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820300.9958Intercalated cell function, kidney innate immunity, and urinary tract infections. Intercalated cells (ICs) in the kidney collecting duct have a versatile role in acid-base and electrolyte regulation along with the host immune defense. Located in the terminal kidney tubule segment, ICs are among the first kidney cells to encounter bacteria when bacteria ascend from the bladder into the kidney. ICs have developed several mechanisms to combat bacterial infections of the kidneys. For example, ICs produce antimicrobial peptides (AMPs), which have direct bactericidal activity, and in many cases are upregulated in response to infections. Some AMP genes with IC-specific kidney expression are multiallelic, and having more copies of the gene confers increased resistance to bacterial infections of the kidney and urinary tract. Similarly, studies in human children demonstrate that those with history of UTIs are more likely to have single-nucleotide polymorphisms in IC-expressed AMP genes that impair the AMP's bactericidal activity. In murine models, depleted or impaired ICs result in decreased clearance of bacterial load following transurethral challenge with uropathogenic E. coli. A 2021 study demonstrated that ICs even act as phagocytes and acidify bacteria within phagolysosomes. Several immune signaling pathways have been identified in ICs which may represent future therapeutic targets in managing kidney infections or inflammation. This review's objective is to highlight IC structure and function with an emphasis on current knowledge of IC's diverse innate immune capabilities.202438227050
621410.9957Central role of toll-like receptor 4 signaling and host defense in experimental pneumonia caused by Gram-negative bacteria. Toll-like receptor 4 (TLR4) has been identified as a receptor for lipopolysaccharide. However, the precise role of TLR4 in regulating gene expression in response to an infection caused by gram-negative bacteria has not been fully elucidated. The role of TLR4 signaling in coordinating gene expression was assessed by gene expression profiling in lung tissue in a mouse model of experimental pneumonia with a low-dose infection of Klebsiella pneumoniae. We analyzed four mouse strains: C57BL/6 mice, which are resistant to bacterial dissemination; 129/SvJ mice, which are susceptible; C3H/HeJ mice, which are susceptible and have defective TLR4 signaling; and their respective control strain, C3H/HeN (intermediate resistance). At 4 h after infection, C57BL/6 and C3H/HeN mice demonstrated the greatest number of genes, with 67 shared induced genes which were TLR4 dependent and highly associated with the resistance phenotype. These genes included cytokine and chemokine genes required for neutrophil activation or recruitment, growth factor receptors, MyD88 (a critical adaptor protein for TLR signaling), and adhesion molecules. TLR4 signaling accounted for over 74% of the gene expression in the C3H background. These data suggest that early TLR4 signaling controls the vast majority of gene expression in the lung in response to an infection caused by gram-negative bacteria and that this subsequent gene expression determines survival of the host.200515618193
617320.9956Mutation in crrB encoding a sensor kinase increases expression of the RND-type multidrug efflux pump KexD in Klebsiella pneumoniae. BACKGROUND: RND-type multidrug efflux systems in Gram-negative bacteria protect them against antimicrobial agents. Gram-negative bacteria generally possess several genes which encode such efflux pumps, but these pumps sometimes fail to show expression. Generally, some multidrug efflux pumps are silent or expressed only at low levels. However, genome mutations often increase the expression of such genes, conferring the bacteria with multidrug-resistant phenotypes. We previously reported mutants with increased expression of the multidrug efflux pump KexD. We aimed to identify the cause of KexD overexpression in our isolates. Furthermore, we also examined the colistin resistant levels in our mutants. METHODS: A transposon (Tn) was inserted into the genome of Klebsiella pneumoniae Em16-1, a KexD-overexpressing mutant, to identify the gene(s) responsible for KexD overexpression. RESULTS: Thirty-two strains with decreased kexD expression after Tn insertion were isolated. In 12 of these 32 strains, Tn was identified in crrB, which encodes a sensor kinase of a two-component regulatory system. DNA sequencing of crrB in Em16-1 showed that the 452nd cytosine on crrB was replaced by thymine, and this mutation changed the 151st proline into leucine. The same mutation was found in all other KexD-overexpressing mutants. The expression of crrA increased in the mutant overexpressing kexD, and the strains in which crrA was complemented by a plasmid showed elevated expression of kexD and crrB from the genome. The complementation of the mutant-type crrB also increased the expression of kexD and crrA from the genome, but the complementation of the wild-type crrB did not. Deletion of crrB decreased antibiotic resistance levels and KexD expression. CrrB was reported as a factor of colistin resistance, and the colistin resistance of our strains was tested. However, our mutants and strains carrying kexD on a plasmid did not show increased colistin resistance. CONCLUSION: Mutation in crrB is important for KexD overexpression. Increased CrrA may also be associated with KexD overexpression.202337331490
