Skip to main content
Download PDF

Proteomic analysis of carbapenem-resistant Klebsiella pneumoniae outer membrane vesicles under the action of phages combined with tigecycline

Annals of Clinical Microbiology and Antimicrobials volume 23, Article number: 73 (2024) Cite this article

Abstract

Background

Klebsiella pneumoniae is the most commonly encountered pathogen in clinical practice. Widespread use of broad-spectrum antibiotics has led to the current global dissemination of carbapenem-resistant K. pneumoniae, which poses a significant threat to antibacterial treatment efficacy and public health. Outer membrane vesicles (OMVs) have been identified as carriers capable of facilitating the transfer of virulence and resistance genes. However, the role of OMVs in carbapenem-resistant K. pneumoniae under external pressures such as antibiotic and phage treatments remains unclear.

Methods

To isolate and purify OMVs under the pressure of phages and tigecycline, we subjected K. pneumoniae 0692 harboring plasmid-mediated blaNDM-1 and blaKPC-2 genes to density gradient separation. The double-layer plate method was used to isolate MJ1, which efficiently lysed K. pneumoniae 0692 cells. Transmission electron microscopy (TEM) was used to characterize the isolated phages and extract OMV groups for relevant morphological identification. Determination of protein content of each OMV group was conducted through bicinchoninic acid assay (BCA) and proteomic analysis.

Results

K. pneumoniae 0692 released OMVs in response to different environmental stimuli, which were characterized through TEM as having the typical structure and particle size of OMVs. Phage or tigecycline treatment alone resulted in a slight increase in the mean protein concentration of OMVs secreted by K. pneumoniae 0692 compared to that in the untreated group. However, when phage treatment was combined with tigecycline, there was a significant reduction in the average protein concentration of OMVs compared to tigecycline treatment alone. Proteomics showed that OMVs encapsulated numerous functional proteins and that under different external stresses of phages and tigecycline, the proteins carried by K. pneumoniae 0692-derived OMVs were significantly upregulated or downregulated compared with those in the untreated group.

Conclusions

This study confirmed the ability of OMVs to carry abundant proteins and highlighted the important role of OMV-associated proteins in bacterial responses to phages and tigecycline, representing an important advancement in microbial resistance research.

Background

Klebsiella pneumoniae is a Gram-negative opportunistic pathogen that causes extremely serious infections with very high morbidity and mortality rates. Carbapenems are considered the last line of defense against drug-resistant Gram-negative bacterial infections in clinical practice. However, the latest monitoring data released by CHINET in August 2023 showed that the resistance rate of K. pneumoniae to carbapenems was expected to increase from 2.9 to 26.2% between 2005 and 2023 [1]. Therefore, carbapenem-resistant K. pneumoniae is recognized as one of the major pathogens causing hospital-acquired infections worldwide, posing a great challenge to hospital infection control measures [2].

As antimicrobial resistance (AMR) has become a growing public health threat, there is now a pressing need for alternative therapies, which has revitalized phage research [3]. As a type of viral particle prevalent in nature, phages can specifically infect host bacteria and are becoming a favorable treatment option for infections in clinical practice [4]. Phages infect their host bacteria by binding to their outer membrane or one of its components [5]. Outer membrane vesicles (OMVs) consist of the outer membrane of Gram-negative bacteria, which may play an important role in the interaction between bacteria and phages. Moreover, OMVs are spherical vesicles consisting of phospholipid (PL) bilayer membrane structures and membrane contents released externally by most Gram-negative bacteria [6, 7]. Ranging in diameter from 20 to 200 nm, OMVs may contain PLs, outer membrane proteins, lipopolysaccharide, DNA, RNA, bacterial metabolites, and signaling molecules, among other components [8, 9]. As part of the secretory delivery system, OMVs allow the dissemination of bacterial components into the environment, facilitating cell-to-cell interactions without the need for direct contact [10]. Therefore, OMVs play a key role in the response to environmental pressures such as phages and antibiotics.

Earlier work by Loeb showed that in the presence of a T4 phage in Escherichia coli, the production and release of OMVs were significantly increased [11]. Furthermore, the production of OMVs when phages are adsorbed onto bacterial surfaces has also been linked to bacterial defense and protection mechanisms [12]. Notably, antibiotics combined with phages for multidrug-resistant Pseudomonas aeruginosa exhibit synergistic activity. Despite the presence of acquired resistance mechanisms in bacteria, including overexpression of efflux pumps and expression of protein channels, phages can reduce the production of OMVs, which may contribute to the reduction of resistance and increase the efficacy of antibiotics [13].

