Cationic Antimicrobial Peptides Resistance Research Articles

The Effect of the PhoP/PhoQ System on the Regulation of Multi-Stress Adaptation Induced by Acid Stress in Salmonella Typhimurium.

Acidic stress in beef cattle slaughtering abattoirs can induce the acid adaptation response of in-plant contaminated Salmonella. This may further lead to multiple resistance responses threatening public health. Therefore, the acid, heat, osmotic and antibiotic resistances of Salmonella typhimurium (ATCC14028) were evaluated after a 90 min adaption in a pH = 5.4 "mild acid" Luria-Bertani medium. Differences in such resistances were also determined between the ∆phoP mutant and wild-type Salmonella strains to confirm the contribution of the PhoP/PhoQ system. The transcriptomic differences between the acid-adapted and ∆phoP strain were compared to explore the role of the PhoP/Q two-component system in regulating multi-stress resistance. Acid adaptation was found to increase the viability of Salmonella to lethal acid, heat and hyperosmotic treatments. In particular, acid adaptation significantly increased the resistance of Salmonella typhimurium to Polymyxin B, and such resistance can last for 21 days when the adapted strain was stored in meat extract medium at 4 °C. Transcriptomics analysis revealed 178 up-regulated and 274 down-regulated genes in the ∆phoP strain. The Salmonella infection, cationic antimicrobial peptide (CAMP) resistance, quorum sensing and two-component system pathways were down-regulated, while the bacterial tricarboxylic acid cycle pathways were up-regulated. Transcriptomics and RT-qPCR analyses revealed that the deletion of the phoP gene resulted in the down-regulation of the expression of genes related to lipid A modification and efflux pumps. These changes in the gene expression result in the change in net negative charge and the mobility of the cell membrane, resulting in enhanced CAMP resistance. The confirmation of multiple stress resistance under acid adaptation and the transcriptomic study in the current study may provide valuable information for the control of multiple stress resistance and meat safety.

Metagenomic gut microbiome analysis of Japanese patients with multiple chemical sensitivity/idiopathic environmental intolerance

BackgroundAlthough the pathology of multiple chemical sensitivity (MCS) is unknown, the central nervous system is reportedly involved. The gut microbiota is important in modifying central nervous system diseases. However, the relationship between the gut microbiota and MCS remains unclear. This study aimed to identify gut microbiota variations associated with MCS using shotgun metagenomic sequencing of fecal samples.MethodsWe prospectively recruited 30 consecutive Japanese female patients with MCS and analyzed their gut microbiomes using shotgun metagenomic sequencing. The data were compared with metagenomic data obtained from 24 age- and sex-matched Japanese healthy controls (HC).ResultsWe observed no significant difference in alpha and beta diversity of the gut microbiota between the MCS patients and HC. Focusing on the important changes in the literatures, at the genus level, Streptococcus, Veillonella, and Akkermansia were significantly more abundant in MCS patients than in HC (p < 0.01, p < 0.01, p = 0.01, respectively, fold change = 4.03, 1.53, 2.86, respectively). At the species level, Akkermansia muciniphila was significantly more abundant (p = 0.02, fold change = 3.3) and Faecalibacterium prausnitzii significantly less abundant in MCS patients than in HC (p = 0.03, fold change = 0.53). Functional analysis revealed that xylene and dioxin degradation pathways were significantly enriched (p < 0.01, p = 0.01, respectively, fold change = 1.54, 1.46, respectively), whereas pathways involved in amino acid metabolism and synthesis were significantly depleted in MCS (p < 0.01, fold change = 0.96). Pathways related to antimicrobial resistance, including the two-component system and cationic antimicrobial peptide resistance, were also significantly enriched in MCS (p < 0.01, p < 0.01, respectively, fold change = 1.1, 1.2, respectively).ConclusionsThe gut microbiota of patients with MCS shows dysbiosis and alterations in bacterial functions related to exogenous chemicals and amino acid metabolism and synthesis. These findings may contribute to the further development of treatment for MCS.Trial registrationThis study was registered with the University Hospital Medical Information Clinical Trials Registry as UMIN000031031. The date of first trial registration: 28/01/2018.