621330.9955Use of a Dictyostelium model for isolation of genetic loci associated with phagocytosis and virulence in Klebsiella pneumoniae. Phagocytosis resistance is an important virulence factor in Klebsiella pneumoniae. Dictyostelium has been used to study the interaction between phagocytes and bacteria because of its similarity to mammalian macrophages. In this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2,500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by Dictyostelium. In the 29 mutants, six loci were identified; three were capsular synthesis genes. Of the other three, one was related to carnitine metabolism, one encoded a subunit of protease (clpX), and one encoded a lipopolysaccharide O-antigen transporter (wzm). Deletion and complementation of these genes showed that only ΔclpX and Δwzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae. Furthermore, we demonstrated that clpP, which exists in an operon with clpX, was also involved in resistance to phagocytosis. The transcriptional profile of ΔclpX was examined by microarray analysis and revealed a 3-fold lower level of expression of capsular synthesis genes. Therefore, we have identified genes involved in resistance to phagocytosis in K. pneumoniae using Dictyostelium, and this model is useful to explore genes associated with resistance to phagocytosis in heavily encapsulated bacteria.201121173313
820440.9954Cecropins contribute to Drosophila host defense against a subset of fungal and Gram-negative bacterial infection. Cecropins are small helical secreted peptides with antimicrobial activity that are widely distributed among insects. Genes encoding Cecropins are strongly induced upon infection, pointing to their role in host defense. In Drosophila, four cecropin genes clustered in the genome (CecA1, CecA2, CecB, and CecC) are expressed upon infection downstream of the Toll and Imd pathways. In this study, we generated a short deletion ΔCecA-C removing the whole cecropin locus. Using the ΔCecA-C deficiency alone or in combination with other antimicrobial peptide (AMP) mutations, we addressed the function of Cecropins in the systemic immune response. ΔCecA-C flies were viable and resisted challenge with various microbes as wild-type. However, removing ΔCecA-C in flies already lacking 10 other AMP genes revealed a role for Cecropins in defense against Gram-negative bacteria and fungi. Measurements of pathogen loads confirm that Cecropins contribute to the control of certain Gram-negative bacteria, notably Enterobacter cloacae and Providencia heimbachae. Collectively, our work provides the first genetic demonstration of a role for Cecropins in insect host defense and confirms their in vivo activity primarily against Gram-negative bacteria and fungi. Generation of a fly line (ΔAMP14) that lacks 14 immune inducible AMPs provides a powerful tool to address the function of these immune effectors in host-pathogen interactions and beyond.202234791204
819750.9954Specific host genes required for the killing of Klebsiella bacteria by phagocytes. The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.200616367873
977760.9953Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production. Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.201020855724
883770.9953Phage resistance formation and fitness costs of hypervirulent Klebsiella pneumoniae mediated by K2 capsule-specific phage and the corresponding mechanisms. INTRODUCTION: Phage is promising for the treatment of hypervirulent Klebsiella pneumoniae (hvKP) infections. Although phage resistance seems inevitable, we found that there still was optimization space in phage therapy for hvKP infection. METHODS: The clinical isolate K. pneumoniae FK1979 was used to recover the lysis phage ΦFK1979 from hospital sewage. Phage-resistant bacteria were obtained on LB agar and used to isolate phages from sewage. The plaque assay, transmission electron microscopy (TEM), multiplicity of infection test, one-step growth curve assay, and genome analysis were performed to characterize the phages. Colony morphology, precipitation test and scanning electron microscope were used to characterize the bacteria. The absorption test, spot test