Detecting and analyzing the expression types and levels of bacterial OMV-associated proteins will be useful for determining the optimal phage-antibiotic combinations for organisms with specific acquired resistance mechanisms and identifying the key mechanisms behind the relationship between phage-combined antibiotics and OMVs. In this study, we focused on a clinically isolated carbapenem-resistant K. pneumoniae strain that was resistant to all antibiotics tested, except tigecycline, to elucidate the protein composition and biological functions of OMVs under pressure from drugs and phages. These findings may contribute to the prevention and control of carbapenem-resistant K. pneumoniae infections, help in understanding the pathogenic mechanisms involved, and promote the development of OMVs.

Methods

Bacterial strains

The K. pneumoniae 0692 strain used in this study, harboring two carbapenemases, NDM-1 and KPC-2, was isolated from the First People’s Hospital of Yunnan Province, China. It was identified simultaneously using the VITEK 2 Compact and VITEK MS systems (bioMérieux, Lyon, France).

Bacterial drug-resistant gene detection

Plasmid DNA was extracted using a plasmid extraction kit (Tiangen, Beijing, China). The blaNDM and blaKPC plasmids were identified using polymerase chain reaction (PCR) and sequencing.

DNA extraction and PCR

The extraction of genomic DNA from K. pneumoniae 0692 isolates was performed with a bacterial genomic DNA extraction kit (Tiangen, Beijing, China). Table 1 lists the specific primers. Set up the PCR reaction system and amplification programme according to the Master Mix instructions (TSINGKE, China).

Table 1 List of primers used in this study
Full size table

Whole-genome sequencing of K. pneumoniae 0692 and phages

The genomic DNA of the clinically isolated K. pneumoniae 0692 strain was extracted using the Bacterial Genomic DNA Extraction Kit (Tiangen, China) and sequenced using the PacBio Sequel II/PacBio Sequel IIe and Illumina NovaSeq PE150 sequencing platforms. Similarly, phage genomic DNA was extracted using the Phage λ Genomic DNA Extraction Kit (Yuanye Biology, China) and sequenced using Illumina NovaSeq sequencing platforms following the manufacturer’s instructions. The sequencing results were compared with NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Determination of the minimum inhibitory concentrations (MICs)

The MICs of antimicrobial agents were determined using the microdilution susceptibility testing method. The following antibiotics were tested: imipenem, meropenem, ertapenem, tigecycline, ceftazidime/avibactam, cefoxitin, amikacin, and aztreonam. MIC results were interpreted as specified by Clinical Laboratory Standards Institute (CLSI) guidelines [14], except for tigecycline, which followed the interpretation defined by the US Food and Drug Administration (susceptible: MIC ≤ 2 mg/L; resistant: MIC ≥ 8 mg/L). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality controls.

Isolation and purification of phages

Phage isolation and purification were performed using the double-layer plate method, as previously described [15]. In brief, 100 μL of K. pneumoniae 0692 microbial suspension from an overnight culture and 100 μL of untreated sewage filtered through a 0.22 μm membrane filter (Thermo Fisher Scientific, USA) were mixed with 10 mL of fresh LB medium (Coolaber, China). The mixture was incubated in a shaking incubator at 160 rpm and 37 °C, and the resulting culture solution was filtered through a 0.22 μm filter to retain the filtrate. The lower plate of LB solid medium (2% agar) was prepared, and the upper plate was prepared by mixing 100 μL of overnight cultured K. pneumoniae 0692 and 100 μL of culture filtrate in semi-solid LB medium (0.5% agar), then spreading the plate and incubating at 37 °C overnight. Transparent round phage spots on top of the background of the host bacterium were observed. Single phage spots were picked and mixed into the K. pneumoniae 0692 microbial suspension to prepare double-layer plates for further purification. At least three rounds of purification were performed to efficiently lyse K. pneumoniae 0692. Plates with 30–300 phage spots were selected for counting. Titer (potency) = (number of phage spots × dilution × 10) PFU/mL.

