Antibiotic Resistance Proteins Research Articles

Rapid evolutionary change in trait correlations of single proteins

Many organismal traits are genetically determined and covary in evolving populations. The resulting trait correlations can either help or hinder evolvability – the ability to bring forth new and adaptive phenotypes. The evolution of evolvability requires that trait correlations themselves must be able to evolve, but we know little about this ability. To learn more about it, we here study two evolvable systems, a yellow fluorescent protein and the antibiotic resistance protein VIM-2 metallo beta-lactamase. We consider two traits in the fluorescent protein, namely the ability to emit yellow and green light, and three traits in our enzyme, namely the resistance against ampicillin, cefotaxime, and meropenem. We show that correlations between these traits can evolve rapidly through both mutation and selection on short evolutionary time scales. In addition, we show that these correlations are driven by a protein’s ability to fold, because single mutations that alter foldability can dramatically change trait correlations. Since foldability is important for most proteins and their traits, mutations affecting protein folding may alter trait correlations mediated by many other proteins. Thus, mutations that affect protein foldability may also help shape the correlations of complex traits that are affected by hundreds of proteins.

Screening of Potential Phytochemicals for the Identified Candidate Virulence Proteins in Enterobacter huaxiensis

ABSTRACT: Enterobacter huaxiensis, a novel gram-negative bacterium of the family Enterobacteriaceae, was recovered from the blood of the patients at West China Hospital. The present study aims to predict potential lead molecules against the identified virulence-associated antibiotic-resistant protein for drug designing. Two virulence-associated antibiotic-resistance proteins belonging to the OqxAB efflux protein family of the RND superfamily were identified in the pathogen using bioinformatics tools/databases. Based on the structure prediction by homology modeling and validations, the RND transporter permease subunit, OqxB was selected as the potential target for lead identification. The binding pocket of the target protein was calculated using CASTp. A total of 204 phytochemicals were screened virtually to obtain compounds that had better binding affinity, drug-likeness and pharmacokinetic potential to be used as safe ligands against the target protein. Among these, Chrysoeriol, Isopimaric acid, Baicalein and Biochanin A were found to be within the permissible range of Lipinski rule of five for drug-likeness, possessing better ADMET properties, and lower target-protein binding energy (less than -8.0 kcal/mol). Ligand-protein docking showed stable non-covalent interactions between active site residues and ligands. Thus, these compounds may be considered potential inhibitors of the target protein that may inactivate the efflux pump and restore antibiotic sensitivity.

The Proteome of Extracellular Membrane Vesicles from Bacillus pumilus 3-19

—Production of extracellular membrane vesicles plays an important role in communication in bacterial populations and in bacteria–host interactions. Vesicles as carriers of various regulatory and signaling molecules may be potentially used as disease biomarkers and promising therapeutic agents, including vaccine preparations. The composition of membrane vesicles has been deciphered for a limited number of Gram-negative and Gram-positive bacteria. In this work, for the first time, extracellular membrane vesicles of a streptomycin-resistant strain Bacillus pumilus 3-19, a producer of extracellular guanyl-preferring ribonuclease binase, are isolated, visualized, and characterized by their genome and proteome composition. It has been established that there is no genetic material in the vesicles and the spectrum of the proteins differs depending on the phosphate content in the culture medium of the strain. Vesicles from a phosphate-deficient medium carry 49 unique proteins in comparison with 101 from a medium with the high phosphate content. The two types of vesicles had 140 mutual proteins. Flagellar proteins, RNase J, which is the main enzyme of RNA degradosomes, phosphatases, peptidases, iron transporters, signal peptides, were identified in vesicles. Antibiotic resistance proteins and amyloid-like proteins whose genes are present in B. pumilus 3-19 cells are absent. Phosphate deficiency-induced binase was found only in vesicles from a phosphate-deficient medium.

Endophytic Bacterium Flexivirga meconopsidis sp. nov. with Plant Growth-Promoting Function, Isolated from the Seeds of Meconopsis integrifolia.