and efficiency of plating (EOP) assay were used to identify the sensitivity of bacteria to phages. Whole genome sequencing (WGS) was used to identify gene mutations of phage-resistant bacteria. The gene expression levels were detected by RT-qPCR. Genes knockout and complementation of the mutant genes were performed. The change of capsules was detected by capsule quantification and TEM. The growth kinetics, serum resistance, biofilm formation, adhesion and invasion to A549 and RAW 264.7 cells, as well as G. mellonella and mice infection models, were used to evaluate the fitness and virulence of bacteria. RESULTS AND DISCUSSION: Here, we demonstrated that K2 capsule type sequence type 86 hvKP FK1979, one of the main pandemic lineages of hvKP with thick capsule, rapidly developed resistance to a K2-specific lysis phage ΦFK1979 which was well-studied in this work to possess polysaccharide depolymerase. The phage-resistant mutants showed a marked decrease in capsule expression. WGS revealed single nucleotide polymorphism (SNP) in genes encoding RfaH, galU, sugar glycosyltransferase, and polysaccharide deacetylase family protein in the mutants. RfaH and galU were further identified as being required for capsule production and phage sensitivity. Expressions of genes involved in the biosynthesis or regulation of capsule and/or lipopolysaccharide significantly decreased in the mutants. Despite the rapid and frequent development of phage resistance being a disadvantage, the attenuation of virulence and fitness in vitro and in vivo indicated that phage-resistant mutants of hvKP were more susceptible to the immunity system. Interestingly, the newly isolated phages targeting mutants changed significantly in their plaque and virus particle morphology. Their genomes were much larger than and significantly different from that of ΦFK1979. They possessed much more functional proteins and strikingly broader host spectrums than ΦFK1979. Our study suggests that K2-specific phage has the potential to function as an antivirulence agent, or a part of phage cocktails combined with phages targeting phage-resistant bacteria, against hvKP-relevant infections.202337538841
481880.9953Complement Susceptibility in Relation to Genome Sequence of Recent Klebsiella pneumoniae Isolates from Thai Hospitals. The capacity to resist the bactericidal action of complement (C') is a strong but poorly understood virulence trait in Klebsiella spp. Killing requires activation of one or more C' pathways, assembly of C5b-9 membrane attack complexes (MACs) on the surface of the outer membrane (OM), and penetration of MACs into the target bilayer. We interrogated whole-genome sequences of 164 Klebsiella isolates from three tertiary hospitals in Thailand for genes encoding surface-located macromolecules considered to play a role in determination of C' resistance. Most isolates (154/164) were identified as Klebsiella pneumoniae, and the collection conformed to previously established population structures and antibiotic resistance patterns. The distribution of sequence types (STs) and capsular (K) types were also typical of global populations. The majority (64%) of isolates were resistant to C', and the remainder were either rapidly or slowly killed. All isolates carried genes encoding capsular polysaccharides (K antigens), which have been strongly linked to C' resistance. In contrast to previous reports, there were no differences in the amount of capsule produced by C'-resistant isolates compared to C'-susceptible isolates, nor was there any correlation between serum reactivity and the presence of hypermucoviscous capsules. Similarly, there were no correlations between the presence of genes specifying lipopolysaccharide O-side chains or major OM proteins. Some virulence factors were found more frequently in C'-resistant isolates but were considered to reflect clonal ST expansion. Thus, no single gene accounts for the C' resistance of the isolates sequenced in this study.IMPORTANCE Multidrug-resistant Klebsiella pneumoniae is responsible for an increasing proportion of nosocomial infections, and emerging hypervirulent K. pneumoniae clones now cause severe community-acquired infections in otherwise healthy individuals. These bacteria are adept at circumventing immune defenses, and most survive and grow in serum; their capacity to avoid C'-mediated destruction is correlated with their invasive potential. Killing of Gram-negative bacteria occurs following activation of the C' cascades and stable deposition of C5b-9 MACs onto the OM. For Klebsiella, studies with mutants and conjugants have invoked capsules, lipopolysaccharide O-side chains, and OM proteins as determinants of C' resistance, although the precise roles of the macromolecules are unclear. In this study, we sequenced 164 Klebsiella isolates with different C' susceptibilities to identify genes involved in resistance. We conclude that no single OM constituent can account for resistance, which is likely to depend on biophysical properties of the target bilayer.201830404929