Isolation and purification of OMVs

OMVs were prepared through density gradient ultracentrifugation in the absence or presence of 2 μg/mL tigecycline (MedChemExpress, Monmouth Junction, NJ, USA) and phage, following previously established protocols with minor adjustments [16]. Briefly, single colonies grown on LB agar plates were inoculated into 20 mL of LB broth medium (OD600 = 0.4). Subsequently, 1 mL of the microbial suspension was inoculated into 300 mL of antibiotic (2 μg/mL tigecycline) or phage-containing (1 mL of phage [1.42 × 1010 PFU/mL] solution added to 300 mL of LB medium) solutions and then incubated at 37 °C with shaking at 150 rpm for 16 h. Cultures were taken at 8000 rpm for 30 min at 4 °C to remove bacteria and cellular debris. The supernatant after centrifugation was collected and filtered using a 0.22 μm filter to remove fine debris. After the supernatant was ultracentrifuged at 25,000 rpm for 50 min at 4 °C in a P100AT2 rotor (Hitachi CP-100WX, Japan), the pellet was resuspended in 8 mL of phosphate-buffered saline (PBS) (Servicebio, China). OptiPrep™ (Sigma-Aldrich, Germany) was used to prepare 10%, 20%, and 30% density gradient solutions, which were distributed into discrete 10–30% density gradient layers. The resuspended OMV samples were placed on the density gradient and centrifuged at 4 °C and 25,000 rpm for 2 h. The OMV layer was collected and purified by washing with a tenfold volume of PBS solution at 25,000 rpm and 4 °C for 30 min. The resulting pellet was resuspended with 5 mL of PBS to obtain purified OMVs. The purified OMV solution was passed through a 0.22 μm membrane filter and filtered again. External DNA and proteins were removed using 2U DNase I (Biosharp, China) and 100 μg/mL Protease K (Biosharp, China). Purified OMV solution was inoculated onto Columbia blood plates to verify sterility.

Transmission electron microscopy

Purified phages and OMVs were observed through transmission electron microscopy (TEM). Samples were prepared by applying 10 μL of phage or OMVs suspended in PBS onto a carbon-coated copper grid for 5 min. After drying, the samples were stained with 2% uranyl acetate (Bio-Rad, Hercules, CA, USA) for 2 min, and the excess liquid was blotted. Observations were performed using a JEM-1400 Plus TEM (JEOL, Tokyo, Japan), operating at 80 kV.

Protein quantification of OMVs

Approximately 50 μL of OMVs were treated with 2U of DNase I (Biosharp, China) and 100 μg/mL of Protease K (Biosharp, China), followed by the addition of 50 μL of RIPA lysate (Beyotime, China). The mixture was then incubated at 4 °C for 30 min and subsequently centrifuges at 10,000×g at 4 °C for 15 min. A bicinchoninic acid (BCA) protein assay kit (Biosharp, China) was used to determine the protein concentration of OMVs samples, according to the manufacturer’s protocols.

Mass spectrometry assay of OMVs

The mass spectrometry experimental analysis process involved protein extraction, peptide enzymolysis, liquid chromatography-tandem mass spectrometry, and database retrieval. Briefly, SDT buffer (4% SDS, 100 mM Tris–HCl, pH 7.6; Bio-Rad, USA) was directly added to the different samples. DTT (with a final concentration of 40 mM; Bio-Rad, USA) was added to each sample individually and mixed at 600 rpm for 1.5 h (37 °C). After the samples were cooled to room temperature, iodoacetamide (Bio-Rad, USA) was added at a final concentration of 20 mM to block the reduced cysteine residues. Subsequently, the samples were incubated for 30 min in the dark, then transferred to filters (MicroCon units, 10 kDa). The filters were washed thrice with 100 μL UA buffer, then twice with 100 μL 25 mM NH4HCO3 (Sigma, Germany) buffer. Finally, trypsin was added to the samples (at a trypsin: protein (wt/wt) ratio of 1:50; Promega, Madison, WI, USA) and incubated at 37 °C for 15–18 h (overnight). The resulting peptides were collected as the filtrate. The peptides from each sample were analyzed using an Orbitrap™ Astral™ mass spectrometer (Thermo Scientific, USA) connected to a Vanquish Neo system liquid chromatograph (Thermo Scientific, USA) in data-independent acquisition (DIA) mode. DIA data were analyzed using DIA-NN 1.8.1 software.

