The effect of erythromycin in Macrolide-Resistant Bordetella pertussis: Inhibitory on Growth, Toxin Expression, and Virulence

The effect of erythromycin in Macrolide-Resistant Bordetella pertussis: Inhibitory on Growth, Toxin Expression, and Virulence | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The effect of erythromycin in Macrolide-Resistant Bordetella pertussis: Inhibitory on Growth, Toxin Expression, and Virulence Kaichong Jiang, Yang Luan, Wei Wang, Da Xue, Shuyue Tang, Xiaokang Peng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3933379/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The macrolide-resistant Bordetella pertussis (MRBp) has appeared in Asian and even been prevalent in China. Causing of the antibiotic sensitivity test is not carried out in the clinic application, macrolide is still the first choice of antibiotic when MRBp infection. The macrolide therapy for pertussis needs to be revised. Macrolide is always shown a positive effect on other macrolide-resistant bacterium infenction in clinical application. However, the mechanism of macrolide on MRBp is unclear.This study conducted a representative isolate BP19147 ( ptxP1 / fhaB3 -MRBp) under a series of sub-inhibitory concentrations of erythromycin. We measured the growth curve, biofilm formation and autoaggregation assay under SS broth. The relative genes expression was detected by RT-qPCR. The proteomics was detected by label-fee DIA. The MR isolate BP19147 is inhibited by sub-MIC of erythromycin and has a concentration-dependent effect. From the proteomics results, the Ptx, FHA, and pertactin do not show a statistical difference ( p >0.05). Other virulence factors (including Dnt, Cya, and et al) show the statistical difference ( p <0.05). In the KEGG enrichment, the BvgAS system, biofilm formation, and some adaptive systems are inhibited by erythromycin. The sub-MIC of erythromycin may reduce the virulence of MRBp, which will provide a theoretical basis for the rational use of erythromycin for MRBp infection and help the development of new antibiotics. Biological sciences/Microbiology Health sciences/Diseases/Infectious diseases Bordetella pertussis Resistance Erythromycin Virulence Factors Proteomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Antibiotic resistance detected in vitro always affects the clinical application. However, resistant bacterium with some antibiotics, such as macrolides, also have the possibility to improve the clinical outcome. The effect of antibiotics on the resistant bacterium has yet to be delineated fully. Bordetella pertussis (Bp) is a gram-negative bacterium causing the acute respiratory infectious disease, pertussis. Macrolides were the first choice in treating pertussis for more than 50 years. However, macrolide-resistant Bordetella pertussis (MRBp) has been prevalent in China since the 2010s. Except for macrolides, sulfamethoxazole and trimethoprim (TMP-SMZ) was the alternate antibiotics if macrolides were intolerant or with resistant isolates infection. In fact, few antibiotics are available in clinical application except macrolides among infants because of the adverse effects or contraindications of TMP-SMZ 1 . Furthermore, the antimicrobial susceptibility test was rare performed in clinical laboratory leads to macrolides still as the first choice of antibiotic even in MRBp infection. Until now, a recent clinical report has shown that macrolide still has a specific clearance effect on MRBp, but it needs a longer cycle 2 . As a representative macrolide, erythromycin with concentrations lower than minimal inhibitory concentration (MIC) could change the expression pattern of virulence genes, and 0.1–0.5× MIC have a strong signal effect in some bacterium effect 3 , 4 . But little has been known regarding the mechanisms of macrolides in resistant isolates, including in MRBp. In this study, we aim to study the effect of sub-inhibitory concentration (sub-MIC) of erythromycin on Bp through adaptive characteristics, virulence gene expression, and proteomics. To provide a theoretical basis for alternative treatment of antibiotic-resistant bacteria through erythromycin's anti-virulence treatment of MRBp. RESULTS Effects of Different Concentrations of Erythromycin on the Growth Characteristics of Bp The MIC value of BP19147 ( ptxP1 / fhaB3 -MRBp) is 500 mg/L. In the standard SS culture and the SS added with 1/2 MIC (250 mg/L), 1/4 MIC (125 mg/L), 1/8 MIC (62.5 mg/L) and 1/16 MIC (31.25 mg/L) concentrations of erythromycin, the growth curve of BP19147 within 48 h is shown in Fig. 1 (A). Erythromycin inhibits the proliferation of BP19147, and the higher the concentration of erythromycin, the strongerly inhibited was observed. In Fig. 1 (B) and (C), the autoaggregation and biofilm formation ability of BP19147 show the same trend. In general, erythromycin can inhibit the growth ability of MRBp. RT-qPCR Detection of Virulence-related and Growth Characteristic-related Genes In order to study the effect of sub-MIC concentration of erythromycin on the gene expression of various toxin factors in BP19147, the bacterial RNA was extracted when the isolate was grown to the logarithmic phase and then was reverse transcribed into cDNA for qPCR detection. It was found that sub-MIC erythromycin inhibited the expression of most virulence factors. The qPCR results are shown in Fig. 2 . The expression of ptxS1 , prn and fhaB genes do not show a statistical difference in the five groups. Erythromycin significantly inhibits five genes: dnt , ptxB , brkA , cyaB , plcD , and plcA . It presents a concentration-dependent pattern, with the lowest expression in the 1/2 MIC group and the highest in the SS group. Identification and Annotation of Proteins The total number of protein identifications and distribution in each sample are shown in Fig. 3 (A). The total number of proteins is 2196, while the number of proteins in the remaining samples ranges from 2147 to 2185. According to the results annotated in Fig. 3 (B) Subcellular location database, the total protein contains the highest number of mitochondria with 734, while the peroxisome data is the lowest with 15. Based on classification according to the GO database, in Fig. 3 (D), 29.35% (943/3213) of their biological process are accomplished by multiple molecular activities, 31.56% (1014/3213) of cellular components, 39.09% (1256/3213) of molecular function work as "catalysis" or "transport". As shown in Figs. 3 (C) and (E), according to the results annotated in the KEGG and Eggnog databases, proteins in the metabolic functional category have the highest type and quantity among protein functions. In KEGG, we focus on the two-component system, which serves as a virulence regulatory system for Bp and is highly correlated with the secretion and colonization of various toxins in the genus. There are 115 proteins associated with the system. According to the EggNOG database annotation results, the toxins in Bp, including invasive adenylate cyclase (Cya), filamentous haemagglutinin (FHA), pertacin, tracheal colonization factor (tcfA), Bordetella resistance to