63090.9952Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43. BACKGROUND: The cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria. METHODS: In this study, the genetic determinants for capsular polysaccharide level and lipopolysaccharide modification involved in polymyxin B resistance of the opportunistic pathogen Klebsiella pneumoniae were characterized. The expressional control of the genes responsible for the resistance was assessed by a LacZ reporter system. The PmrD connector-mediated regulation for the expression of pmr genes involved in polymyxin B resistance was also demonstrated by DNA EMSA, two-hybrid analysis and in vitro phosphor-transfer assay. RESULTS: Deletion of the rcsB, which encoded an activator for the production of capsular polysaccharide, had a minor effect on K. pneumoniae resistance to polymyxin B. On the other hand, deletion of ugd or pmrF gene resulted in a drastic reduction of the resistance. The polymyxin B resistance was shown to be regulated by the two-component response regulators PhoP and PmrA at low magnesium and high iron, respectively. Similar to the control identified in Salmonella, expression of pmrD in K. pneumoniae was dependent on PhoP, the activated PmrD would then bind to PmrA to prolong the phosphorylation state of the PmrA, and eventually turn on the expression of pmr for the resistance to polymyxin B. CONCLUSIONS: The study reports a role of the capsular polysaccharide level and the pmr genes for K. pneumoniae resistance to polymyxin B. The PmrD connector-mediated pathway in governing the regulation of pmr expression was demonstrated. In comparison to the pmr regulation in Salmonella, PhoP in K. pneumoniae plays a major regulatory role in polymyxin B resistance.201020653976
4819100.9952Gram-Negative Bacteria. Gram-negative bacteria (GNB) are among the world's most significant public health problems due to their high resistance to antibiotics. These microorganisms have significant clinical importance in hospitals because they put patients in the intensive care unit (ICU) at high risk and lead to high morbidity and mortality. Two large groups, Enterobacteriaceae and the non-fermenters, are responsible for most clinical isolates; nevertheless, other clinically concerning gram-negative organisms exist, including but not limited to Neisseria, Haemophilus spp., Helicobacter pylori, and Chlamydia trachomatis. Enterobacteriaceae   Enterobacteriaceae are a heterogeneous group widely dispersed in nature. They account for about 80% of gram-negative isolates with a myriad of disease-causing general/species in humans, including urinary tract infections, pneumonia, diarrhea, meningitis, sepsis, endotoxic shock, and many others. The general/species that frequently affect humans are Escherichia, Proteus, Enterobacter, Klebsiella, Citrobacter, Yersinia, Shigella, and Salmonella, among others. Laboratory characterization is an essential component when it comes to microorganisms; therefore, it is imperative to expose characteristics of Enterobacteriaceae, which are bacilli, non-sporulated, have variable motility, grow in the presence and absence of oxygen, ferment organisms of glucose, are cytochrome oxidase negative, and can reduce nitrate to nitrite. Non-Fermenters The non-fermenter, gram-negative bacilli (BNF) have a lower frequency of isolation when compared to Enterobacteriaceae; however, they are a relevant group since they cause severe, fatal infections, especially in the hospital environment. They also cause opportunistic diseases in ICU patients who undergo invasive procedures. The main BNF microorganisms that cause human disease are Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cepacia, Burkholderia pseudomallei, Stenotrophomonas., Alcaligenes, and Moraxella. These stand out for being aerobic and non-sporulated; they are incapable of fermenting sugars, using them through the oxidative route. The critical issue regarding BNF, when it comes to the antimicrobial sensitivity profile, is undoubtedly their intrinsic resistance since they produce a variety of genes with multiple mechanisms capable of mitigating the microbicidal action. Thus, it stands out in P. aeruginosa, cephalosporinase of type AmpC, and efflux systems that confer resistance to b-lactams. The most frequent are MexAB-OprM; and loss of OprD (which gives impermeability to the bacterial cell due to the loss of porin.) Acinetobacter baumannii naturally produces AmpC cephalosporinase and oxacillinase (OXA), leaving it spontaneously immune to many drugs. The genetic ingenuity of this microorganism goes further, and it combines high impermeability with genetic plasticity, combining with the resistance of mechanisms such as extended-spectrum b-lactamases (ESBL). The Stenotrophomonas exhibit a pattern of intrinsic multi-resistance, especially in patients who have had contact with carbapenems. Thus, Stenotrophomonas present several efflux pumps and produce two carbapenemases – L1 (resistance to all carbapenems) and L2 (cephalosporinase).  