Results

Bacterial drug-resistant gene detection

As demonstrated in Fig. 1 (Supplementary Material 2), DNA purified from the K. pneumoniae 0692 isolate exhibited specific amplified products for KPC and NDM (The lengths of the PCR products were KPC-752 bp and NDM-595 bp, Supplementary Material 1). Moreover, Whole-genome sequencing results were compared with NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that K. pneumoniae 0692 harbored both blaNDM-1 and blaKPC-2.

Fig. 1
figure 1

PCR profile of the strain used in this study. The primers used to perform PCR are KPC (left), NDM (middle) and the marker DL2000 (right), respectively

Full size image

Antimicrobial susceptibility testing

K. pneumoniae 0692 was resistant to cephalosporins, carbapenems, aminoglycosides, and β-lactams/β-lactamase inhibitors. However, this strain was sensitive to tigecycline with a MIC of 1 μg/mL. The results of the drug sensitivity tests are presented in Table 2. Based on the drug sensitivity results, tigecycline was administered to induce OMVs production.

Table 2 Results of MIC determination
Full size table

Isolation and biological characterization of phages

K. pneumoniae 0692 phages, named MJ1, were isolated from the sewage of the First People’s Hospital of Yunnan Province, using K. pneumoniae 0692 as the host. The phage plaque formed by MJ1 had a transparent appearance and a diameter of 3–4 mm. The phage concentration was determined to be approximately 1.42 × 1010 PFU/mL by titer (Fig. 2A, B). Morphologically, MJ1 displayed a polyhedral stereosymmetric head with a diameter of approximately 100 nm and a long tail, as observed through TEM. The genome length of phage MJ1 measured 111,163 bp in length, with a G+C% content of 44.72%, and comprised 1,385 open reading frames. According to the virus classification standards outlined by the International Committee on Taxonomy of Viruses in 2022, MJ1 phages are classified within the Myoviridae family.

Fig. 2
figure 2

A Isolation and purification via the bilayer plate method to obtain phage spots of lysed K. pneumoniae 0692. B Transmission electron observation of phage morphology. Scale bar: 200 nm

Full size image

Detection of OMVs

K. pneumoniae 0692 was cultured with or without 2 μg/mL tigecycline as well as phages, and purified OMVs were extracted from K. pneumoniae 0692 cultured under different conditions using density gradient centrifugation (Fig. 3A–D). TEM showed confirmed the spherical nature of the purified OMVs. No bacteria were detected under a light microscope, and cultures of the OMVs on Columbia blood culture plates showed no bacterial growth, indicating successful purification (Fig. 3). The mean particle size and protein concentration of each OMV group is shown in Table 3. The protein concentration in OMVs increased 1.33-, 4.38-, and 3.03-fold when K. pneumoniae 0692 was treated with phages, tigecycline, and phages with tigecycline, respectively. The average diameter of OMVs decreased after treatment with tigecycline, regardless of whether it was combined with phages.

Fig. 3
figure 3

A OMVs extracted without treatment (M). B OMVs extracted following treatment with 2 μg/mL tigecycline (MT). C OMVs extracted following phage treatment (MP). D OMVs extracted following co-treatment with 2 μg/mL tigecycline and phage (MTP). Scale bar: 200 nm

Full size image
Table 3 The mean particle size and protein concentration of OMVs
Full size table

The results are summarized in Table 3. The mean particle size of each OMV group was analyzed through TEM, and the mean protein concentration of the OMVs was determined using a BCA protein assay kit (Biosharp, China).

Proteomic analysis of different OMVs

Proteins from OMVs were digested on a gel with trypsin and extracted from the gel for proteomic analysis. Differential protein expression in OMVs from group M was compared with that of other groups separately. The proteomic analysis revealed a significant decrease in the gpmA protein across all groups. Formate-dependent phosphoribosylglycinamide formyltransferase (purT) protein expression was significantly upregulated in the MT group, whereas the 60 kDa chaperonin (groEL) protein was significantly upregulated in the MP group compared to that in the untreated group. Simultaneous treatment with tigecycline and phages resulted in significant upregulation of purT, groEL and thioredoxin (trxA) proteins (Fig. 4). The subcellular localization of the differentially expressed proteins in OMVs detected in this study was determined using an online subcellular structure prediction website (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) [17], as shown in Table 4. Among the identified proteins, the most significantly enriched differentially expressed proteins were associated with cellular metabolic processes, biosynthesis, intracellular structures, and nucleic acid binding.