killing (Brk), and LPS, were annotated on cellular processes and signalling, with a total of 357 protein annotation results for this function. Quantitative Results of Protein The heat map of protein intensity distribution is shown in Fig. 4 (A), and the value is the result of log10 processing. In general, there are differences between each group; with the greater the concentration difference of erythromycin, the more significant the difference. For instance, the protein intensity between the 1/2 MIC group and 1/4 MIC group is closer than that between the 1/2 MIC group and the SS group. The Hierarchical clustering heat map among each group is shown in Fig. 4 (B). And the clustering results showed the same trend. Moreover, the PCA results show that the clustering of samples within the group is good, and individual differences are minor, as shown in Fig. 4 (C). The inter-group differences are still consistent with the differences in erythromycin concentration. At the same time, the sample repeatability was analyzed based on the coefficient of the variation (CV), and the results are shown in Fig. 4 (D), indicating good intra-group and inter-group repeatability. Differential Protein Statistics We conducted differential protein (DF) screening between each two groups, mainly calculated through univariate analysis. Using fold change (FC) > 1.5 times and T-test statistics, DFs were screened out with p < 0.05. The total DF quantity statistics are shown in Fig. 4 (E). We also quantitatively display the DFs between groups through volcanic maps, with log2 as the ratio of the comparison groups in the horizontal axis so that the ratio is symmetrically distributed. Log2 (ratio) > 0 indicates high expression proteins, log2 (ratio) < 0 indicates low expression proteins, and pairwise proteomic comparison of volcanic maps is Supplementary Figure S1 . The difference between the number of DFs between groups and the concentration of erythromycin increases. In the comparison between the 1/2 MIC group and the SS group, 321 up-regulated proteins and 309 down-regulated proteins are included. However, in the comparison between the 1/16 MIC group and the 1/8 MIC group, there are only 21 up-regulated proteins and 14 down-regulated proteins. In order to study the virulence factors of Bp, we retrieve the BvgAS, toxin, antibiotic, hemolysin, phospholipase, and TTSS secret of all inter-group DFs, and the search results are shown in Supplementary Table S2 . There is no difference in the two crucial component proteins, BvgA and BvgS, of the BvgAS system. Five kinds of exotoxins: dermonecrotic toxin (Dnt), pertussis toxin (Ptx), Cya, phospholipase A (plcA) and phospholipase D(plcD) are all affected by erythromycin. The expression of Dnt protein was inhibited, suggesting that the ability of Bp affected by erythromycin to change cell morphology was reduced. The upregulation of exotoxin expression is affected, including Ptx, plcA, and plcD, which may promote the pathogenesis of host infection. Cya protein is that the expression of the CyaB region is inhibited by a higher concentration of erythromycin (over 1/4 MIC of erythromycin), and the hemolysis ability of Bp affected by a higher concentration of erythromycin decreases. Among the adherence functional proteins, FHA and pertactin are unaffected. Only the tcfA is inhibited by erythromycin, and the colonization ability of Bp will be reduced. The expression of LPS (the protein in the pathway) that plays the role of Immune modulation is up-regulated under the stimulation of erythromycin, and the affected Bp will further promote the host's inflammatory response. Moreover, it suppresses the two TTSS secrets of Bp: type II secret type III secret, and the two types of antibiotic efflux pumps for Bp: drug metabolite transporter (DMT) and resistance-nodulation-cell division (RND). KEGG and GO Databases Annotation of the Significant Virulence Factors The main virulence factors of Bp were annotated based on the KEGG database. Enriching the relevant virulence factor entries between groups and comparing the differences in enrichment, the results are shown in Supplementary Table S3 . The four pathways of mapko02020, mapko01100, mapko01110, and mapko02010 will be affected to varying degrees at each concentration of erythromycin. Among them, mapko02020: Two-component system is the BvgAS system, which regulates multiple virulence factors and is affected by erythromycin concentration. The greater the concentration difference, the greater the enrichment. In addition, the same trend is also shown in mapko01100: Metabolic pathways and its subordinate pathway mapko01110: Biosynthesis of secondary metals. The higher the concentration of erythromycin, the stronger the inhibitory effect on metabolic pathways and the BvgAS system of Bp. The RND antibiotic efflux pump, biofilm, Cya, and LPS-related pathways of Bp were all inhibited by erythromycin. The higher the concentration of erythromycin, the stronger the inhibition of Bp. As Fig. 5 , the erythromycin also inhibits the tricarboxylic acid cycle (TCA) and the glyoxylate cycle (GAC). Except for the shown in the figure, any other enzyme and accessory factor are also inhibited. Under other concentrations, some of these enzyme and accessory factors are reduced or no longer inhibited. However, the TCA and GAC are all inhibited. Similarly, the main virulence factors of Bp were annotated in the GO database, and their enrichment levels were compared. The results are shown in Supplementary Figure S2 . Compared with standard SS culture, the cellular component functional proteins of BP were promoted by erythromycin, and the virulent functional proteins of Bp: T3SS, outer membrane, and pathogenesis were inhibited by erythromycin. The result of the comparison among groups is that the more significant the difference in erythromycin concentration, the higher the protein count and enrichment factor. Methods Bacterial Strain, Detection of MIC Value, Culture Method and Growth Curve Source of Strain and Detection of MIC Value The strains used in this study were one clinical Bordetella pertussis strain isolated from 2019 named BP19147 belonging to ptxP1 / fhaB3 with high-level macrolides resistance (the MIC of erythromycin > 256 mg/L, detected by E-test). Due to the need to identify the exact MIC, the agar dilution method is used: erythromycin thinner for each drug concentration is added to Bordet-Gengou (BG) agar. After thoroughly mixing, pour a plate on the horizontal table. Cultivate the strain in Stainer and Scholte (SS) medium for 24 hours, take 100µl of McFarland to 0.5, and inoculate it on a BG plate containing different concentrations of erythromycin. Incubate for 3 to 5 days, and determine the MIC value of the strain based on its growth status Culture Method and Growth Curve Determination Take the frozen glycerol strain from the refrigerator at -80 ℃, inoculate it into the carbon agar plate medium, culture it at 37 ℃ for five days, and then transfer it to the SS liquid medium, with FeSO 4 .7H 2 O, L-cysteine, L-ascorbic acid, Nicotinic acid, L-glutathione reduced, and cyclodextrin