Preclinical Alzheimer’s disease is associated with distinct gut microbial metagenomic profiles

AbstractBackgroundAlterations in gut microbiome in individuals with Alzheimer’s disease (AD) may contribute to development or exacerbation of AD pathology via the microbiota‐gut‐brain axis. Composition and diversity of gut microbiota have been studied previously in AD dementia; here we investigate the gut microbial genes involved and their association with AD pathology prior to development of dementia.MethodCerebrospinal fluid (CSF; N = 98) and fecal samples (N = 333) were collected from participants enrolled in the Wisconsin Registry for Alzheimer’s Prevention and Wisconsin Alzheimer’s Disease Research Center studies. CSF biomarkers of AD, β‐amyloid (Aβ)42/Aβ40 and phosphorylated‐tau‐181 (phospho‐tau [181P]), were quantified using robust prototype assays from the Roche NeuroToolKit (Roche Diagnostics International Ltd, Rotkreuz, Switzerland). Fecal samples were analyzed by whole genome sequencing. Sequences were filtered, quality controlled, and profiled for taxonomy and functional classification at species level using MetaPhlAn 4.0/HUMAnN 3.0 software. Compositional/functional differential abundance, alpha‐/beta‐diversity, correlation between feature abundance and sample metadata, gene‐set enrichment (GSE), and network analyses were performed using R packages (phyloseq/vegan/DESeq2/NetCoMi/rstatix/clusterProfiler). Analyses compared clinical status and biomarker groups.ResultThe relative abundance of phylum Firmicutes and Bacteroidetes decreased and increased, respectively, in individuals with AD versus cognitively unimpaired (CU) individuals. In CU individuals, functional profiles differed in Aβ‐positive (A+) and phospho‐tau [181P]‐positive (T+) groups versus Aβ‐ and phospho‐tau [181P]‐negative (A−T−) individuals. In the microbial metagenome, 64 genes were significantly increased (p &lt; 0.05) in the A+T+ group versus the A−T− group; 21/64 genes had ≥1 significant correlation with the AD biomarkers (e.g., toxic protein SymE positively correlated with phospho‐tau [181P] and negatively correlated with Aβ42/Aβ40 in CU individuals, suggesting that it may be a contributor to pathophysiological metabolic pathways in AD). Three KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways identified in GSE analyses, including fatty acid metabolism/degradation and cationic antimicrobial peptide resistance, were significantly associated (p &lt; 0.05) with genes that were enriched in the AD population versus CU individuals.ConclusionIndividuals with AD have distinct differential abundance in microbiota composition, in addition to an increase and enrichment of genes associated with AD pathogenesis pathways. These genes were associated with AD biomarkers prior to development of AD dementia.

The erythromycin polyketide compound TMC-154 stimulates ROS generation to exert antibacterial effects against Streptococcus pyogenes

The erythromycin polyketide compound TMC-154 is a secondary metabolite that is isolated from the rhizospheric fungus Clonostachys rogersoniana associated with Panax notoginseng, which possesses antibacterial activity. However, its antibacterial mechanism has not been investigated thus far. In this study, proteomics coupled with bioinformatics approaches was used to explore the antibacterial mechanism of TMC-154. KEGG pathway enrichment analysis indicated that eight signaling pathways were associated with TMC-154, including oxidative phosphorylation, cationic antimicrobial peptide (CAMP) resistance, benzoate degradation, heme acquisition systems, glycine/serine and threonine metabolism, beta-lactam resistance, ascorbate and aldarate metabolism, and phosphotransferase system (PTS). Cell biology experiments confirmed that TMC-154 could induce reactive oxygen species (ROS) generation in Streptococcus pyogenes; moreover, TMC-154-induced antibacterial effects could be blocked by the inhibition of ROS generation with the antioxidant N-acetyl L-cysteine. In addition, TMC-154 combined with ciprofloxacin or chloramphenicol had synergistic antibacterial effects. These findings indicate the potential of TMC-154 as a promising drug to treat S. pyogenes infections. SignificanceStreptococcus pyogenes is a nearly ubiquitous human pathogen that causes a variety of diseases ranging from mild pharyngitis and skin infection to fatal sepsis and toxic heat shock syndrome. With the increasing incidence of known antibiotic resistance, there is an urgent need to find novel drugs with good antibacterial activity against S. pyogenes. In this study, we found that TMC-154, a secondary metabolite from the fungus Clonostachys rogersoniana, inhibited the growth of various bacteria, including Staphylococcus aureus, S. pyogenes, Streptococcus mutans, Pseudomonas aeruginosa and Vibrio parahemolyticus. Proteomic analysis combined with cell biology experiments revealed that TMC-154 stimulated ROS generation to exert antibacterial effects against S. pyogenes. This study provides potential options for the treatment of S. pyogenes infections in the future.