Fig. 4
figure 4

Heatmap of significantly differentially expressed proteins. The results of hierarchical clustering were represented as a tree heat map. The horizontal axis represents the samples with different colors to indicate sample grouping information. The vertical axis represents differentially expressed proteins, with significant changes denoted by vertical coordinates. Significantly differentially expressed proteins across samples were normalized using the Z-score method and are presented in the heatmap with no colors. The red color indicates significantly upregulated proteins, the green color represents significantly downregulated proteins, and the grey color represents no protein quantification information

Full size image
Table 4 Subcellular localization of differential proteins detected in this study
Full size table

Discussion

Carbapenem-resistant K. pneumoniae stands as a prominent clinical pathogen, with NDM-1 and KPC-2 being the prevailing carbapenems posing significant threats to global public health [18]. Exploring the mechanisms of drug resistance in carbapenem-resistant K. pneumoniae and new combinations of therapies is conducive to further improving outcomes and developing cures for infections caused by carbapenem-resistant K. pneumoniae.

In this study, we isolated OMVs from K. pneumoniae 0692 alone and with the addition of phages and tigecycline. TEM examination revealed morphological differences in OMVs, particularly in particle size (Fig. 3). Notably, treatment with tigecycline led to a reduction in the average particle size of OMVs. We hypothesized that the smaller OMVs secreted in response to tigecycline treatment could potentially alter the outer membrane structure, thereby inducing partial defects in OMVs synthesis.

In the presence of phages, tigecycline, and phages with tigecycline, the protein concentration in OMVs increased by 1.33-, 4.38-, and 3.03-fold. To elucidate the changes in the proteins in OMVs, proteomic analyses of different groups of OMVs were performed. We observed significant differences in the protein expression in OMVs after induction with tigecycline, phages, or both. Four differentially expressed proteins were identified: gpmA, purT, groEL, and trxA (Table 4). These proteins are associated with energy metabolism processes, catalytic activities, and chaperone functions, suggesting their potential importance in phages and drugs (Fig. 5).

Fig. 5
figure 5

Schematic diagram depicting significantly altered OMV proteins gpmA, purT, groEL, and trxA, which are involved in are prosess to environment pressure. The figure was designed using Biorender

Full size image

In the MT, MP, and MTP groups, we observed a significant down-regulation of the 2, 3-bisphosphoglycerate-dependent phosphoglycerate mutase protein after tigecycline and phage treatment. This enzyme, encoded by gpmA, is known to be involved in glycolysis and plays a crucial role in bacterial energy metabolism [19]. In addition, gpmA functions as a member of the bacterial oxidative stress defense mechanism and influences bacterial tolerance to the external environment [20]. Previous studies have isolated actinomycin D, a potent natural anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) agent, from Streptomyces maritimus. Actinomycin D has demonstrated potential anti-MRSA activity by downregulating the mRNA expression level of the key differential protein gpmA, thereby inducing a novel metabolic pathway that promotes antibiotic susceptibility in MRSA [21]. Because this protein is associated with biofilm formation and antibiotic susceptibility, we hypothesized that tigecycline in combination with phages may exert antibacterial effects by influencing bacterial energy metabolism and biofilm formation.

When treated with tigecycline alone (MT group), the significantly differentially expressed protein was found to be formate-dependent purT, which showed significant upregulation in its expression after tigecycline induction compared with that in the untreated group. Notably, purT from E. coli encodes a glycine amide ribonuclease (or purT-converting enzyme) that belongs to the ATP-grasp superfamily and requires the co-catalysis of Mg2+ ATP and formate to promote the formylation of glycine amide ribonucleotides for purine biosynthesis [22, 23]. Furthermore, cytoHubba analysis based on transcriptomic profiles demonstrated that purT is the hub gene for resistance to fluoroquinolones, and that the upregulation of purT affects bacterial resistance to antibiotics [24]. In this study, we hypothesized that in response to the effects of tigecycline, the K. pneumoniae 0692 strain releases purT enzymes to improve its survival.