Strain Q11T of an irregular coccoid Gram-positive bacterium, aerobic and non-motile, was isolated from Meconopsis integrifolia seeds. Strain Q11T grew optimally in 1% (w/v) NaCl, pH 7, at 30 °C. Strain Q11T is most closely related to Flexivirga, as evidenced by 16S rRNA gene analysis, and shares the highest similarity with Flexivirga aerilata ID2601ST (99.24%). Based on genome sequence analysis, the average nucleotide identity and digital DNA-DNA hybridization values of strains Q11T and D2601ST were 88.82% and 36.20%, respectively. Additionally, strain Q11T showed the abilities of nitrogen fixation and indole acetic acid production and was shown to promote maize growth under laboratory conditions. Its genome contains antibiotic resistance genes (the vanY gene in the vanB cluster and the vanW gene in the vanI cluster) and extreme environment tolerance genes (ectoine biosynthetic gene cluster). Shotgun proteomics also detected antibiotic resistance proteins (class A beta-lactamases, D-alanine ligase family proteins) and proteins that improve plant cold tolerance (multispecies cold shock proteins). Strain Q11T was determined to be a novel species of the genus Flexivirga, for which the name Flexivirga meconopsidis sp. nov. is proposed. The strain type is Q11T (GDMCC 1.3002T = JCM 36020 T).

Characterization of Cronobacter sakazakii and Cronobacter malonaticus Strains Isolated from Powdered Dairy Products Intended for Consumption by Adults and Older Adults.

The objective of this study was to characterize Cronobacter spp. and related organisms isolated from powder dairy products intended for consumption by adults and older adults using whole-genome sequencing (WGS), and to identify genes and traits that encode antibiotic resistance and virulence. Virulence (VGs) and antibiotic resistance genes (ARGs) were detected with the Comprehensive Antibiotic Resistance Database (CARD) platform, ResFinder, and MOB-suite tools. Susceptibility testing was performed using disk diffusion. Five presumptive strains of Cronobacter spp. were identified by MALDI-TOF MS and ribosomal MLST. Three C. sakazakii strains were of the clinical pathovar ST1, one was ST31, and the remaining isolate was C. malonaticus ST60. In addition, Franconibacter helveticus ST345 was identified. The C. sakazakii ST1 strains were further distinguished using core genome MLST based on 2831 loci. Moreover, 100% of the strains were resistant to cefalotin, 75% to ampicillin, and 50% to amikacin. The C. sakazakii ST1 strains were multiresistant (MDR) to four antibiotics. Additionally, all the strains adhered to the N1E-115 cell line, and two invaded it. Eighteen ARGs mainly involved in antibiotic target alteration and antibiotic efflux were detected. Thirty VGs were detected and clustered as flagellar proteins, outer membrane proteins, chemotaxis, hemolysins, and genes involved in metabolism and stress. The pESA3, pSP291-1, and pCMA1 plasmids were detected, and the prevalent mobile genetic elements (MGEs) were ISEsa1, ISEc52, and IS26. The isolates of C. sakazakii and C. malonaticus exhibited multiresistance to antibiotics, harbored genes encoding various antibiotic resistance proteins, and various virulence factors. Consequently, these contaminated powdered dairy products pose a risk to the health of hypersensitive adults.

Metaproteomic Analysis of Gut Resistome in the Cecal Microbiota of Fattening Pigs Raised without Antibiotics.