et al. The bacterial solution was cultured in a shaking table at 230 rpm/min for 24 hours. Add fresh and complete SS culture medium to the bacterial suspension after the completion of SS culture, adjust the OD 600 to 0.1, and cultivate. Extract the bacterial suspension at (2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h) for OD 600 measurement, and draw a standard curve. Detection of Autoaggregation assay and Biofilm Formation Ability Autoaggregation assay After being washed by PBS, the liquid suspension growing for 24 hours is added to the liquid culture medium without additives. Each sample is divided into two tubes (3ml each) and left at room temperature. At different time points (2h, 6 h, 12h, 24h). Detection of bacterial liquid OD 600 . One tube carefully aspirates the liquid level for detection, and the other tube evenly mixes the liquid for detection. The autoaggregation index (AI) calculation for each node, AI= (1-OD 600 liquid level /OD 600 mix ). Biofilm Formation Ability Use crystal violet micropore to detect the biofilm-forming ability: Adjust the cultured bacterial solution to OD 595 to 0.1, and inoculate 100 µL. After standing at 36 ℃ for 24 hours in a 96 sterile plate, remove the suspended liquid, clean it three times with PBS, and add fresh SS medium again. Incubate for 96 hours and replace with fresh SS medium every 24 hours. After culture and cleaning, add 0.1% crystal violet to the dye, dissolve it with absolute ethanol, and then measure the OD 595 absorbance. In order to figure out the dynamic changes of biofilm, four repeated tests were used to determine crystal violet staining at 24h, 48h, 72h, and 96h, respectively. RT-qPCR Detection of Genes Related to Toxin and Colonization Characteristics For the cultivated bacterial solution, the Shanghai Biotech Bacteria Total RNA Isolation Kit and MightyScript First Strand cDNA Synthesis Master Mix were used to extract total bacterial RNA according to the instructions and perform reverse transcription. Perform qPCR detection on the virulence and colonization-related genes of Bp, and the primer sequence is shown in Supplementary Table S1 . Proteomics and bioinformatics analysis Total Protein Extraction and Detection Each sample has 3 biological replicates. Proteins in the sample are extracted and enzymatically hydrolyzed, then enriched and separated into peptide segments detected by high-performance liquid chromatography-tandem high-resolution mass spectrometry, generating a large amount of mass spectrometry data. Quality control of protein extraction by using the Bradford method and the SDS-PAGE. Protein Identification, Quanntification and Annoatation Utilizing DIA-NN (v1.8) software ( https://www.nature.com/articles/s41592-019-0638-x ), identify the proteins in the sample under the following conditions: False positive pattern (PSM FDR) < 0.01, Protein FDR < 0.01. The database reference is Bordetella pertussis Tohama I, and the database sequence is https://www.ncbi.nlm.nih.gov/nuccore/NC_002929.2.Utilize multiple functional databases (Subcellular location, GO, EggNOG, and KEGG) to annotate the identified proteins and their functional classification. In order to understand the protein strength distribution, sample relationship, and other information, the software DIA-NN is used to evaluate all protein strength information quantified by sample identification, including protein strength distribution, sample relationship, and repeatability analysis. Statistical Analysis Statistical analysis was performed in R (version 4.0.0). The raw protein intensity will be normalized by the method "medium", Hierarchical clustering was performed using the pheatmap package. Principal component analysis (PCA) was performed using the metaX package. T-test was used for statistical differential analysis, and a cut of P ≤ 0.05 and fold change ≥ 1.2 was used to select statistically differential expressed proteins. Hypergeometric-based enrichment analysis with KEGG Pathway, Gene Ontology and Reactome Pathway were performed to annotate protein sequences. WoLF PSORT performed subcellular localization analysis. Transcription factor annotation was based on AnimalTFDB/PlantTFDB. The difference between samples is calculated using the Euclidean distance to build a clustering tree. Take the first two principal components for PCA analysis, use the coefficient of variation to calculate differences within phenotypic groups, and compare between groups. Discussion Culturing of B.pertussis is fastidious and time-consuming for at least 3 days. Thus, the antibiotic sensitivity test was only sometimes performed in clinical laboratories. As the first choice of antibiotic for more than 50 years, macrolide has been resistant in more than 95% of prevailing isolates of B.pertussi s in China. However, the macrolides were still the first chosen antibiotic in pertussis therapy even though the resistance status is not known and seems to have a positive effect in clinical application 2 . Thus, a deeper understanding of the mechanisms of macrolide against not only sensitive isolates but also resistant isolates will promote the proposal of effective alternative treatments. To our limited knowledge, this is the first report trying to recover the potential mechanism of erythromycin in the MRBp. Erythromycin, an essential and common macrolide antibiotic, exerts its antibacterial activity by inhibiting the protein synthesis of target bacteria 5 . Meanwhile, erythromycin also has an anti-inflammatory function, regulating airway secretion and immune regulation-related antimicrobial effects 6 . As for pertussis with MRBp, it is reported macrolides, including erythromycin, still have the potential to clear the MRBp from the nasopharynx (NP). In this study, we selected a representational isolate of MRBp, BP19147, as the target research bacteria. We tried to reveal the adaptive characteristics and proteomics (especially virulence factor-related proteins) of MRBp under the pressure of different concentrations of erythromycin. It was obvious that a concentration-dependent inhibitory effect of erythromycin on the growth curve, biofilm formation ability, and autoaggregation ability in BP19147. Our previous research found that, unlike the predominant ptxP1 / fhaB3 -MRBp in China, the predominant strain in Europe and America is ptxP3 / fhaB1 -MSBp (macrolide-sensitive Bordetella pertussis ) 7 . Nevertheless, the strain ptxP1 / fhaB3 -MRBp selected in this study with hyperbiofilm forming ability is still inhibited by sub-MIC erythromycin. There are also consistent results in other studies, such as macrolide can inhibit the synthesis of alginate (the main component of the biofilm) and reduce the number of live bacteria of antibiotic-sensitive pseudomonas aeruginosa in the biofilm 8 . Therefore, sub-MIC erythromycin may inhibit the adaptability of MRBp. The two components of the BvgAS system, the BvgA and BvgS, critical virulence regulatory systems of Bp, showed no significant differences between the groups at the protein level through the RT-qPCR and proteome in this study. However, there were differences