Calcium-mediated modulation of Pseudomonas fluorescens biofilm formation

Biofilm formation is usually affected by many environmental factors, including divalent cations. The purpose of the current work was to analyze how calcium (Ca2+) affects the biofilm formation of dairy Pseudomonas fluorescens isolates by investigating their growth, swarming motility, biofilm-forming capacity, extracellular polymeric substance production, and biofilm structures. Moreover, the regulation mechanism of Ca2+ involved in its biofilm formation was explored through RNA-sequencing analysis. This work revealed that supplementation of 5, 10, 15, and 20 mM Ca2+ significantly reduced the swarming motility of P. fluorescens strains (P.F2, P.F4, and P.F17), but the biofilm-forming ability and polysaccharide production were increased after the supplementation of 5 and 10 mM Ca2+. By the supplementation of Ca2+, complex structures with more cell clusters glued together in P. fluorescens P.F4 biofilms were confirmed by scanning electron microscopy, and increased biomass and coverage of P. fluorescens P.F4 biofilms were observed by confocal laser scanning microscopy. In addition, RNA-sequencing results showed that P. fluorescens P.F4 showed a transcriptional response to the supplementation of 10 mM Ca2+, and a total of 137 genes were significantly expressed. The differential genes were represented in 4 upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (nonribosomal peptide structures, quorum sensing, biosynthesis of siderophore group nonribosomal peptides, and phenylalanine metabolism), and 4 downregulated KEGG pathways (flagellar assembly, amino sugar and nucleotide sugar metabolism, nitrotoluene degradation, and cationic antimicrobial peptide resistance). The results indicate that Ca2+ might serve as an enhancer to substantially trigger the biofilm formation of dairy P. fluorescens isolates in the dairy industry.

Multiomics analysis reveals the genetic and metabolic characteristics associated with the low prevalence of dental caries

ABSTRACT Background Despite poor oral hygiene, the Baiku Yao (BKY) ethnic group in China presents a low prevalence of dental caries, which may be related to genetic susceptibility. Due to strict intra-ethnic marriage rule, this ethnic has an advantage in studying the interaction between genetic factors and other regulatory factors related to dental caries. Methods Peripheral blood from a caries-free adult male was used for whole genome sequencing, and the BKY assembled genome was compared to the Han Chinese genome. Oral saliva samples were collected from 51 subjects for metabolomic and metagenomic analysis. Multiomics data were integrated for combined analysis using bioinformatics approaches. Results Comparative genomic analysis revealed the presence of structural variations in several genes associated with dental caries. Metabolomic and metagenomic sequencing demonstrated the caries-free group had significantly higher concentration of antimicrobials and higher abundance of core oral health-related microbiota. The functional analysis indicated that cationic antimicrobial peptide resistance and the lipopolysaccharide biosynthesis pathway were enriched in the caries-free group. Conclusions Our study provided new insights into the specific regulatory mechanisms that contribute to the low prevalence of dental caries in the specific population and may provide new evidence for the genetic diagnosis and control of dental caries.

Exopolysaccharide produced by Lactiplantibacillus plantarum Y12 exhibits inhibitory effect on the Shigella flexneri genes expression related to biofilm formation

Shigella is a specific enteric pathogen in humans, causing symptoms of bacterial dysentery. The biofilm formation of S. flexneri contributes to the emergence of multidrug resistance and facilitates the establishment of persistent chronic infections. This study investigated the regulatory effects of Lactiplantibacillus plantarum Y12 exopolysaccharide (L-EPS) on gene expression and its spatial hindrance effects in inhibiting the biofilm formation of S. flexneri. The transcriptome analysis revealed a significant impact of L-EPS on the gene expression profile of S. flexneri, with a total of 968 genes showing significant changes (507 up-regulated and 461 down-regulated). The significantly down-regulated KEGG metabolic pathway enriched in phosphotransferase system, Embden-Meyerhf-Parnas, Citrate cycle, Lipopolysaccharide biosynthesis, Cationic antimicrobial peptide resistance, Two-component system. Moreover, L-EPS significantly down-regulated the gene expression levels of fimbriae synthesis (fimF), lipopolysaccharide synthesis (lptE, lptB), anchor protein repeat domain (arpA), virulence factor (lpp, yqgB), antibiotic resistance (marR, cusB, mdtL, mdlB), heavy metal resistance (zraP), and polysaccharide synthesis (mtgA, mdoB, mdoC). The expression of biofilm regulator factor (bssS) and two-component system suppressor factor (mgrB) were significantly up-regulated. The RT-qPCR results indicated that a major component of L-EPS (L-EPS 2-1) exhibited the gene regulatory effect on the S. flexneri biofilm formation. Furthermore, electrophoresis and isothermal microtitration calorimetry demonstrated that the interaction between L-EPS 2-1 and eDNA is electrostatic dependent on the change in environmental pH, disrupting the stable spatial structure of S. flexneri biofilm. In conclusion, L-EPS inhibited the biofilm formation of S. flexneri through gene regulation and spatial obstruction effects.