In the MP group, we observed that GroEL was significantly upregulated in OMVs. This increase suggests that tigecycline or phage treatment enhances the bacteria's ability to prevent protein misfolding under stressful conditions [25]. Previous studies have proposed that aminoglycosides cause cytosolic protein misfolding, and that chaperonin GroEL/GroES overexpression counters this defect, leading to aminoglycoside resistance [26, 27]. In our study, an increase in GroEL expression was also observed with phage treatment, suggesting that the phage is ineffective when combined with aminoglycosides. Therefore, exploring an optimal therapeutic regimen for phages and antibiotics is essential for addressing bacterial drug resistance.

Furthermore, in the MTP group where tigecycline and phages were administered concurrently, we observed differential expression of not only gpmA and purT proteins but also a third protein, TrxA. The TrxA system is an important contributor to cellular redox balance, regulates cell growth, apoptosis, gene expression, and antioxidant defense in nearly all living cells, and is one of the key regulators of the proliferative potential of cells through the reduction of key proteins [28]. Increased oxidative stress is characterized by highly proliferative, metabolically hyperactive cells, which are forced to mobilize antioxidant enzymes to balance the increase in free radical concentration and prevent irreversible damage and cell death. However, the role of TrxA in antibiotic drug resistance remains unclear [29]. We speculate that TrxA protects organisms from antibiotic and phage attacks by playing a role in redox signaling and acting as a redox-independent cellular chaperone [30]. Bacteriophage T7 can use the TrxA of its host, E. coli, to enhance the processivity of its DNA polymerase, thus increasing the ability of the phage to act on host bacteria [31]. TrxA reductase encapsulated in OMVs was significantly upregulated in the presence of tigecycline and phage compared with that in the untreated group, suggesting that this protein plays an important role in response to the action of external environmental pressure. Further studies into its mechanism of action will enhance our understanding of tigecycline and phage antibacterial mechanisms.

In summary, carbapenem-resistant K. pneumoniae demonstrated the ability to produce and release OMVs under the influence of tigecycline and phages, encapsulating numerous proteins vital for bacterial survival and energy metabolism. These differentially expressed proteins warrant further exploration to decipher the interaction mechanisms among phages, antimicrobial drugs, and bacterial vesicles, offering potential solutions to the pressing issue of AMR.

Conclusions

Our study demonstrated that under external environmental pressures, such as exposure to tigecycline or phages, OMVs derived from K. pneumoniae 0692 can disrupt the cell membrane by encapsulating relevant cell membrane proteins, thereby achieving a synergistic bactericidal effect. We have demonstrated that OMVs play an important role in synergizing the antibacterial effects of tigecycline and can enhance antibiotic effects by transporting protein cargoes while resisting external environmental pressures. Our research highlights the important role of OMVs as “protein vectors” in bacterial responses to environmental changes.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

K. pneumoniae :

Klebsiella pneumoniae

AMR:

Antimicrobial resistance

OMVs:

Outer membrane vesicles

MIC:

Minimum inhibitory concentrations

CLSI:

Clinical & Laboratory Standards Institute

BCA:

Bicinchoninic acid assay

PBS:

Phosphate-buffered saline

OD:

Optical density

TEM:

Transmission electron microscopy

DIA:

Data-independent acquisition

References

  1. CHINET. http://www.chinets.com; 2023.

  2. Gomez-Simmonds A, Uhlemann AC. Clinical implications of genomic adaptation and evolution of carbapenem-resistant Klebsiella pneumoniae. J Infect Dis. 2017;215:S18-s27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Knezevic P, Hoyle NS, Matsuzaki S, Gorski A. Editorial: advances in phage therapy: present challenges and future perspectives. Front Microbiol. 2021;12: 701898.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Khalid A, Lin RCY, Iredell JR. A phage therapy guide for clinicians and basic scientists: background and highlighting applications for developing countries. Front Microbiol. 2020;11: 599906.

    Article  PubMed  Google Scholar 

  5. Stern A, Sorek R. The phage-host arms race: shaping the evolution of microbes. BioEssays. 2011;33(1):43–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kim JY, Suh JW, Kang JS, Kim SB, Yoon YK, Sohn JW. Gram-negative bacteria’s outer membrane vesicles. Infect Chemother. 2023;55(1):1–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Guerrero-Mandujano A, Hernández-Cortez C, Ibarra JA, Castro-Escarpulli G. The outer membrane vesicles: secretion system type zero. Traffic. 2017;18(7):425–32.