in the enrichment factors between the KEGG pathway enriched groups, which were concentration-dependent. The reason may be that sub-MIC erythromycin will not affect BvgA and BvgS but will inhibit the phosphorylation level of the system. Related studies have also pointed out that when Bp is under unsuitable conditions (such as in MgSO4, nicotinic, or low-temperature environments), Bvg (+) will transform into Bvg (-), and the phosphorylation level of the BvgAS system shows low levels 9 . Under Bvg (-), the expression of virulence-repressed genes (vrgs) is up-regulated, and the level of toxin secretion is significantly reduced 10 . The T3SS, the toxic efflux pump of Bp, has an inject effector function and modulates the pathway to enhance colonization. Some studies suggest that T3SS, as a needle-like, is used to inject cytotoxic effector into host cells and is not activated. However, other studies have isolated T3SS protein in clinical isolates and laboratory environments 11 . The results of this study indicate that erythromycin inhibits the proteins of the T3SS efflux pump of BP19147. This may reduce the toxicity and colonization ability of Bp. A similar effect of macrolides was also reported in Escherichia coli O157 12 . Regarding the specific toxin of Bp, Ptx, FHA, and pertactin do not show statistical differences in proteomics. Other exotoxins, Dnt and Cya, are inhibited by erythromycin. In fact, we observed that the hemolysis ability of BP19147 is also inhibited by erythromycin in the BG plate (data not shown). A recent study reported that macrolides can reduce the hemolysis of Escherichia coli 13 . Tcfa is also inhibited, suggesting that the colonization ability of Bp may be inhibited by erythromycin. A recent study reported that azithromycin therapy can significantly reduce the colonization ability of Pseudomonas aeruginosa in patients with severe chronic obstructive pulmonary disease 14 . The DMT and RND related to the antibiotic are affected by erythromycin, and the protein expression is up-regulated. Except for these toxin proteins, any KEGG pathways (such as quorum sensing and biofilm formation) also have essential regulation functions, while pathogenic bacteria exert pathogenicity 15 . Moreover, the BvgAS system is also a critical virulence regulation system in Bp. Some studies have pointed out that antibiotics can inhibit the quorum-sensing effect of bacteria, and the downstream effect is usually related to virulence-related characteristics and biofilm formation 16 . Our data indicate that the enrichment factor of the three virulence-related pathways (quorum sensing, the BvgAS system, and biofilm formation) are affected by erythromycin and shows a positive gradient trend. Except for the virulence-related pathway, the sub-MIC erythromycin inhibits the metabolic pathways(mapko01100) of Bp. Recent evidence suggests that biofilm cells tend to complete the TCA, and planktonic cells push forward the GAC in Bp. The two factors may increase cell survivability and lead to persistent infection 15 . However, in our study, these two pathways both are inhibited. It may impact on the virulence of Bp. Some recent studies performed on the low-lever TAC and GAC will inhibit the virulence of Mycobacterium tuberculosis and Salmonella 17 – 19 . A limitation of our research is that this study focuses on the mechanism of sub-MIC in MRBp in vitro. Lacking the clinical effect and possible mechanism of macrolides in MRBp in vivo, including the anti-inflammatory function and immune regulation function of erythromycin. To provide a theoretical basis for the treatment of clinical antibiotic-resistant bacterial infections, especially in MRBp, in the following research, we will focus on the changes in Bp bacterial load and blood drug concentration during erythromycin treatment, further monitoring various physiological and immune indicators of patients with MSBp and MRBp infection. To sum, our study found that some proteins, including colonization, hemolysis, and other functional proteins of MRBp, are inhibited by erythromycin with concentration-dependent. Moreover, the expression level of the BvgAS in the virulence regulation system and the T3SS virulence efflux pump is also inhibited. Our results demonstrate that erythromycin has inhibitory effects on the toxicity of MRBp, and further clinical case-control studies need to be conducted to verify. Conclusion In conclusion, the sub-MIC erythromycin inhibits the growth ability, biofilm formation ability and any toxins of MRBp. Moreover, the inhibiting ability has a concentration-dependent effect. Thus, the sub-MIC of erythromycin may reduce the virulence of MRBp. It will provide a theoretical basis for the rational use of macrolide for MRBp infection and help the development of new antibiotics. Declarations Funding This work was supported by the [National Natural Science Foundation of China] under Grant [number 8217081223], and [Shaanxi Health and Family Planning Commission] under Grant [number 2021E002]. Ethical approval Not required. Competing Interests The authors declare that there are no conflicts of interest. Author Contribution Kaichong Jiang and Yuan Luan: Contributed to the Investigation and Writing - Original Draft. Wei Wang: Contributed to the isolate of clinical strains. Da Xue and Shuyue Tang: Contributed to the Formal analysis. Xiaokang Peng: Contributed to the Funding acquisition. Xiaoguai Liu: Contributed to the study design. Zengguo Wang: Contributed to the study design, Project administration and Funding acquisition. Data availability The datasets generated during this study are available from the corresponding author upon reasonable request. References Tiwari, T., Murphy, T. V., Moran, J. & National Immunization Program, C. D. C. Recommended antimicrobial agents for the treatment and postexposure prophylaxis of pertussis: 2005 CDC Guidelines. MMWR Recomm Rep 54 , 1-16 (2005). Mi, Y. M. et al. Effect of Macrolides and beta-lactams on Clearance of Bordetella pertussis in the Nasopharynx in Children With Whooping Cough. 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The whole-cell proteome shows the characteristics of macrolides-resistant Bordetella pertussis in China linked to the biofilm formation. Arch Microbiol 205 , 219, doi:10.1007/s00203-023-03566-0 (2023). Yasuda, H., Ajiki, Y., Koga, T., Kawada, H. & Yokota, T. Interaction between biofilms formed by Pseudomonas aeruginosa and clarithromycin. Antimicrob Agents Chemother 37 , 1749-1755, doi:10.1128/AAC.37.9.1749 (1993). Herrou, J. et al. Periplasmic domain of the sensor-kinase BvgS reveals a new paradigm for the Venus flytrap mechanism. Proc Natl Acad Sci U S A 107 , 17351-17355, doi:10.1073/pnas.1006267107 (2010). Merkel, T. J., Barros, C. & Stibitz, S. Characterization of the bvgR locus of Bordetella pertussis. J Bacteriol 180 , 1682-1690, doi:10.1128/JB.180.7.1682-1690.1998 (1998). Yahalom, A., Shaked, H., Ruthstein, S. & Chill, J. H. Inherent Minor Conformer of Bordetella Effector BteA Directs Chaperone-Mediated Unfolding. 