Genome analysis of clinical genotype Vibrio vulnificus isolated from seafood in Mangaluru Coast, India provides insights into its pathogenicity

Vibrio vulnificus an opportunistic human pathogen native to marine/estuarine environment, is one of the leading causes of death due to seafood consumption and exposure of wounds to seawater worldwide. The present study involves the whole genome sequence analysis of an environmental strain of V. vulnificus (clinical genotype) isolated from seafood along the Mangaluru coast of India. The sequenced genome data was subjected to in-silico analysis of phylogeny, virulence genes, antimicrobial resistance determinants, and secretary proteins using suitable bioinformatics tools. The sequenced isolate had an overall genome length of 4.8 Mb and GC content of 46% with 4400 coding DNA sequences. The sequenced strain belongs to a new sequence type (Multilocus sequence typing) and was also found to branch with a phylogenetic lineage that groups the most infectious strains of V. vulnificus. The seafood isolate had complete genes involved in conferring serum resistance yet showed limited serum resistance. The study identified several genes against the antibiotics that are commonly used in their treatment, highlighting the need for alternative treatments. Also, the secretory protein analysis revealed genes associated with major pathways like ABC transporters, two-component systems, quorum sensing, biofilm formation, cationic antimicrobial peptide (CAMP) resistance, and others that play a critical role in the pathogenesis of the V. vulnificus. To the best of our knowledge, this is the first report of a detailed analysis of the genomic information of a V. vulnificus isolated from the Indian subcontinent and provides evidence that raises public health concerns about the safety of seafood.

ArnB mediates CAMP resistance and in vivo colonization in the fish pathogen Edwardsiella piscicida

Adoption of a live attenuated vaccine (LAV) will be an effective disease management strategy in farmed fish. Given the natural existence of cationic antimicrobial peptides (CAMP) in fish, the CAMP susceptible strain has been a promising LAV candidate against the bacterial disease of fish. Edwardsiella piscicida, the aetiological agent of fish edwardsiellosis, has caused massive economic losses in aquaculture industries. However, the mechanism underlying the CAMP resistance of E. piscicida is not entirely clear. In this study, the arnB gene was identified to contribute to E. piscicida resistance to CAMP, such as polymyxin B and polymyxin E. The expression of arnB was inhibited by Mg2+ ions and activated by PhoP. Moreover, ∆arnB had impaired capability for in vivo colonization and had reduced virulence in turbot. Together, our findings demonstrated that arnB was required for CAMP resistance and in vivo colonization in E. piscicida, suggesting that ∆arnB might be a prospective potential LAV candidate against edwardsiellosis.

Proteomic analysis of Staphylococcus aureus exposed to bacteriocin XJS01 and its bio-preservative effect on raw pork loins

Antibacterial mechanism of bacteriocins against foodborne S. aureus is still to be explored, particularly in proteomics, and a deep and comprehensive study on application of bacteriocins for preservation of raw pork is required. Here, proteomic mechanism of Lactobacillus salivarius bacteriocin XJS01 against foodborne S. aureus 2612:1606BL1486 (S. aureus_26) and its preservation effect on raw pork loins stored at 4 °C for 12 days was investigated. The results showed that 301 differentially abundant proteins (DAPs) were identified between XJS01-treated and -free groups (control group) using Tandem mass tag (TMT) quantitative proteomics technology, which were primarily involved in amino acids and carbohydrate metabolism, cytolysis, defense response, cell apoptosis, cell killing, adhesion, and oxygen utilization of S. aureus_26. Bacterial secretion system (SRP) and cationic antimicrobial peptide resistance may be key pathways to maintain protein secretion and counteract the deleterious effects on S. aureus_26 caused by XJS01. In addition, XJS01 could significantly improve the preservation of raw pork loins by the evaluation results of sensory and antibacterial activity on the meat surface. Overall, this study showed that XJS01 induced a complex organism response in S. aureus, and it could be potential pork preservative.

Urbanization led to the abundance of Gram-negative, chemo-organo-heterotrophs, and antibiotic resistance genes in the downstream regions of the Ganga River water of India.