These mechanisms, associated or separate, restrict the treatment options to an alarming level. Sulfamethoxazole-trimethoprim remains the mainstay of treatment.  Antibiotic Resistance These organisms have a range of mechanisms to prevent the action of many antimicrobials used in clinical medicine. Some of the mechanisms of resistance include efflux pumps, alteration of the drug binding site and membrane permeability, degradation enzymes, and the conformational change of the drug culminating in its inactivation. GNB have two membranes, an external and an internal. The external membrane expresses a potent immune response inducer, lipopolysaccharide (LPS), which is composed of three units: a hydrophilic polysaccharide, O antigen, and a hydrophobic domain known as lipid A. Lipid A are responsible for the higher endotoxic activity of these bacteria. However, the LPS is heterogeneous in the various bacterial groups, and some bacteria manifest this antigen weakly due to genetic changes and are not recognized by Toll-like receptors. In contrast, there are BGN groups that can trigger such a response in large proportions. Thus, LPS can trigger the innate immune response through Toll-like receptors 4 (TLR4), which occurs in many immune cells such as monocytes, macrophages, dendritic cells, and neutrophils. The resulting activation of the innate immune response mediated by LPS together with TLR4 receptors culminates in an exacerbated response with the production of cytokines, chemokines, and interferons and their suppression. Enterobacteriaceae diffuse their plasmids by conjugation, which gives rise to resistance to almost all existing antibiotics. The family of enzymes carbapenemase – KPC, NDM-1, IMP, VIM, OXA-48 – is undoubtedly one of the most significant health challenges of the century, given the potential for dissemination between species and mortality rates due to infections caused by bacteria with such plasmids. Colistin, one of the few antibiotics that still treat multiresistant infections, already has a mobile resistance gene, mcr-1, and Enterobacteriaceae has a crucial role in the spread of this gene, with worldwide reports. Moreover, a further concern is that they usually associate these genes with other resistance genes (CTX-M, NDM, IMP), producing resistance to cephalosporins and carbapenems, enhancing the deleterious effects caused by these microorganisms.202530855801
8206110.9952Spontaneous and transient defence against bacteriophage by phase-variable glucosylation of O-antigen in Salmonella enterica serovar Typhimurium. As natural killers of bacteria, bacteriophages have forced bacteria to develop a variety of defence mechanisms. The alteration of host receptors is one of the most common bacterial defence strategies against phage infection, which completely blocks phage attachment but comes at a potential fitness cost to the bacteria. Here, we report the cost-free, transient emergence of phage resistance in Salmonella enterica subspecies enterica serovar Typhimurium through a phase-variable modification of the O-antigen. Phage SPC35 typically requires BtuB as a host receptor but also uses the Salmonella O12-antigen as an adsorption-assisting apparatus for the successful infection of S. Typhimurium. The α-1,4-glucosylation of galactose residues in the O12-antigen by phase variably expressed O-antigen glucosylating genes, designated the (LT) (2) gtrABC1 cluster, blocks the adsorption-assisting function of the O12-antigen. Consequently, it confers transient SPC35 resistance to Salmonella without any mutations to the btuB gene. This temporal switch-off of phage adsorption through phase-variable antigenic modification may be widespread among Gram-negative bacteria-phage systems.201222928771
5067120.9951Stepwise Evolution of a Klebsiella pneumoniae Clone within a Host Leading to Increased Multidrug Resistance. Five bla(CTX-M-14)-positive Klebsiella pneumoniae isolates (KpWEA1, KpWEA2, KpWEA3, KpWEA4-1, and KpWEA4-2) were consecutively obtained from a patient with relapsed acute myeloid leukemia who was continuously administered antimicrobials. Compared with KpWEA1 and KpWEA2, KpWEA3 showed decreased susceptibility to antimicrobials, and KpWEA4-1 and KpWEA4-2 (isolated from a single specimen) showed further-elevated multidrug-resistance (MDR) phenotypes. This study aims to clarify the clonality of the five isolates and their evolutionary processes leading to MDR by comparison of these complete genomes. The genome comparison