Improper use of antibiotics in swine could reduce commensal bacteria and possibly increase pathogen infections via the gut resistome. This study aimed to compare the metaproteomic profiles of the gut resistome and related metabolism in the cecal microbiota of fattening pigs raised under antibiotic-free (ABF) conditions with those of ordinary industrial pigs (controls [CTRL]). The top three relatively abundant microbes in both groups were Escherichia coli, Ruminococcus, and Lactobacillus, followed by Bacteroides and Bifidobacterium. E. coli, Lactobacillus, and Bacteroides were found to be increased in the CTRL group, whereas Ruminococcus and Clostridium were greater in the ABF group. The highest abundances of antibiotic resistance proteins (log2 expression levels [ELs] of >10) were found to be for tetracycline resistance (Tetr) and aminoglycoside resistance (AMGr) proteins found in Bacteroides, with a significant increase in the CTRL group. High Tetr (ELs of 5.32) was found in Ruminococcus in the CTRL group, although pigs in both groups had never received tetracycline, possibly reflecting the influence of environments in farms. In E. coli, AMGr and β-lactamase family proteins were observed in both groups (ELs of 3 to 6), whereas multidrug resistance protein MdtL was significantly expressed in the CTRL group (ELs of around 3). In the ABF group, CRISPR-associated endonucleases Cas1 and Cas9, which function to defend against viruses, were markedly observed in Ruminococcus and Lactobacillus, respectively, with ELs of 8.6 and 4.15, respectively. In conclusion, this study demonstrated that CRISPR-associated endonucleases were markedly observed in the ABF group, whereas higher levels of Tetr, AMGr, and multidrug resistance protein MdtL was markedly observed in dominant bacterial species in the CTRL group. IMPORTANCE In order to control and reduce antibiotic use in animals, the Department of Livestock Development, Thailand, has launched a campaign for antibiotic-free livestock production. The present study has shown for the first time that CRISPR-associated endonucleases Cas1 and Cas9, which function to defend against viruses, were markedly observed in Ruminococcus and Lactobacillus, respectively, in ceca of pigs raised without antibiotics (ABF). The highest abundances of antibiotic resistance proteins were for tetracycline (Tetr) and aminoglycoside resistance (AMGr) proteins found in Bacteroides, with a significant increase in the controls. In E. coli, the microbe with the highest relative abundance, AMGr and β-lactamase family proteins were observed in both groups, whereas multidrug resistance protein MdtL was significantly expressed in the controls. Pigs in both ABF and control groups had never received tetracycline, possibly reflecting the influence of farm environments. We suggest that pigs raised without antibiotics may have more beneficial microorganisms for the gut than pigs raised with antibiotics.

Experimental Evolution of the TolC-Receptor Phage U136B Functionally Identifies a Tail Fiber Protein Involved in Adsorption through Strong Parallel Adaptation.

Bacteriophages have received recent attention for their therapeutic potential to treat antibiotic-resistant bacterial infections. One particular idea in phage therapy is to use phages that not only directly kill their bacterial hosts but also rely on particular bacterial receptors, such as proteins involved in virulence or antibiotic resistance. In such cases, the evolution of phage resistance would correspond to the loss of those receptors, an approach termed evolutionary steering. We previously found that during experimental evolution, phage U136B can exert selection pressure on Escherichia coli to lose or modify its receptor, the antibiotic efflux protein TolC, often resulting in reduced antibiotic resistance. However, for TolC-reliant phages like U136B to be used therapeutically, we also need to study their own evolutionary potential. Understanding phage evolution is critical for the development of improved phage therapies as well as the tracking of phage populations during infection. Here, we characterized phage U136B evolution in 10 replicate experimental populations. We quantified phage dynamics that resulted in five surviving phage populations at the end of the 10-day experiment. We found that phages from all five surviving populations had evolved higher rates of adsorption on either ancestral or coevolved E. coli hosts. Using whole-genome and whole-population sequencing, we established that these higher rates of adsorption were associated with parallel molecular evolution in phage tail protein genes. These findings will be useful in future studies to predict how key phage genotypes and phenotypes influence phage efficacy and survival despite the evolution of host resistance. IMPORTANCE Antibiotic resistance is a persistent problem in health care and a factor that may help maintain bacterial diversity in natural environments. Bacteriophages ("phages") are viruses that specifically infect bacteria. We previously discovered and characterized a phage called U136B, which infects bacteria through TolC. TolC is an antibiotic resistance protein that helps bacteria pump antibiotics out of the cell. Over short timescales, phage U136B can be used to evolutionarily "steer" bacterial populations to lose or modify the TolC protein, sometimes reducing antibiotic resistance. In this study, we investigate whether U136B itself evolves to better infect bacterial cells. We discovered that the phage can readily evolve specific mutations that increase its infection rate. This work will be useful for understanding how phages can be used to treat bacterial infections.

Effects of successional sulfadiazine exposure on biofilm in moving bed biofilm reactor: Secretion of extracellular polymeric substances, community activity and functional gene expression

The effects of sulfadiazine (SDZ) on responses of biofilm in a moving bed biofilm reactor were explored with emphasis on the changes in extracellular polymeric substances (EPS) and functional genes. It was found that 3 to 10 mg/L SDZ reduced the protein (PN) and polysaccharide (PS) contents of EPS by 28.7%-55.1% and 33.3%-61.4%, respectively. The EPS maintained high ratio of PN to PS (10.3–15.1), and the major functional groups within EPS remained unaffected to SDZ. Bioinformatics analysis showed that SDZ significantly altered the community activity such as increased expression of s_Alcaligenes faecali. Totally, the biofilm held high SDZ removal rates, which were ascribed to the self-protection by secreted EPS, and genes levels upregulation of antibiotic resistance and transporter protein. Collectively, this study provides more details on the biofilm community exposure to an antibiotic and highlights the role of EPS and functional genes in antibiotic removal.