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Chem Rev 111 , 5492-5505, doi:10.1021/cr2000509 (2011). Munoz-Elias, E. J. & McKinney, J. D. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat Med 11 , 638-644, doi:10.1038/nm1252 (2005). Bhusal, R. P. et al. Acetyl-CoA-mediated activation of Mycobacterium tuberculosis isocitrate lyase 2. Nat Commun 10 , 4639, doi:10.1038/s41467-019-12614-7 (2019). Cohen, H. et al. The ancestral stringent response potentiator, DksA has been adapted throughout Salmonella evolution to orchestrate the expression of metabolic, motility, and virulence pathways. Gut Microbes 14 , 1997294, doi:10.1080/19490976.2021.1997294 (2022). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3933379","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":275274332,"identity":"05478d5c-758c-43ab-837a-dde9c959cd08","order_by":0,"name":"Kaichong Jiang","email":"","orcid":"","institution":"National Regional Children's Medical Center (Northwest), Xi'an Children's Hospital, Affiliated Children's Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Kaichong","middleName":"","lastName":"Jiang","suffix":""},{"id":275274333,"identity":"3cb8a4b6-e4a0-431c-aacb-fb98b751c2e6","order_by":1,"name":"Yang Luan","email":"","orcid":"","institution":"Xi'an Municipal Center for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Luan","suffix":""},{"id":275274334,"identity":"20e837eb-04a1-42c7-8d2f-9442fcf344f8","order_by":2,"name":"Wei Wang","email":"","orcid":"","institution":"National Regional Children's Medical Center (Northwest), Xi'an Children's Hospital, Affiliated Children's Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":275274335,"identity":"2fa9f363-e0de-4e7b-8cfa-ecf335d176eb","order_by":3,"name":"Da Xue","email":"","orcid":"","institution":"Shaanxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Da","middleName":"","lastName":"Xue","suffix":""},{"id":275274336,"identity":"b5924460-968c-4632-bcef-d659914e7626","order_by":4,"name":"Shuyue Tang","email":"","orcid":"","institution":"Shaanxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuyue","middleName":"","lastName":"Tang","suffix":""},{"id":275274337,"identity":"e23ff6f1-520b-495f-aa96-d0b35a2aa4a2","order_by":5,"name":"Xiaokang Peng","email":"","orcid":"","institution":"National Regional Children's Medical Center (Northwest), Xi'an Children's Hospital, Affiliated Children's Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Xiaokang","middleName":"","lastName":"Peng","suffix":""},{"id":275274338,"identity":"9b5c0395-a18b-4020-be91-7d26847f505b","order_by":6,"name":"Xiaoguai Liu","email":"","orcid":"","institution":"National Regional Children's Medical Center (Northwest), Xi'an Children's Hospital, Affiliated Children's Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoguai","middleName":"","lastName":"Liu","suffix":""},{"id":275274339,"identity":"6846a689-dcc2-4e3f-874c-4711b74b3f41","order_by":7,"name":"Zengguo Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDACZubGAwkQJuODhAobYrQwNsC0MBs8OJNGjDVALVAWm+TDtkOENRgcB2p5ULONweD88WsVCWwHGPjbuxPwazkMctix2wwGB86U3UjgucMgcebsBrxazMBa2G4zmB3sSbuRIPGMwUAilxgt/4BaDvOkFSQYHCZSS2IbUMsx9mMMCQlEaLEHa+m7zWB/hodZIuFAGg9Bv0j2Hz748Me320DG8Ycff/6zkeNv78WvBQbqGxh4DEAMHqKUQwH7A1JUj4JRMApGwQgCAG8RU0mb0N/4AAAAAElFTkSuQmCC","orcid":"","institution":"National Regional Children's Medical Center (Northwest), Xi'an Children's Hospital, Affiliated Children's Hospital of Xi'an Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Zengguo","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-02-06 09:15:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3933379/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3933379/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51754403,"identity":"1c12cd95-f1a5-4bda-9feb-2fcf966a9772","added_by":"auto","created_at":"2024-02-28 14:08:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":537646,"visible":true,"origin":"","legend":"\u003cp\u003e(A) the growth curve of BP19147 in different concentrations of erythromycin, (B) the autoaggregation assay of BP19147 in different concentrations of erythromycin, (C) the biofilm formation of BP19147 in different concentrations of erythromycin\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3933379/v1/09721682665cfee770320cb9.png"},{"id":51754404,"identity":"a167b6a7-dcef-462e-8137-a1a8c03d4013","added_by":"auto","created_at":"2024-02-28 14:08:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":815580,"visible":true,"origin":"","legend":"\u003cp\u003ethe virulence factors of qPCR detection, “*” mean \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, (A) dnt, (B) ptxB, (C) brkA, (D) cyaB, (E) plcD, (F) plcA, (G) ptxS1, (H) prn, (I) fhaB.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3933379/v1/13aba5d42471b17375f44055.png"},{"id":51754405,"identity":"c48bfd0c-56f5-4369-93ca-5472e148d040","added_by":"auto","created_at":"2024-02-28 14:08:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1319642,"visible":true,"origin":"","legend":"\u003cp\u003eBP19147 in different concentrations of erythromycin (A) total number of protein identification and distribution in each sample number, (B) subcellular location database annotation, (C) KEGG database annotation, (D) Go database annotation, (E) EggNOG database annotation\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3933379/v1/54d81a3c81b4de72519b301d.png"},{"id":51754410,"identity":"fa5a7335-7d2c-4ad5-a087-e05ca43ceada","added_by":"auto","created_at":"2024-02-28 14:08:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1219369,"visible":true,"origin":"","legend":"\u003cp\u003eConduct overall evaluation on all protein strength information obtained from sample identification (A) protein strength distribution, (B) Hierarchical Clustering, (C) principal component analysis of phenotypic group analysis, (D)repeatability analysis. (E)the differential protein statics of BP19147 in different cultivation states.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3933379/v1/622991b983753f96eb95c781.png"},{"id":51754408,"identity":"b47c86d4-2732-42c1-801d-560e684a5549","added_by":"auto","created_at":"2024-02-28 14:08:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":558746,"visible":true,"origin":"","legend":"\u003cp\u003eMajor changed core metabolic reactions from comparing the tricarboxylic acid cycle (TCA) and the glyoxylate cycle (GAC) generated from SS and 1/2 MIC BP protein expression data. The blue arrow represents GAC, and the yellow arrow represents TAC. The green means down-regulated.