revealed KpWEA1 was the antecedent of the other four isolates, and KpWEA4-1 and KpWEA4-2 independently emerged from KpWEA3. Increasing levels of MDR were acquired by gradual accumulation of genetic alterations related to outer membrane protein expression: the loss of OmpK35 and upregulation of AcrAB-TolC occurred in KpWEA3 due to ramA overexpression caused by a mutation in ramR; then OmpK36 was lost in KpWEA4-1 and KpWEA4-2 by different mechanisms. KpWEA4-2 further acquired colistin resistance by the deletion of mgrB. In addition, we found that exuR and kdgR, which encode repressors of hexuronate metabolism-related genes, were disrupted in different ways in KpWEA4-1 and KpWEA4-2. The two isolates also possessed different amino acid substitutions in AtpG, which occurred at very close positions. These genetic alterations related to metabolisms may compensate for the deleterious effects of major porin loss. Thus, our present study reveals the evolutionary process of a K. pneumoniae clone leading to MDR and also suggests specific survival strategies in the bacteria that acquired MDR by the genome evolution. IMPORTANCE Within-host evolution is a survival strategy that can occur in many pathogens and is often associated with the emergence of novel antimicrobial-resistant (AMR) bacteria. To analyze this process, suitable sets of clinical isolates are required. Here, we analyzed five Klebsiella pneumoniae isolates which were consecutively isolated from a patient and showed a gradual increase in the AMR level. By genome sequencing and other analyses, we show that the first isolate was the antecedent of the later isolates and that they gained increased levels of antimicrobial resistance leading to multidrug resistance (MDR) by stepwise changes in the expression of outer membrane proteins. The isolates showing higher levels of MDR lost major porins but still colonized the patient's gut, suggesting that the deleterious effects of porin loss were compensated for by the mutations in hexuronate metabolism-related genes and atpG, which were commonly detected in the MDR isolates.202134817239
6162130.9951The resistance of BALB/cJ mice to Yersinia pestis maps to the major histocompatibility complex of chromosome 17. Yersinia pestis, the causative agent of plague, has been well studied at the molecular and genetic levels, but little is known about the role that host genes play in combating this highly lethal pathogen. We challenged several inbred strains of mice with Y. pestis and found that BALB/cJ mice are highly resistant compared to susceptible strains such as C57BL/6J. This resistance was observed only in BALB/cJ mice and not in other BALB/c substrains. Compared to C57BL/6J mice, the BALB/cJ strain exhibited reduced bacterial burden in the spleen and liver early after infection as well as lower levels of serum interleukin-6. These differences were evident 24 h postinfection and became more pronounced with time. Although a significant influx of neutrophils in the spleen and liver was exhibited in both strains, occlusive fibrinous thrombi resulting in necrosis of the surrounding tissue was observed only in C57BL/6J mice. In an effort to identify the gene(s) responsible for resistance, we measured total splenic bacteria in 95 F(2) mice 48 h postinfection and performed quantitative trait locus mapping using 58 microsatellite markers spaced throughout the genome. This analysis revealed a single nonrecessive plague resistance locus, designated prl1 (plague resistance locus 1), which coincides with the major histocompatibility complex of chromosome 17. A second screen of 95 backcrossed mice verified that this locus confers resistance to Y. pestis early in infection. Finally, eighth generation backcrossed mice harboring prl1 were found to maintain resistance in the susceptible C57BL/6J background. These results identify a novel genetic locus in BALB/cJ mice that confers resistance to Y. pestis.200818573896
202140.9951Surface Anchoring of the Kingella kingae Galactan Is Dependent on the Lipopolysaccharide O-Antigen. Kingella kingae is a leading cause of bone and joint infections and other invasive diseases in young children. A key K. kingae virulence determinant is a secreted exopolysaccharide that mediates resistance to serum complement and neutrophils and is required for full pathogenicity. The K. kingae exopolysaccharide is a galactofuranose homopolymer called galactan and is encoded by the pamABC genes in the pamABCDE locus. In this study, we sought to define the mechanism by which galactan is tethered on the bacterial surface, a prerequisite for mediating evasion of host immune mechanisms. We found that the pamD and pamE genes encode glycosyltransferases and are required for synthesis of an atypical lipopolysaccharide (LPS) O-antigen. The LPS O-antigen in turn is required for anchoring of galactan, a