The present investigation assesses the bacterial microbiome and antibiotic resistance genes (ARGs) of the river Ganga from Uttarakhand (upstream region; US group) and Uttar Pradesh (downstream region; DS group) regions using a 16S rRNA amplicon-based metagenomic approach. Gram-negative, aerobic, and chemo-organotrophic bacteria made up the majority of the bacterial genera during the overall analysis. Physicochemical analysis revealed a higher concentration of nitrate and phosphate in the downstream sites of the Ganga River. The prevalence of Gemmatimonas, Flavobacterium, Arenimonas, and Verrucomicrobia in the water of the DS region indicates a high organic load. Pseudomonas and Flavobacterium emerged as the most prevalent genera among the 35 significantly different shared genera (p-value < 0.05) in the US and DS regions, respectively. Overall antibiotic resistance analysis of the samples showed the dominance of β-lactam resistance (33.92%) followed by CAMP (cationic antimicrobial peptide) resistance (27.75%), and multidrug resistance (19.17%), vancomycin resistance (17.84%), and tetracycline resistance (0.77%). While comparing, the DS group exhibited a higher abundance of ARGs over the US group, where the CAMP resistance and β-lactam ARGs were dominant in the respective regions. The correlation (p-value < 0.05) analysis showed that most bacteria exhibit a significant correlation with tetracycline resistance followed by the phenicol antibiotic. The present findings draw attention to the need for regulated disposal of multiform human-derived wastes into the Ganga River to reduce the irrepressible ARGs dissemination.

Dissemination of sulfonamide resistance genes in digester microbiome during anaerobic digestion of food waste leachate

The preeminence of sulfonamide drug resistance genes in food waste (FW) and the increased utilization of high-strength organic FW in anaerobic digestion (AD) to enhance methane production have raised severe public health concerns in wastewater treatment plants worldwide. In this regard, the dissemination patterns of different sulfonamide resistance genes (sul1 and sul2) and their impact on the digester core microbiota during AD of FW leachate (FWL) were evaluated. The presence of various sulfonamide antibiotics (SAs) in FWL digesters improved the final methane yield by 37 % during AD compared with FWL digesters without SAs. Microbial population shifts towards hydrolytic, acidogenic, and acetogenic bacteria in the phyla Actinobacteriota, Bacteroidota, Chloroflexi, Firmicutes, Proteobacteria, and Synergistota occurred due to SA induced substrate digestion and absorption through active transport; butanoate, propanoate, and pyruvate metabolism; glycolysis; gluconeogenesis; the citrate cycle; and pentose phosphate pathway. The initial dominance of Methanosaeta (89–96 %) declined to 47–53 % as AD progressed and shifted towards Methanosarcina (40 %) in digesters with the highest SA concentrations at the end of AD. Dissemination of sul1 depended on class 1 integron gene (intl1)-based horizontal gene transfer to pathogenic members of Chloroflexi, Firmicutes, and Patescibacteria, whereas sul2 was transmitted to Synergistota independent of intl1. Low susceptibility and ability to utilize SAs during methanogenesis shielded methanogenic archaea against selection pressure, thus preventing them from interacting with sul or intl1 genes, thereby minimizing the risk of antibiotic resistance development. The observed emergence of cationic antimicrobial peptide, vancomycin, and β-lactam resistance in the core microbiota during AD of FWL in the presence of SAs suggests that multidrug resistance caused by bacterial transformation could lead to an increase in the environmental resistome through wastewater sludge treatment.

Zinc pyrithione induced volatile fatty acids promotion derived from sludge anaerobic digestion: Interrelating the affected steps with microbial metabolic regulation and adaptive responses

The massive use of zinc pyrithione (ZPT, as broad-spectrum bactericides) resulted in its high levels in waste activated sludge (WAS) and affected subsequent WAS treatment. This work revealed the effects of ZPT on the volatile fatty acids (VFAs) during WAS anaerobic digestion, in which VFAs yield was enhanced by approximately 6-9 folds (from 353 mg COD/L in control to 2526-3318 mg COD/L with low level of ZPT (20-50 mg/g TSS)). The ZPT occurred in WAS enabled the acceleration of solubilization, hydrolysis and acidification processes while inhibited the methanogenesis. Also, the low ZPT contributed to the enrichment of functional hydrolytic-acidifying microorganisms (e.g., Ottowia and Acinetobacter) but caused the reduction of methanogens (e.g., Methanomassiliicoccus and Methanothrix). Meta-transcriptomic analysis demonstrated that the critical genes relevant to extracellular hydrolysis (i.e. CLPP and ZapA), membrane transport (i.e. gltI, and gltL), substrates metabolisms (i.e. fadj, and acd), and VFAs biosynthesis (i.e. porB and porD) were all upregulated by 25.1-701.3% with low level of ZPT. Specifically, the ZPT stimulus on amino acids metabolism for VFAs transformation was prominent over carbohydrates. Moreover, the functional species enabled to regulate the genes in QS and TCS systems to maintain favorable cell chemotaxis to adapt the ZPT stress. The cationic antimicrobial peptide resistance pathway was upregulated to blunt ZPT with the secretion of more lipopolysaccharide and activate proton pumps to maintain ions homeostasis to antagonize the ZPT toxicity for high microbial activities, the abundance of related genes was up-regulated by 60.5 to 524.5%. This work enlightened environmental behaviors of emerging pollutants on WAS anaerobic digestion process with interrelations of microbial metabolic regulation and adaptive responses.