    Article  CAS  PubMed  Google Scholar 

  8. Roier S, Zingl FG, Cakar F, Durakovic S, Kohl P, Eichmann TO, et al. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria. Nat Commun. 2016;7:10515.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Skotland T, Llorente A, Sandvig K. Lipids in extracellular vesicles: what can be learned about membrane structure and function? Cold Spring Harb Perspect Biol. 2023;15(8): a041415.

    Article  CAS  PubMed  Google Scholar 

  10. van Niel G, D’Angelo G, Raposo G. Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol. 2018;19(4):213–28.

    Article  PubMed  Google Scholar 

  11. Loeb MR, Kilner J. Release of a special fraction of the outer membrane from both growing and phage T4-infected Escherichia coli B. Biochim Biophys Acta. 1978;514(1):117–27.

    Article  CAS  PubMed  Google Scholar 

  12. Li M, Zhou H, Yang C, Wu Y, Zhou X, Liu H, et al. Bacterial outer membrane vesicles as a platform for biomedical applications: an update. J Control Release. 2020;323:253–68.

    Article  CAS  PubMed  Google Scholar 

  13. Holger DJ, Lev KL, Kebriaei R, Morrisette T, Shah R, Alexander J, et al. Bacteriophage-antibiotic combination therapy for multidrug-resistant Pseudomonas aeruginosa: in vitro synergy testing. J Appl Microbiol. 2022;133(3):1636–49.

    Article  CAS  PubMed  Google Scholar 

  14. Wayne P. CLSI. Performance standards for antimicrobial susceptibility testing. CLSI supplement M100. 32nd ed. Clinical and Laboratory Standards Institute; 2022.

    Google Scholar 

  15. Zhang X, Qian C, Tang M, Zeng W, Kong J, Fu C, et al. Carbapenemase-loaded outer membrane vesicles protect Pseudomonas aeruginosa by degrading imipenem and promoting mutation of antimicrobial resistance gene. Drug Resist Updat. 2023;68: 100952.

    Article  CAS  PubMed  Google Scholar 

  16. Hua Y, Wang J, Huang M, Huang Y, Zhang R, Bu F, et al. Outer membrane vesicles-transmitted virulence genes mediate the emergence of new antimicrobial-resistant hypervirulent Klebsiella pneumoniae. Emerg Microbes Infect. 2022;11(1):1281–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Shen HB, Chou KC. Gneg-mPLoc: a top-down strategy to enhance the quality of predicting subcellular localization of Gram-negative bacterial proteins. J Theor Biol. 2010;264(2):326–33.

    Article  CAS  PubMed  Google Scholar 

  18. Huang Y, Li J, Wang Q, Tang K, Cai X, Li C. Detection of carbapenem-resistant hypervirulent Klebsiella pneumoniae ST11-K64 co-producing NDM-1 and KPC-2 in a tertiary hospital in Wuhan. J Hosp Infect. 2023;131:70–80.

    Article  CAS  PubMed  Google Scholar 

  19. Lee J, Lee J, Cho Y, Choi J, Han SW. A putative 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase is involved in the virulence, carbohydrate metabolism, biofilm formation, twitching halo, and osmotic tolerance in Acidovorax citrulli. Front Plant Sci. 2022;13:1039420.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Roth M, Goodall ECA, Pullela K, Jaquet V, François P, Henderson IR, et al. Transposon-directed insertion-site sequencing reveals glycolysis gene gpmA as part of the H(2)O(2) defense mechanisms in Escherichia coli. Antioxidants (Basel). 2022;11(10):2053.

    Article  CAS  PubMed  Google Scholar 

  21. Xia X, Liu J, Huang L, Zhang X, Deng Y, Li F, et al. Molecular details of actinomycin D-treated MRSA revealed via high-dimensional data. Mar Drugs. 2022;20(2):114.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Thoden JB, Firestine SM, Benkovic SJ, Holden HM. PurT-encoded glycinamide ribonucleotide transformylase. Accommodation of adenosine nucleotide analogs within the active site. J Biol Chem. 2002;277(26):23898–908.