Genes Vary Greatly in Their Propensity for Collateral Fitness Effects of Mutations.

Mutations can have deleterious fitness effects when they decrease protein specific activity or decrease active protein abundance. Mutations will also be deleterious when they cause misfolding or misinteractions that are toxic to the cell (i.e., independent of whether the mutations affect specific activity and abundance). The extent to which protein evolution is shaped by these and other collateral fitness effects is unclear in part because little is known of their frequency and magnitude. Using deep mutational scanning (DMS), we previously found at least 42% of missense mutations in the TEM-1 β-lactamase antibiotic resistance gene cause deleterious collateral fitness effects. Here, we used DMS to comprehensively determine the collateral fitness effects of missense mutations in three genes encoding the antibiotic resistance proteins New Delhi metallo-β-lactamase (NDM-1), chloramphenicol acetyltransferase I (CAT-I), and 2″-aminoglycoside nucleotidyltransferase (AadB). AadB (20%), CAT-I (0.9%), and NDM-1 (0.2%) were less susceptible to deleterious collateral fitness effects than TEM-1 (42%) indicating that genes have different propensities for these effects. As was observed with TEM-1, all the studied deleterious aadB mutants increased aggregation. However, aggregation did not correlate with collateral fitness effects for many of the deleterious mutants of CAT-I and NDM-1. Select deleterious mutants caused unexpected phenotypes to emerge. The introduction of internal start codons in CAT-1 caused loss of the episome and a mutation in aadB made its cognate antibiotic essential for growth. Our study illustrates how the complexity of the cell provides a rich environment for collateral fitness effects and new phenotypes to emerge.

Protein Therapeutic Target Candidates Against Acinetobacter baumannii, a Pathogen of Concern to Planetary Health: A Network-Based Integrative Omics Drug Discovery Approach.

Acinetobacter baumannii, an opportunistic gram-negative pathogen responsible for several nosocomial infections, has developed resistance to various antibiotics. Proteins involved in the two-component system (TCS), virulence, and antibiotic resistance (AR), help this pathogen in regulating antibiotic susceptibility and virulence mechanisms. The present study reports a network-based integrative omics approach to drug discovery to identify key regulatory proteins as therapeutic candidates against A. baumannii. We collected data on the TCS, virulence, and AR proteins from various databases (P2CS, VFDB, ARDB, and PAIDB), which were subjected to network, host-pathogen, and gene expression data analysis. Network analysis identified 43 hubs, and 10 proteins were found to be interacting with human proteins associated with vital pathways. Of the 53 (43 + 10) pathogen proteins, 46 had no orthologs in the human host. Twelve proteins, namely, RpfC, Wzc, OmpR, EnvZ, BfmS, PilG, histidine kinase, ABC 3 transport family protein, outer membrane porin OprD family, CsuD, Pgm, and LpxA, were differentially expressed in the resistant strain. We propose these proteins as key regulators that warrant evaluation as therapeutic target candidates in the future. Furthermore, structure prediction of ABC 3 transport family protein was performed as a case study. The findings from this study are poised to facilitate and inform drug discovery and development against A. baumannii.

A contrast of Pb(II), Cd(II), and Cu(II) toxicities to Aspergillus niger through biochemical, morphological, and genetic investigations

The toxicity of metals to microorganisms is highly correlated with the type of metal used. However, the differences in the resistance mechanisms of filamentous fungi to multiple metals remain unclear. In this study, we investigated the responses of Aspergillus niger to three toxic metals, i.e., Pb2+, Cd2+, and Cu2+. Fungal growth and metabolism indices showed that A. niger had a higher tolerance to Pb2+ (>1000 mg L−1) than to Cu2+ (300 mg L−1) and Cd2+ (50 mg L−1). An appropriate Pb2+ concentration (<500 mg L−1) stimulated fungal growth and metabolic activity, whereas Cd2+ and Cu2+ stress showed continuously negative influences on fungal physiological parameters, such as biomass and secretion of oxalic acid. A. niger responded to Pb stress by constructing a new border layer around its cell wall. This pathway was also confirmed using RNA-seq analysis, i.e., the gene encoding cell wall α-1,3-glucan synthase was upregulated. This upregulation subsequently promoted the production of polysaccharides, which are the main components that support fungal cell walls. In contrast, the expression of genes encoding both AAA family ATPase and efflux pump antibiotic resistance proteins for Cd2+ and Cu2+ was significantly downregulated. Therefore, these findings elucidated the relatively complete fungal responses to different metal stresses.