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3933379/v1/28246badf65ceaaf29970bf7.png"},{"id":52600045,"identity":"46366624-4610-43d2-b3fe-b40f3f821a47","added_by":"auto","created_at":"2024-03-13 12:51:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1182386,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3933379/v1/fa632545-a0c7-4d43-81fb-bd1a076225da.pdf"},{"id":51754406,"identity":"3b3f0e4f-a51b-4794-9f7f-6fb10bcfa58e","added_by":"auto","created_at":"2024-02-28 14:08:58","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3695800,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3933379/v1/a6450019195d05130dc4adc5.tif"},{"id":51754411,"identity":"a7d97595-02d6-4d00-a5a9-b5fbcdac4b82","added_by":"auto","created_at":"2024-02-28 14:08:58","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4449704,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3933379/v1/0391408902e92458aa45a664.tif"},{"id":51754409,"identity":"05137f28-3117-4fe4-9a10-c20ae05fa338","added_by":"auto","created_at":"2024-02-28 14:08:58","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2500105,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-3933379/v1/aa1d3a41bd29ee76cb6920e3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of erythromycin in Macrolide-Resistant Bordetella pertussis: Inhibitory on Growth, Toxin Expression, and Virulence","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntibiotic resistance detected in vitro always affects the clinical application. However, resistant bacterium with some antibiotics, such as macrolides, also have the possibility to improve the clinical outcome. The effect of antibiotics on the resistant bacterium has yet to be delineated fully.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBordetella pertussis\u003c/em\u003e (Bp) is a gram-negative bacterium causing the acute respiratory infectious disease, pertussis. Macrolides were the first choice in treating pertussis for more than 50 years. However, macrolide-resistant \u003cem\u003eBordetella pertussis\u003c/em\u003e (MRBp) has been prevalent in China since the 2010s.\u003c/p\u003e \u003cp\u003eExcept for macrolides, sulfamethoxazole and trimethoprim (TMP-SMZ) was the alternate antibiotics if macrolides were intolerant or with resistant isolates infection. In fact, few antibiotics are available in clinical application except macrolides among infants because of the adverse effects or contraindications of TMP-SMZ\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Furthermore, the antimicrobial susceptibility test was rare performed in clinical laboratory leads to macrolides still as the first choice of antibiotic even in MRBp infection.\u003c/p\u003e \u003cp\u003eUntil now, a recent clinical report has shown that macrolide still has a specific clearance effect on MRBp, but it needs a longer cycle\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. As a representative macrolide, erythromycin with concentrations lower than minimal inhibitory concentration (MIC) could change the expression pattern of virulence genes, and 0.1\u0026ndash;0.5\u0026times; MIC have a strong signal effect in some bacterium effect\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. But little has been known regarding the mechanisms of macrolides in resistant isolates, including in MRBp.\u003c/p\u003e \u003cp\u003eIn this study, we aim to study the effect of sub-inhibitory concentration (sub-MIC) of erythromycin on Bp through adaptive characteristics, virulence gene expression, and proteomics. To provide a theoretical basis for alternative treatment of antibiotic-resistant bacteria through erythromycin's anti-virulence treatment of MRBp.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Different Concentrations of Erythromycin on the Growth Characteristics of Bp\u003c/h2\u003e \u003cp\u003eThe MIC value of BP19147 (\u003cem\u003eptxP1\u003c/em\u003e/\u003cem\u003efhaB3\u003c/em\u003e-MRBp) is 500 mg/L. In the standard SS culture and the SS added with 1/2 MIC (250 mg/L), 1/4 MIC (125 mg/L), 1/8 MIC (62.5 mg/L) and 1/16 MIC (31.25 mg/L) concentrations of erythromycin, the growth curve of BP19147 within 48 h is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (A). Erythromycin inhibits the proliferation of BP19147, and the higher the concentration of erythromycin, the strongerly inhibited was observed. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (B) and (C), the autoaggregation and biofilm formation ability of BP19147 show the same trend. In general, erythromycin can inhibit the growth ability of MRBp.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR Detection of Virulence-related and Growth Characteristic-related Genes\u003c/h2\u003e \u003cp\u003eIn order to study the effect of sub-MIC concentration of erythromycin on the gene expression of various toxin factors in BP19147, the bacterial RNA was extracted when the isolate was grown to the logarithmic phase and then was reverse transcribed into cDNA for qPCR detection. It was found that sub-MIC erythromycin inhibited the expression of most virulence factors. The qPCR results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The expression of \u003cem\u003eptxS1\u003c/em\u003e, \u003cem\u003eprn\u003c/em\u003e and \u003cem\u003efhaB\u003c/em\u003e genes do not show a statistical difference in the five groups. Erythromycin significantly inhibits five genes: \u003cem\u003ednt\u003c/em\u003e, \u003cem\u003eptxB\u003c/em\u003e, \u003cem\u003ebrkA\u003c/em\u003e, \u003cem\u003ecyaB\u003c/em\u003e, \u003cem\u003eplcD\u003c/em\u003e, and \u003cem\u003eplcA\u003c/em\u003e. It presents a concentration-dependent pattern, with the lowest expression in the 1/2 MIC group and the highest in the SS group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and Annotation of Proteins\u003c/h2\u003e \u003cp\u003eThe total number of protein identifications and distribution in each sample are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (A). The total number of proteins is 2196, while the number of proteins in the remaining samples ranges from 2147 to 2185. According to the results annotated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (B) Subcellular location database, the total protein contains the highest number of mitochondria with 734, while the peroxisome data is the lowest with 15. Based on classification according to the GO database, in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (D), 29.35% (943/3213) of their biological process are accomplished by multiple molecular activities, 31.56% (1014/3213) of cellular components, 39.09% (1256/3213) of molecular function work as \"catalysis\" or \"transport\". As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (C) and (E), according to the results annotated in the KEGG and Eggnog databases, proteins in the metabolic functional category have the highest type and quantity among protein functions. In KEGG, we focus on the two-component system, which serves as a virulence regulatory system for Bp and is highly correlated with the secretion and colonization of various toxins in the genus. There are 115 proteins associated with the system. According to the EggNOG database annotation results, the toxins in Bp, including invasive adenylate cyclase (Cya), filamentous haemagglutinin (FHA), pertacin, tracheal colonization factor (tcfA), Bordetella resistance to killing (Brk), and LPS, were annotated on cellular processes and signalling, with a total of 357 protein annotation results for this function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Results of Protein\u003c/h2\u003e \u003cp\u003eThe heat map of protein intensity distribution is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (A), and the value is the result of