Rose bud extract as a natural antimicrobial agent against Staphylococcus aureus: Mechanisms and application in maintaining pork safety

Pingyin rose bud extract (RBE) may serve as a green antibacterial agent replacing chemically synthesized agents, but its potential antibacterial activity and mechanisms were not well studied. In the present study, RBE component was analyzed through untargeted metabolomics. Then, Staphylococcus aureus was taken as research subject, and the action mechanisms of RBE in Luria–Bertani broth and its elimination efficiency in pork were studied. The results indicated that fatty acids were the most abundant category of antibacterial substances. The minimum inhibitory concentration and minimum bactericidal concentration of RBE against S. aureus were 0.19 and 0.39 mg/mL, respectively. Cell wall and membrane disruptions were observed in S. aureus treated with RBE. 865 differentially expressed genes (DEGs) were identified through transcriptome analysis (≥2-fold change, P < 0.05); DEGs related to two-component systems, DNA replication, and cationic antimicrobial peptide resistance were significantly affected. RBE can control the contamination of S. aureus in cooked pork without adversely impacting odor, pH, and color. Findings from this study revealed that RBE demonstrated good antibacterial efficacy against S. aureus, and can be used as a natural antimicrobial agent in cooked pork for maintaining food safety.

Comparative Proteomics of Outer Membrane Vesicles from Polymyxin-Susceptible and Extremely Drug-Resistant Klebsiella pneumoniae.

Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria serve as transporters for the delivery of cargo such as virulence and antibiotic resistance factors. OMVs play a key role in the defense against membrane-targeting antibiotics such as the polymyxin B. Herein, we conducted comparative proteomics of OMVs from paired Klebsiella pneumoniae ATCC 700721 polymyxin-susceptible (polymyxin B MIC = 0.5 mg/L) and an extremely resistant (polymyxin B MIC ≥128 mg/L), following exposure to 2 mg/L of polymyxin B. Comparative profiling of the OMV subproteome of each strain revealed proteins from multiple perturbed pathways, particularly in the polymyxin-susceptible strain, including outer membrane assembly (lipopolysaccharide, O-antigen, and peptidoglycan biosynthesis), cationic antimicrobial peptide resistance, β-lactam resistance, and quorum sensing. In the polymyxin-susceptible strain, polymyxin B treatment reduced the expression of OMV proteins in the pathways related to adhesion, virulence, and the cell envelope stress responses, whereas, in the polymyxin-resistant strain, the proteins involved in LPS biosynthesis, RNA degradation, and nucleotide excision repair were significantly overexpressed in response to polymyxin B treatment. Intriguingly, the key polymyxin resistance enzymes 4-amino-4-deoxy-l-arabinose transferase and the PhoPQ two-component protein kinase were significantly downregulated in the OMVs of the polymyxin-susceptible strain. Additionally, a significant reduction in class A β-lactamase proteins was observed following polymyxin B treatment in the OMVs of both strains, particularly the OMVs of the polymyxin-susceptible strain. These findings shed new light on the OMV subproteome of extremely polymyxin resistant K. pneumoniae, which putatively may serve as active decoys to make the outer membrane more impervious to polymyxin attack. IMPORTANCE OMVs can help bacteria to fight antibiotics not only by spreading antibiotic resistance genes but also by acting as protective armor against antibiotics. By employing proteomics, we found that OMVs have a potential role in shielding K. pneumoniae and acting as decoys to polymyxin attack, through declining the export of proteins (e.g., 4-amino-4-deoxy-l-arabinose transferase) involved in polymyxin resistance. Furthermore, polymyxin B treatment of both strains leads to shedding of the OMVs with perturbed proteins involved in outer membrane remodeling (e.g., LPS biosynthesis) as well as pathogenic potential of K. pneumoniae (e.g., quorum sensing). The problematic extended spectrum beta-lactamases SHV and TEM were significantly reduced in both strains, suggesting that polymyxin B may act as a potentiator to sensitize the bacterium to β-lactam antibiotics. This study highlights the importance of OMVs as "molecular mules" for the intercellular transmission and delivery of resistance and cellular repair factors in the bacterial response to polymyxins.