Molecular identification and differential proteomics of drug resistant Salmonella Typhi

This study aimed to elucidate differentially expressed proteins in drug resistant Salmonella Typhi. Among 100 samples, S. typhi were identified in 43 samples. In drug susceptibility profile, 95.3% (41/43), 80% (35/43) and 70% (30/43) resistances were observed against Nalidixic acid, Ampicillin, and Chloramphenicol respectively. No resistance was observed against Imipenum and Azithromycin while only 11% (5/43) isolates were found resistant to Ceftriaxone. Mass spectrometric differential analysis resulted in 23 up-regulated proteins in drug resistant isolates. Proteins found up-regulated are involved in virulence (vipB, galU, tufA, and lpp1), translation (rpsF, rpsG, rplJ, and rplR), antibiotic resistance (zwf, phoP, and ompX), cell metabolism (metK, ftsZ, pepD, and secB), stress response (ridA, rbfA, and dps), housekeeping (gapA and eno) and hypothetical proteins including ydfZ, t1802, and yajQ. These proteins are of diverse nature and functions but highly interconnected. Further characterization may be helpful for elucidation of new biomarker proteins and therapeutic drug targets.

MP4: a machine learning based classification tool for prediction and functional annotation of pathogenic proteins from metagenomic and genomic datasets

Bacteria can exceptionally evolve and develop pathogenic features making it crucial to determine novel pathogenic proteins for specific therapeutic interventions. Therefore, we have developed a machine-learning tool that predicts and functionally classifies pathogenic proteins into their respective pathogenic classes. Through construction of pathogenic proteins database and optimization of ML algorithms, Support Vector Machine was selected for the model construction. The developed SVM classifier yielded an accuracy of 81.72% on the blind-dataset and classified the proteins into three classes: Non-pathogenic proteins (Class-1), Antibiotic Resistance Proteins and Toxins (Class-2), and Secretory System Associated and capsular proteins (Class-3). The classifier provided an accuracy of 79% on real dataset-1, and 72% on real dataset-2. Based on the probability of prediction, users can estimate the pathogenicity and annotation of proteins under scrutiny. Tool will provide accurate prediction of pathogenic proteins in genomic and metagenomic datasets providing leads for experimental validations. Tool is available at: http://metagenomics.iiserb.ac.in/mp4.

Abstract 15608: Validation of Enhancer/Silencer Activity of the Intracranial Aneurysm-Associated SNP at SOX17 in Primary Human Endothelia

Introduction: GWAS have identified multiple SNPs that occur more commonly in those with intracranial aneurysms. Many SNPs are located in noncoding genomic regions, suggesting that genetic risk may operate on functional regulatory elements that influence gene expression. However, regulatory elements are most often only indirectly identified through methods such as DNAse hypersensitivity without direct assessment of activity. Hypothesis: We hypothesize CRISPR mediated ablation of putative regulatory elements located within intergenic loci associated with IA will result in mRNA transcript modulation of genes located within the respective topology associated domain. Methods: We investigated a SNP within a haplotype block near SOX17. The haplotype was predicted to have active enhance activity, and to mediate SOX17 expression, based on assessment of its topologically associated domain. Lentiviral constructs (each with unique antibiotic resistance and fluorescent protein reporters) for the expression of Cas9 nuclease and two gRNAs flanking a putative element located at chr8:54412191-54413910 were transfected into the Lenti-X viral propagating cell line (Figure, left). Viral supernatants were transduced into primary HUVEC cells by spinfection with 5 ug/mL polybrene. After antibiotic selection and fluorescence validation, mRNA was isolated, and transcripts quantified through PCR. Results: Lentiviral transduction of HUVECs resulted in CRISPR-mediated removal of the enhancer element located within the haplotype block chr8:54397171-54415556 and resulted in a substantial increase of the SOX17 gene mRNA located within its topology associated domain (Figure, right). Conclusions: CRISPR mediated ablation was used to efficiently remove and validate a genomic element with putative regulatory activity in HUVECs. The methods will be employed to study additional regulatory elements with IA associations as well as other primary vascular cells.