log10 processing. In general, there are differences between each group; with the greater the concentration difference of erythromycin, the more significant the difference. For instance, the protein intensity between the 1/2 MIC group and 1/4 MIC group is closer than that between the 1/2 MIC group and the SS group. The Hierarchical clustering heat map among each group is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (B). And the clustering results showed the same trend. Moreover, the PCA results show that the clustering of samples within the group is good, and individual differences are minor, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (C). The inter-group differences are still consistent with the differences in erythromycin concentration. At the same time, the sample repeatability was analyzed based on the coefficient of the variation (CV), and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (D), indicating good intra-group and inter-group repeatability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDifferential Protein Statistics\u003c/h2\u003e \u003cp\u003eWe conducted differential protein (DF) screening between each two groups, mainly calculated through univariate analysis. Using fold change (FC)\u0026thinsp;\u0026gt;\u0026thinsp;1.5 times and T-test statistics, DFs were screened out with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The total DF quantity statistics are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (E). We also quantitatively display the DFs between groups through volcanic maps, with log2 as the ratio of the comparison groups in the horizontal axis so that the ratio is symmetrically distributed. Log2 (ratio)\u0026thinsp;\u0026gt;\u0026thinsp;0 indicates high expression proteins, log2 (ratio)\u0026thinsp;\u0026lt;\u0026thinsp;0 indicates low expression proteins, and pairwise proteomic comparison of volcanic maps is Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The difference between the number of DFs between groups and the concentration of erythromycin increases. In the comparison between the 1/2 MIC group and the SS group, 321 up-regulated proteins and 309 down-regulated proteins are included. However, in the comparison between the 1/16 MIC group and the 1/8 MIC group, there are only 21 up-regulated proteins and 14 down-regulated proteins.\u003c/p\u003e \u003cp\u003eIn order to study the virulence factors of Bp, we retrieve the BvgAS, toxin, antibiotic, hemolysin, phospholipase, and TTSS secret of all inter-group DFs, and the search results are shown in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. There is no difference in the two crucial component proteins, BvgA and BvgS, of the BvgAS system. Five kinds of exotoxins: dermonecrotic toxin (Dnt), pertussis toxin (Ptx), Cya, phospholipase A (plcA) and phospholipase D(plcD) are all affected by erythromycin. The expression of Dnt protein was inhibited, suggesting that the ability of Bp affected by erythromycin to change cell morphology was reduced. The upregulation of exotoxin expression is affected, including Ptx, plcA, and plcD, which may promote the pathogenesis of host infection. Cya protein is that the expression of the CyaB region is inhibited by a higher concentration of erythromycin (over 1/4 MIC of erythromycin), and the hemolysis ability of Bp affected by a higher concentration of erythromycin decreases. Among the adherence functional proteins, FHA and pertactin are unaffected. Only the tcfA is inhibited by erythromycin, and the colonization ability of Bp will be reduced. The expression of LPS (the protein in the pathway) that plays the role of Immune modulation is up-regulated under the stimulation of erythromycin, and the affected Bp will further promote the host's inflammatory response. Moreover, it suppresses the two TTSS secrets of Bp: type II secret type III secret, and the two types of antibiotic efflux pumps for Bp: drug metabolite transporter (DMT) and resistance-nodulation-cell division (RND).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eKEGG and GO Databases Annotation of the Significant Virulence Factors\u003c/h2\u003e \u003cp\u003eThe main virulence factors of Bp were annotated based on the KEGG database. Enriching the relevant virulence factor entries between groups and comparing the differences in enrichment, the results are shown in Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e. The four pathways of mapko02020, mapko01100, mapko01110, and mapko02010 will be affected to varying degrees at each concentration of erythromycin. Among them, mapko02020: Two-component system is the BvgAS system, which regulates multiple virulence factors and is affected by erythromycin concentration. The greater the concentration difference, the greater the enrichment. In addition, the same trend is also shown in mapko01100: Metabolic pathways and its subordinate pathway mapko01110: Biosynthesis of secondary metals. The higher the concentration of erythromycin, the stronger the inhibitory effect on metabolic pathways and the BvgAS system of Bp. The RND antibiotic efflux pump, biofilm, Cya, and LPS-related pathways of Bp were all inhibited by erythromycin. The higher the concentration of erythromycin, the stronger the inhibition of Bp. As Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the erythromycin also inhibits the tricarboxylic acid cycle (TCA) and the glyoxylate cycle (GAC). Except for the shown in the figure, any other enzyme and accessory factor are also inhibited. Under other concentrations, some of these enzyme and accessory factors are reduced or no longer inhibited. However, the TCA and GAC are all inhibited.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, the main virulence factors of Bp were annotated in the GO database, and their enrichment levels were compared. The results are shown in Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Compared with standard SS culture, the cellular component functional proteins of BP were promoted by erythromycin, and the virulent functional proteins of Bp: T3SS, outer membrane, and pathogenesis were inhibited by erythromycin. The result of the comparison among groups is that the more significant the difference in erythromycin concentration, the higher the protein count and enrichment factor.