Escape and functional alterations of microbial aerosol particles containing Pseudomonas sp. during wastewater treatment

Wastewater treatment plants (WWTP) are considered sources of bioaerosols emission that negatively affects the surrounding atmosphere. This study focused on Pseudomonas sp. Emissions in bioaerosols from a WWTP that adopts the A2O treatment process, and their inactivation through ultraviolet (UV) radiation. High-throughput sequencing was used to assay the microbial population, and functional composition profiles were predicted using 16 S rRNA sequencing data with PICRUSt2. Recorded emission levels of airborne bacteria and Pseudomonas sp. In WWTP were 130 ± 83–6113 ± 3015 CFU/m3 and 0–6431 ± 1945 CFU/m3, respectively. Bioaerosol emissions presented site-related and temporal variation. Over 80% of Pseudomonas sp. Were attached to coarse particles with sizes over 2.1 μm. Bioaerosol concentration and particle-size distribution in the air were closely related to ambient temperature, relative humidity, light intensity, and wind speed. Exposure to 45.67 μW/cm3 UV radiation led to a significant decline in bioaerosol concentrations in the air, and reduction rate reached 89.16% and 95.77% for airborne bacteria and Pseudomonas sp., respectively. The results suggested that UV radiation can be an effective method in reducing bioaerosols. Compared with other bacteria, Pseudomonas stutzeri and Bacillus sp. Are more resistant to UV radiation. The abundance of antibiotic resistance genes noticeably receded when exposed to UV irradiation. The relative abundance of cationic antimicrobial peptide resistance, categorized under human diseases in KEGG (level 3), significantly decreased in Pseudomonas sp. After 120 min of UV irradiation. This study provides a novel insight into the control of bioaerosol emissions carrying pathogenic bacteria.

Molecular Characteristics and Quantitative Proteomic Analysis of Klebsiella pneumoniae Strains with Carbapenem and Colistin Resistance.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are usually multidrug resistant (MDR) and cause serious therapeutic problems. Colistin is a critical last-resort therapeutic option for MDR bacterial infections. However, increasing colistin use has led to the emergence of extensively drug-resistant (XDR) strains, raising a significant challenge for healthcare. In order to gain insight into the antibiotic resistance mechanisms of CRKP and identify potential drug targets, we compared the molecular characteristics and the proteomes among drug-sensitive (DS), MDR, and XDR K. pneumoniae strains. All drug-resistant isolates belonged to ST11, harboring blaKPC and hypervirulent genes. None of the plasmid-encoded mcr genes were detected in the colistin-resistant XDR strains. Through a tandem mass tag (TMT)-labeled proteomic technique, a total of 3531 proteins were identified in the current study. Compared to the DS strains, there were 247 differentially expressed proteins (DEPs) in the MDR strains and 346 DEPs in the XDR strains, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that a majority of the DEPs were involved in various metabolic pathways, which were beneficial to the evolution of drug resistance in K. pneumoniae. In addition, a total of 67 DEPs were identified between the MDR and XDR strains. KEGG enrichment and protein–protein interaction network analysis showed their participation in cationic antimicrobial peptide resistance and two-component systems. In conclusion, our results highlight the emergence of colistin-resistant and hypervirulent CRKP, which is a noticeable superbug. The DEPs identified in our study are of great significance for the exploration of effective control strategies against infections of CRKP.