The Physiological Functions of AbrB on Sporulation, Biofilm Formation and Carbon Source Utilization in Clostridium tyrobutyricum.

As a pleiotropic regulator, Antibiotic resistant protein B (AbrB) was reported to play important roles in various cellular processes in Bacilli and some Clostridia strains. In Clostridium tyrobutyricum, abrB (CTK_C 00640) was identified to encode AbrB by amino acid sequence alignment and functional domain prediction. The results of abrB deletion or overexpression in C. tyrobutyricum showed that AbrB not only exhibited the reported characteristics such as the negative regulation on sporulation, positive effects on biofilm formation and stress resistance but also exhibited new functions, especially the negative regulation of carbon metabolism. AbrB knockout strain (Ct/ΔabrB) could alleviate glucose-mediated carbon catabolite repression (CCR) and enhance the utilization of xylose compared with the parental strain, resulting in a higher butyrate titer (14.79 g/L vs. 7.91 g/L) and xylose utilization rate (0.19 g/L·h vs. 0.02 g/L·h) from the glucose and xylose mixture. This study confirmed the pleiotropic regulatory function of AbrB in C. tyrobutyricum, suggesting that Ct/ΔabrB was the potential candidate for butyrate production from abundant, renewable lignocellulosic biomass mainly composed of glucose and xylose.

P024 Genetic determinants of antifungal drug resistance in Fusarium solani species complex

 Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMDue to its challenging diagnosis and treatment, fungal keratitis is one of the most serious kinds of corneal infection. The Fusarium solani species complex is responsible for nearly half of all fungal keratitis cases. Fusarium infection is difficult to treat because of the increased antifungal resistance of Fusarium species.ObjectiveTo check the antifungal susceptibility in keratitis-causing isolates of F. solani species complex.To find the genetic determinants of resistance using whole genome sequencing.MethodologyPrior to AFST and according to CLSI, clinical isolates of Fusarium species (n = 5) were cultured on potato dextrose agar for 7 days at room temperature. Fungal spores were harvested using 0.8% NaCl and antifungal drug susceptibility testing of 6 different antifungal drugs were tested using CLSI standards broth microdilution method and minimum inhibitory concentrations were obtained. After 24 and 48 h, broth microdilution plates were manually examined. The wells for growth control were also examined. The minimum inhibitory concentration of anti-fungal drugs was the lowest dose that inhibits growth completely (compared to the growth control well). Minimal effective concentration (MEC) was also determined against antifungal drugs. Whole genome sequencing was done using Illumina Hiseqx10. Bioinformatics analysis was done using different bioinformatics tools and software (FASTQC, SPAdes, OmicsBox, AUGUSTUS, etc.).ResultsIn this study the antifungal drug susceptibility from average MIC value were found as fluconazole (512 μg/ml) > itraconazole (32 μg/ml) > amphotericin B (8 μg/ml) > natamycin (8 μg/ml) > posaconazole (2 μg/ml) > voriconazole (1 μg/ml). Fluconazole had a higher MIC value (512 g/ml) in all isolates, however, voriconazole was shown to be more sensitive, with a lower MIC range (0.25-4 g/ml). Glyoxalase/Bleomycin resistance protein, mfs—multidrug resistance transporter, fusaric acid resistance protein, efflux pump antibiotic resistance protein, and copper resistance protein were found by genome-based analysis.ConclusionAs a conclusion of this study, we observed antifungal drug resistance in Fusarium spp., which is often used to treat keratitis in patients, employing fluconazole, itraconazole, and amphotericin B. Resistance to first-line azoles was as a gene variant which contributed to antifungal drug resistance. This study using the phenotypic and genotypic characterization of drug resistance patterns will help to combat antifungal drug resistance.

Identification of Antibiotic Resistance Proteins via MiCId's Augmented Workflow. A Mass Spectrometry-Based Proteomics Approach.