novel mechanism for association of an exopolysaccharide with the bacterial surface. IMPORTANCE Kingella kingae is an emerging pediatric pathogen and produces invasive disease by colonizing the oropharynx, invading the bloodstream, and disseminating to distant sites. This organism produces a uniquely multifunctional exopolysaccharide called galactan that is critical for virulence and promotes intravascular survival by mediating resistance to serum and neutrophils. In this study, we established that at least some galactan is anchored to the bacterial surface via a novel structural interaction with an atypical lipopolysaccharide O-antigen. Additionally, we demonstrated that the atypical O-antigen is synthesized by the products of the pamD and pamE genes, located downstream of the gene cluster responsible for galactan biosynthesis. This work addresses how the K. kingae exopolysaccharide can mediate innate immune resistance and advances understanding of bacterial exopolysaccharides and lipopolysaccharides.202236069736
9760150.9951Mutations leading to ceftolozane/tazobactam and imipenem/cilastatin/relebactam resistance during in vivo exposure to ceftazidime/avibactam in Pseudomonas aeruginosa. Identifying resistance mechanisms to novel antimicrobials informs treatment strategies during infection and antimicrobial development. Studying resistance that develops during the treatment of an infection can provide the most clinically relevant mutations conferring resistance, but cross-sectional studies frequently identify multiple candidate resistance mutations without resolving the driver mutation. We performed whole-genome sequencing of longitudinal Pseudomonas aeruginosa from a patient whose P. aeruginosa developed imipenem/cilastatin/relebactam and ceftolozane/tazobactam resistance during ceftazidime/avibactam treatment. This analysis determined new mutations that arose in isolates resistant to both imipenem/cilastatin/relebactam and ceftolozane/tazobactam. Mutations in penicillin-binding protein 3 ftsI, the MexAB-OprM repressor nalD, and a virulence regulator pvdS were found in resistant isolates. Importantly, drug efflux was not increased in the resistant isolate compared to the most closely related susceptible isolates. We conclude that mutations in peptidoglycan synthesis genes can alter the efficacy of multiple antimicrobials. IMPORTANCE: Antibiotic resistance is a significant challenge for physicians trying to treat infections. The development of novel antibiotics to treat resistant infections has not been prioritized for decades, limiting treatment options for infections caused by many high-priority pathogens. Cross-resistance, when one mutation provides resistance to multiple antibiotics, is most problematic. Mutations that cause cross-resistance need to be considered when developing new antibiotics to guide developers toward drugs with different targets, and thus a better likelihood of efficacy. This work was undertaken to determine the mutation that caused resistance to three antibiotics for highly resistant Pseudomonas aeruginosa infection treatment while the bacteria were exposed to only one of these agents. The findings provide evidence that drug developers should endeavor to find effective antibiotics with new targets and that medical providers should utilize medications with different mechanisms of action in bacteria that have become resistant to even one of these three agents.202539932323
8838160.9951Dual RNA-seq analysis reveals the interaction between multidrug-resistant Klebsiella pneumoniae and host in a mouse model of pneumonia. BACKGROUND: Multidrug-resistant Klebsiella pneumoniae (MDR-KP) poses a significant global health threat, associated with high morbidity and mortality rates among hospitalized patients. The interaction between MDR-KP and its host is highly complex, and few studies have investigated these interactions from both the pathogen and host perspectives. Here, we explored these interactions in a mouse model of pneumonia using dual RNA-seq analysis. METHODS: PCR identification and antimicrobial susceptibility test were employed to screen for MDR-KP strains. A mouse model of pneumonia was established through aerosolized intratracheal inoculation with high-dose or low-dose bacteria. Bacterial loads, pathological changes, inflammatory cytokine expression, and immune cell infiltration were assessed post-challenge. Dual RNA-seq analysis was conducted on lung tissues following infection. RESULTS: NY13307 was identified as an MDR-KP strain with minimal virulence factor genes and broad-spectrum drug resistance. High-dose bacteria induced more severe pulmonary pathological changes, a significant increase in bacterial load, and notably elevated secretion of inflammatory cytokines compared to low-dose bacteria. Alveolar macrophages and resident interstitial macrophages were identified as the primary sources of these cytokines. Further RNA-seq analysis revealed that, compared to the low-dose group, the high-dose group significantly upregulated hypoxia and pro-inflammatory cytokine-related genes in the host, and siderophore-related genes in the bacteria. Correlation analysis demonstrated a significant association between siderophore-related genes and clusters of genes related to pro-inflammatory cytokines and hypoxia. CONCLUSIONS: In this mouse model of bacterial pneumonia, excessive siderophore expression may trigger the activation of hypoxia signaling pathways and the release of pro-inflammatory cytokines, ultimately reducing survival rates.202540702458