Co-culture with Acinetobacter johnsonii enhances benzalkonium chloride resistance in Salmonella enterica via triggering lipid A modifications

Salmonella enterica is one of the leading causes of foodborne gastroenteritis worldwide. In the food production environment, many bacterial species co-exist on surfaces in biofilm structures, which can act as reservoirs of microbial contamination of food products. Polymicrobial biofilms have been shown to have greater tolerance to antimicrobials, such as disinfectants, however the mechanistic basis of this is poorly understood. In this study, S. enterica subsp. enterica serovar Liverpool was co-cultured in mixed-species biofilms with bacteria isolated from the food production environment and challenged with the cationic biocide disinfectant, benzalkonium chloride (BC). Co-culture with the common environmental bacterium Acinetobacter johnsonii resulted in >200-fold higher resistance of S. Liverpool to BC, compared to mono-culture biofilms. The transcriptional response of S. enterica to biofilm co-culture was determined using a dual RNA-seq strategy. Genes controlled by the PhoPQ and PmrAB two-component systems, involved in lipid A modification and associated with cationic antimicrobial peptide resistance (CAMP) of S. Liverpool, were significantly upregulated. Deletion of either the phoP or pmrA genes resulted in an increase in susceptibility to BC, suggesting that activation of their regulons during co-culture enhances BC resistance. S. Liverpool lipid A profiles changed significantly upon co-culturing, with greater incorporation of both phosphoethanolamine and palmitate, which was dependent upon activation of PhoPQ and PmrAB. We conclude that when grown in the presence of A. johnsonii, S. Liverpool increases its tolerance to cationic BC disinfection by remodelling its cell envelope including reducing the net negative charge of lipid A and increasing lipid A acyl density.

CrrAB regulates PagP-mediated glycerophosphoglycerol palmitoylation in the outer membrane of Klebsiella pneumoniae

The outer membrane (OM) of Gram-negative bacteria is an evolving antibiotic barrier composed of a glycerophospholipid (GP) inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The two-component regulatory system CrrAB has only recently been reported to confer high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Mutations in crrB have been shown to lead to the modification of the lipid A moiety of LPS through CrrAB activation. However, functions of CrrAB activation in the regulation of other lipids are unclear. Work here demonstrates that CrrAB activation not only stimulates LPS modification but also regulates synthesis of acyl-glycerophosphoglycerols (acyl-PGs), a lipid species with undefined functions and biosynthesis. Among all possible modulators of acyl-PG identified from proteomic data, we found expression of lipid A palmitoyltransferase (PagP) was significantly upregulated in the crrB mutant. Furthermore, comparative lipidomics showed that most of the increasing acyl-PG activated by CrrAB was decreased after pagP knockout with CRISPR-Cas9. These results suggest that PagP also transfers a palmitate chain from GPs to PGs, generating acyl-PGs. Further investigation revealed that PagP mainly regulates the GP contents within the OM, leading to an increased ratio of acyl-PG to PG species and improving OM hydrophobicity, which may contribute to resistance against certain cationic antimicrobial peptides resistance upon LPS modification. Taken together, this work suggests that CrrAB regulates the palmitoylation of PGs and lipid A within the OM through upregulated PagP, which functions together to form an outer membrane barrier critical for bacterial survival.

Cell Membrane Remodeling Mediates Polymyxin B Resistance in Klebsiella pneumoniae: An Integrated Proteomics and Metabolomics Study.

Polymyxin B (PB) is introduced into the clinic as the last-line therapy against carbapenem-resistant Klebsiella pneumoniae (CRKP). Unfortunately, increased resistance to PB in Klebsiella pneumoniae (K. pneumoniae) has threatened global health. Resistance of K. pneumoniae to PB was induced by passaging in serial concentrations of PB and determined by microbroth dilution method. Growth characteristics of induced strains including growth curve, reversibility of resistance, and biofilm formation (crystal violet staining method) were measured. This study employed TMT-labeled quantitative proteomics and LC-MS/MS metabolomics analysis to investigate the key biological processes associated with PB resistance in K. pneumoniae. A total of 315 differentially expressed proteins (DEPs) were identified, of which 133 were upregulated and 182 were downregulated in the PB-resistant K. pneumoniae. KEGG enrichment analysis revealed that the DEPs were mainly involved in ATP-binding cassette (ABC) transporters and cationic antimicrobial peptide (CAMP) resistance. Proteins related to central carbon metabolism were inhibited in the PB-resistant K. pneumoniae, but proteins mediating LPS modification were activated. Transcriptional levels of CAMP resistance-related proteins were significantly different between PB-susceptible and -resistant K. pneumoniae. PB treatment led to an increase in reactive oxygen species (ROS) levels of K. pneumoniae. Metabolomics data demonstrated that 23 metabolites were significantly upregulated in PB-resistant K. pneumoniae and 5 were downregulated. The differential metabolites were mainly lipids, including glycerophospholipids, sphingolipids, and fatty acids. Exposure to PB resulted in increased level of phospholipid transport gene mlaF in K. pneumoniae. Our study suggested that membrane remodeling and inhibited central carbon metabolism are conducive to the development of PB resistance in K. pneumoniae.