexpectedly, pretreatment with some of the antibiotics tested enhanced ST258 survival in NHS. For example, a subinhibitory concentration of mupirocin increased survival for 7 of 10 clinical isolates evaluated and there was increased cell-associated CPS for 3 of these isolates compared with untreated controls. Additionally, mupirocin pretreatment caused concomitant reduction in the deposition of the serum complement protein C5b-9 on the surface of these three isolates. Transcriptome analyses with a selected ST258 isolate (34446) indicated that genes implicated in the stringent response and/or serum resistance were upregulated following mupirocin treatment and/or culture in NHS. In conclusion, mupirocin and/or human serum causes changes in the K. pneumoniae transcriptome that likely contribute to the observed decrease in serum susceptibility via a multifactorial process. Whether these responses can be extended more broadly and thus impact clinical outcome in the human host merits further investigation. IMPORTANCE The extent to which commensal bacteria are altered by exposure to subinhibitory concentrations of antibiotics (outside resistance) remains incompletely determined. To gain a better understanding of this phenomenon, we tested the ability of selected antibiotics (at subinhibitory concentrations) to alter survival of ST258 clinical isolates in normal human serum. We found that exposure of ST258 to antibiotics at low concentrations differentially altered gene expression, capsule production, serum complement deposition, and bacterial survival. The findings were isolate and antibiotic dependent but provide insight into a potential confounding issue associated with ST258 infections.

Terpenes Combinations Inhibit Biofilm Formation in Staphyloccocus aureus by Interfering with Initial Adhesion.

The biofilm is a conglomerate of cells surrounded by an extracellular matrix, which contributes to the persistence of infections. The difficulty in removing the biofilm drives the research for new therapeutic options. In this work, the effect of terpenes (−)-trans-Caryophyllene, (S)-cis-Verbenol, (S)-(−)-Limonene, (R)-(+)-Limonene, and Linalool was evaluated, individually and in combinations on bacterial growth, by assay with resazurin; the formation of biofilm, by assay with violet crystal; and the expression of associated genes, by real-time PCR, in two clinical isolates of Staphyloccocus aureus, ST30-t019 and ST5-t311, responsible for more than 90% of pediatric infections by this pathogen in Paraguay. All combinations of terpenes can inhibit biofilm formation in more than 50% without affecting bacterial growth. The most effective combination was (−)-trans-Caryophyllene and Linalool at a 500 μg/mL concentration for each, with an inhibition percentage of 88%. This combination decreased the expression levels of the sdrD, spa, agr, and hld genes associated with the initial cell adhesion stage and quorum sensing. At the same time, it increased the expression levels of the cap5B and cap5C genes related to the production of capsular polysaccharides. The combinations of compounds tested are promising alternatives to inhibit biofilm formation in S. aureus.

CRISPR Contributes to Adhesion, Invasion, and Biofilm Formation in Streptococcus agalactiae by Repressing Capsular Polysaccharide Production.

ABSTRACTThe clustered regularly interspaced palindromic repeat (CRISPR)-associated (Cas) system functions classically as a prokaryotic defense system against invading mobile genetic elements, such as phages, plasmids, and viruses. Our previous study revealed that CRISPR deletion caused increased transcription of capsular polysaccharide (CPS) synthesis-related genes and severely attenuated virulence in the hypervirulent piscine Streptococcus agalactiae strain GD201008-001. Here, we found that CRISPR deficiency resulted in reduced adhesion, invasion, and biofilm formation abilities in this strain by upregulating the production of CPS. However, enhanced CPS production was not responsible for the attenuated phenotype of the ΔCRISPR mutant. RNA degradation assays indicated that inhibited transcription of the cps operon by CRISPR RNA (crRNA) was not due to the base pairing of the crRNA with the cps mRNA but to the repression of the promoter activity of cpsA, which is a putative transcriptional regulator of the capsule locus.IMPORTANCE Beyond protection from invading nucleic acids, CRISPR-Cas systems have been shown to have an important role in regulating bacterial endogenous genes. In this study, we demonstrate that crRNA inhibits the transcription of the cps operon by repressing the activity of promoter PcpsA, leading to increases in the abilities of adhesion, invasion, and biofilm formation in S. agalactiae. This study highlights the regulatory role of crRNA in bacterial physiology and provides a new explanation for the mechanism of crRNA-mediated endogenous gene regulation in S. agalactiae.

Reduced Virulence and Enhanced Host Adaption during Antibiotics Therapy: a Story of a Within-Host Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 Evolution in a Patient with a Serious Scrotal Abscess.

ABSTRACTCarbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated globally and threatened human life. The sequence type (ST) 11 CRKP is a dominant clone in Asia, but how this clone evolves in vivo then adapts to the host and facilitates dissemination remains largely unknown. Here, the genomic dynamics of 4 ST11-CRKP isolates, which were sequentially collected from the urine of a patient with initial serious scrotal abscess and finally recovered without effective medication, were analyzed. Genomic differences were identified and their implications for pathogenesis and host adaptation were investigated. The related transcriptional pathways were further explored by RNA-Seq. Genomic analysis identified 4 to 24 mutations, among which 94% to 100% of them were synonymous or intergenic mutations. During 47 days of antibiotics therapy, CRKP underwent adaptive evolution, including tigecycline resistance and virulence attenuation. Tigecycline resistance was caused by a deletion within the ramR ribosomal binding site, which has been described by us previously. On the other hand, mutations associated with two genes, acyltransferase (act) and ompK26, resulted in the attenuation phenotype of ST11-CRKP. act deficiency reduced the capsular polysaccharide (CPS) production, enhanced biofilm formation, weakened capsular protection, and decreased induction of proinflammatory cytokines. Further RNA-Seq analysis revealed that act influenced the expression of ldhA, bglX, mtnK, and metE which likely participate in capsular synthesis and biofilm formation. ompK26 affected the virulence by its overexpression caused by the deletion of the upstream repressor binding site. This study presents a within-host adaption of ST11-CRKP and suggests an important role of CPS in the adaptive evolution of virulence and persistence of CRKP.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) has disseminated worldwide and can cause life-threatening infections, including pneumonia, bloodstream infections, urinary tract infections, intraabdominal infection, liver abscess, and meningitis. CRKP infection is the leading cause of high mortality in hospitals. The sequence type (ST) 11 CRKP is a dominant clone and accounts for 60% of CRKP infections in China. Recently, the ST11-CRKP with high transmissibility is increasingly identified. Understanding how this clone has evolved is crucial for developing strategies to control its further dissemination. The significance of our research is the identification of the in vivo genomic dynamics of ST11-CRKP and the genetic basis for ST11-CRKP that facilitate persistence and dissemination. Furthermore, our study also highlights the importance of monitoring the within-host evolution of pathogens during the treatment and developing interventions to minimize the potential impact of host adaptation on human health.