Fast and accurate identifications of pathogenic bacteria along with their associated antibiotic resistance proteins are of paramount importance for patient treatments and public health. To meet this goal from the mass spectrometry aspect, we have augmented the previously published Microorganism Classification and Identification (MiCId) workflow for this capability. To evaluate the performance of this augmented workflow, we have used MS/MS datafiles from samples of 10 antibiotic resistance bacterial strains belonging to three different species: Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The evaluation shows that MiCId’s workflow has a sensitivity value around 85% (with a lower bound at about 72%) and a precision greater than 95% in identifying antibiotic resistance proteins. In addition to having high sensitivity and precision, MiCId’s workflow is fast and portable, making it a valuable tool for rapid identifications of bacteria as well as detection of their antibiotic resistance proteins. It performs microorganismal identifications, protein identifications, sample biomass estimates, and antibiotic resistance protein identifications in 6–17 min per MS/MS sample using computing resources that are available in most desktop and laptop computers. We have also demonstrated other use of MiCId’s workflow. Using MS/MS data sets from samples of two bacterial clonal isolates, one being antibiotic-sensitive while the other being multidrug-resistant, we applied MiCId’s workflow to investigate possible mechanisms of antibiotic resistance in these pathogenic bacteria; the results showed that MiCId’s conclusions agree with the published study. The new version of MiCId (v.07.01.2021) is freely available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html.

Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering

Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as “exosome painting,” we show here that CP05 binds equally well to CD63−/− cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.

Class 1 In-Tn5393c array contributed to antibiotic resistance of non-pathogenic Pseudoxanthomonas mexicana isolated from a wastewater bioreactor treating streptomycin

The emergence of antibiotic resistance in retort to environmental pollutants during wastewater treatment still remains elusive. Here, we first to investigate the emergence of antibiotic resistance in an environmental non-pathogenic bacterium, Pseudoxanthomonas mexicana isolated from a lab-scale bioreactor treating wastewater containing streptomycin. The molecular mechanism of antibiotic resistance development was evaluated in its genomic, transcriptional, and proteomic levels. The streptomycin resistant (SR) strain showed strong resistance to streptomycin (MIC > 600 μg/mL) as well to sulfamethoxazole, ampicillin, and kanamycin (≥250 μg/mL). A 13.4 kb class-1-integron array consisting of a new arrangement of gene cassette (IS6100-sul1-aadA2-catB3-aacA1-2-aadB-int1-IS256-int) linked with Tn5393c transposon was identified in the SR strain, which has only been reported in clinical pathogens so far. iTRAQ-LC-MS/MS proteomics revealed 22 up-regulated proteins in the SR strain growing under 100 mg L−1 streptomycin, involving antibiotic resistance, toxin production, stress response, and ribosomal protein synthesis. At the mRNA level, elevated expressions of ARGs (strA, strB, and aadB) and 30S-ribosomal protein genes (rpsA and rpsU) were observed in the SR strain. The results highlighted the genomic plasticity and multifaceted regulatory mechanism employed by P. mexicana in adaptation to high-level streptomycin during biological wastewater treatment.

Long-Term Effects of Soil Remediation with Willow Short Rotation Coppice on Biogeographic Pattern of Microbial Functional Genes.

Short rotation coppice (SRC) is increasingly being adopted for bioenergy production, pollution remediation and land restoration. However, its long-term effects on soil microbial communities are poorly characterized. Here, we studied soil microbial functional genes and their biogeographic pattern under SRC with willow trees as compared to those under permanent grassland (C). GeoChip analysis showed a lower functional gene diversity in SRC than in C soil, whereas microbial ATP and respiration did not change. The SRC soil had lower relative abundances of microbial genes encoding for metal(-oid) resistance, antibiotic resistance and stress-related proteins. This indicates a more benign habitat under SRC for microbial communities after relieving heavy metal stress, consistent with the lower phytoavailability of some metals (i.e., As, Cd, Ni and Zn) and higher total organic carbon, NO3−-N and P concentrations. The microbial taxa–area relationship was valid in both soils, but the space turnover rate was higher under SRC within 0.125 m2, which was possibly linked to a more benign environment under SRC, whereas similar values were reached beyond thisarea. Overall, we concluded that SRC management can be considered as a phytotechnology that ameliorates the habitat for soil microorganisms, owing to TOC and nutrient enrichment on the long-term.