    Article  CAS  PubMed  Google Scholar 

  23. Thoden JB, Firestine S, Nixon A, Benkovic SJ, Holden HM. Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase. Biochemistry. 2000;39(30):8791–802.

    Article  CAS  PubMed  Google Scholar 

  24. Gu Y, Huang L, Wu C, Huang J, Hao H, Yuan Z, et al. The evolution of fluoroquinolone resistance in Salmonella under exposure to sub-inhibitory concentration of enrofloxacin. Int J Mol Sci. 2021;22(22):12218.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hayer-Hartl M, Bracher A, Hartl FU. The GroEL-GroES chaperonin machine: a nano-cage for protein folding. Trends Biochem Sci. 2016;41(1):62–76.

    Article  CAS  PubMed  Google Scholar 

  26. Goltermann L, Good L, Bentin T. Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli. J Biol Chem. 2013;288(15):10483–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Goltermann L, Sarusie MV, Bentin T. Chaperonin GroEL/GroES over-expression promotes aminoglycoside resistance and reduces drug susceptibilities in Escherichia coli following exposure to sublethal aminoglycoside doses. Front Microbiol. 2015;6:1572.

    PubMed  Google Scholar 

  28. Muri J, Kopf M. The thioredoxin system: balancing redox responses in immune cells and tumors. Eur J Immunol. 2023;53(1): e2249948.

    Article  PubMed  Google Scholar 

  29. May HC, Yu JJ, Guentzel MN, Chambers JP, Cap AP, Arulanandam BP. Repurposing auranofin, ebselen, and PX-12 as antimicrobial agents targeting the thioredoxin system. Front Microbiol. 2018;9:336.

    Article  PubMed  PubMed Central  Google Scholar 

  30. AlOkda A, Van Raamsdonk JM. Evolutionarily conserved role of thioredoxin systems in determining longevity. Antioxidants (Basel). 2023;12(4):944.

    Article  CAS  PubMed  Google Scholar 

  31. Lee SJ, Tran NQ, Lee J, Richardson CC. Hydrophobic residue in Escherichia coli thioredoxin critical for the processivity of T7 DNA polymerase. Biochemistry. 2018;57(40):5807–17.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank the Institutional Center for Shared Technologies and Facilities of Kunming Institute of Zoology, Chinese Academy of Sciences for providing us with Transmission Electron Microscopy and Ultracentrifuge. We are grateful to Yingqi Guo for her great help in making TEM samples and taking/analyzing TEM images.

Funding

This work was supported by the National Natural Science Foundation of China (grant No: 82160403), the Young and Middle-aged Academic and Technical Leaders Talent Project (Grant No. 202305AC160023), the Yunnan Science and Technology Commission (grant No: 202101AY070001-247) of the Yunnan Provincial Science and Technology Department and Kunming Medical University.

Author information

Author notes

  1. Jing Mao and Xiaoyu Yang contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Laboratory, The First People’s Hospital of Yunnan Province, Kunming, Yunnan, China

    Jing Mao, Cheng Yan, Fan Wang & Rui Zheng

  2. The Affiliated Hospital of Kunming University of Science and Technology, Kunming, Yunnan, China

    Jing Mao, Xiaoyu Yang, Cheng Yan, Fan Wang & Rui Zheng

  3. Regenerative Medicine Research Center, The First People’s Hospital of Yunnan Province, Kunming, Yunnan, China

    Xiaoyu Yang

  4. The Affiliated Hospital of College of Medical, Kunming University of Science and Technology, Kunming, Yunnan, China

    Jing Mao & Cheng Yan

Authors
  1. Jing Mao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoyu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cheng Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rui Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

RZ and FW designed the study. RZ revised the manuscript. JM drafted the first version of this manuscript. XY analyze the data of Proteomic. JM and CY carried out the experiments. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Fan Wang or Rui Zheng.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mao, J., Yang, X., Yan, C. et al. Proteomic analysis of carbapenem-resistant Klebsiella pneumoniae outer membrane vesicles under the action of phages combined with tigecycline. Ann Clin Microbiol Antimicrob 23, 73 (2024). https://doi.org/10.1186/s12941-024-00734-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12941-024-00734-y

Keywords

Annals of Clinical Microbiology and Antimicrobials

ISSN: 1476-0711

Contact us