633170.9951The sensor kinase PhoQ mediates virulence in Pseudomonas aeruginosa. Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. PhoP-PhoQ is a two-component regulatory system that has been identified as essential for virulence and cationic antimicrobial peptide resistance in several other Gram-negative bacteria. This study demonstrated that mutation of phoQ caused reduced twitching motility, biofilm formation and rapid attachment to surfaces, 2.2-fold reduced cytotoxicity to human lung epithelial cells, substantially reduced lettuce leaf virulence, and a major, 10 000-fold reduction in competitiveness in chronic rat lung infections. Microarray analysis revealed that PhoQ controlled the expression of many genes consistent with these phenotypes and with its known role in polymyxin B resistance. It was also demonstrated that PhoQ controls the expression of many genes outside the known PhoP regulon.200919246741
4821180.9950Enterobacter hormaechei replaces virulence with carbapenem resistance via porin loss. Pathogenic Enterobacter species are of increasing clinical concern due to the multidrug-resistant nature of these bacteria, including resistance to carbapenem antibiotics. Our understanding of Enterobacter virulence is limited, hindering the development of new prophylactics and therapeutics targeting infections caused by Enterobacter species. In this study, we assessed the virulence of contemporary clinical Enterobacter hormaechei isolates in a mouse model of intraperitoneal infection and used comparative genomics to identify genes promoting virulence. Through mutagenesis and complementation studies, we found two porin-encoding genes, ompC and ompD, to be required for E. hormaechei virulence. These porins imported clinically relevant carbapenems into the bacteria, and thus loss of OmpC and OmpD desensitized E. hormaechei to the antibiotics. Our genomic analyses suggest porin-related genes are frequently mutated in E. hormaechei, perhaps due to the selective pressure of antibiotic therapy during infection. Despite the importance of OmpC and OmpD during infection of immunocompetent hosts, we found the two porins to be dispensable for virulence in a neutropenic mouse model. Moreover, porin loss provided a fitness advantage during carbapenem treatment in an ex vivo human whole blood model of bacteremia. Our data provide experimental evidence of pathogenic Enterobacter species gaining antibiotic resistance via loss of porins and argue antibiotic therapy during infection of immunocompromised patients is a conducive environment for the selection of porin mutations enhancing the multidrug-resistant profile of these pathogens.202539977318
8839190.9950Bacteriophage infection drives loss of β-lactam resistance in methicillin-resistant Staphylococcus aureus. Bacteriophage (phage) therapy is a promising means to combat drug-resistant bacterial pathogens. Infection by phage can select for mutations in bacterial populations that confer resistance against phage infection. However, resistance against phage can yield evolutionary trade-offs of biomedical relevance. Here, we report the discovery that infection by certain staphylococcal phages sensitizes different strains of methicillin-resistant Staphylococcus aureus (MRSA) to β-lactams, a class of antibiotics against which MRSA is typically resistant. MRSA cells that survive infection by these phages display significant reductions in minimal inhibitory concentration against different β-lactams compared to uninfected bacteria. Transcriptomic profiling reveals that these evolved MRSA strains possess highly modulated transcriptional profiles, where numerous genes involved in S. aureus virulence are downregulated. Phage-treated MRSA exhibited attenuated virulence phenotypes in the form of reduced hemolysis and clumping. Despite sharing similar phenotypes, whole-sequencing analysis revealed that the different MRSA strains evolved unique genetic profiles during infection. These results suggest complex evolutionary trajectories in MRSA during phage predation and open up new possibilities to reduce drug resistance and virulence in MRSA infections.202540637714