\u003c/p\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Strain, Detection of MIC Value, Culture Method and Growth Curve\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eSource of Strain and Detection of MIC Value\u003c/h2\u003e \u003cp\u003eThe strains used in this study were one clinical \u003cem\u003eBordetella pertussis\u003c/em\u003e strain isolated from 2019 named BP19147 belonging to \u003cem\u003eptxP1\u003c/em\u003e/\u003cem\u003efhaB3\u003c/em\u003e with high-level macrolides resistance (the MIC of erythromycin\u0026thinsp;\u0026gt;\u0026thinsp;256 mg/L, detected by E-test). Due to the need to identify the exact MIC, the agar dilution method is used: erythromycin thinner for each drug concentration is added to Bordet-Gengou (BG) agar. After thoroughly mixing, pour a plate on the horizontal table. Cultivate the strain in Stainer and Scholte (SS) medium for 24 hours, take 100\u0026micro;l of McFarland to 0.5, and inoculate it on a BG plate containing different concentrations of erythromycin. Incubate for 3 to 5 days, and determine the MIC value of the strain based on its growth status\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCulture Method and Growth Curve Determination\u003c/h2\u003e \u003cp\u003eTake the frozen glycerol strain from the refrigerator at -80 ℃, inoculate it into the carbon agar plate medium, culture it at 37 ℃ for five days, and then transfer it to the SS liquid medium, with FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, L-cysteine, L-ascorbic acid, Nicotinic acid, L-glutathione reduced, and cyclodextrin et al. The bacterial solution was cultured in a shaking table at 230 rpm/min for 24 hours.\u003c/p\u003e \u003cp\u003eAdd fresh and complete SS culture medium to the bacterial suspension after the completion of SS culture, adjust the OD\u003csub\u003e600\u003c/sub\u003e to 0.1, and cultivate. Extract the bacterial suspension at (2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h) for OD\u003csub\u003e600\u003c/sub\u003e measurement, and draw a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Autoaggregation assay and Biofilm Formation Ability\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eAutoaggregation assay\u003c/h2\u003e \u003cp\u003eAfter being washed by PBS, the liquid suspension growing for 24 hours is added to the liquid culture medium without additives. Each sample is divided into two tubes (3ml each) and left at room temperature. At different time points (2h, 6 h, 12h, 24h). Detection of bacterial liquid OD\u003csub\u003e600\u003c/sub\u003e. One tube carefully aspirates the liquid level for detection, and the other tube evenly mixes the liquid for detection. The autoaggregation index (AI) calculation for each node, AI= (1-OD\u003csub\u003e600 liquid level\u003c/sub\u003e/OD\u003csub\u003e600 mix\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm Formation Ability\u003c/h2\u003e \u003cp\u003eUse crystal violet micropore to detect the biofilm-forming ability: Adjust the cultured bacterial solution to OD\u003csub\u003e595\u003c/sub\u003e to 0.1, and inoculate 100 \u0026micro;L. After standing at 36 ℃ for 24 hours in a 96 sterile plate, remove the suspended liquid, clean it three times with PBS, and add fresh SS medium again. Incubate for 96 hours and replace with fresh SS medium every 24 hours. After culture and cleaning, add 0.1% crystal violet to the dye, dissolve it with absolute ethanol, and then measure the OD\u003csub\u003e595\u003c/sub\u003e absorbance. In order to figure out the dynamic changes of biofilm, four repeated tests were used to determine crystal violet staining at 24h, 48h, 72h, and 96h, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR Detection of Genes Related to Toxin and Colonization Characteristics\u003c/h2\u003e \u003cp\u003eFor the cultivated bacterial solution, the Shanghai Biotech Bacteria Total RNA Isolation Kit and MightyScript First Strand cDNA Synthesis Master Mix were used to extract total bacterial RNA according to the instructions and perform reverse transcription. Perform qPCR detection on the virulence and colonization-related genes of Bp, and the primer sequence is shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eProteomics and bioinformatics analysis\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003eTotal Protein Extraction and Detection\u003c/h2\u003e \u003cp\u003eEach sample has 3 biological replicates. Proteins in the sample are extracted and enzymatically hydrolyzed, then enriched and separated into peptide segments detected by high-performance liquid chromatography-tandem high-resolution mass spectrometry, generating a large amount of mass spectrometry data. Quality control of protein extraction by using the Bradford method and the SDS-PAGE.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eProtein Identification, Quanntification and Annoatation\u003c/h2\u003e \u003cp\u003eUtilizing DIA-NN (v1.8) software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nature.com/articles/s41592-019-0638-x\u003c/span\u003e\u003cspan address=\"https://www.nature.com/articles/s41592-019-0638-x\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), identify the proteins in the sample under the following conditions: False positive pattern (PSM FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Protein FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003cp\u003eThe database reference is \u003cem\u003eBordetella pertussis\u003c/em\u003e Tohama I, and the database sequence is \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/nuccore/NC_002929.2.Utilize\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/nuccore/NC_002929.2.Utilize\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e multiple functional databases (Subcellular location, GO, EggNOG, and KEGG) to annotate the identified proteins and their functional classification. In order to understand the protein strength distribution, sample relationship, and other information, the software DIA-NN is used to evaluate all protein strength information quantified by sample identification, including protein strength distribution, sample relationship, and repeatability analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed in R (version 4.0.0). The raw protein intensity will be normalized by the method \"medium\", Hierarchical clustering was performed using the pheatmap package. Principal component analysis (PCA) was performed using the metaX package. T-test was used for statistical differential analysis, and a cut of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026ge;\u0026thinsp;1.2 was used to select statistically differential expressed proteins. Hypergeometric-based enrichment analysis with KEGG Pathway, Gene Ontology and Reactome Pathway were performed to annotate protein sequences. WoLF PSORT performed subcellular localization analysis. Transcription factor annotation was based on AnimalTFDB/PlantTFDB. The difference between samples is calculated using the Euclidean distance to build a clustering tree. Take the first two principal components for PCA analysis, use the coefficient of variation to calculate differences within phenotypic groups, and compare between groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCulturing of \u003cem\u003eB.pertussis\u003c/em\u003e is fastidious and time-consuming for at least 3 days. Thus, the antibiotic sensitivity test was only sometimes performed in clinical laboratories. As the first choice of antibiotic for more than 50 years, macrolide has been resistant in more than 95% of prevailing isolates of \u003cem\u003eB.pertussi\u003c/em\u003es in China. However, the macrolides were still the first chosen antibiotic in pertussis therapy even though the resistance status is not known and seems to have a positive effect in clinical application\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Thus, a deeper understanding of the mechanisms of macrolide against not only sensitive isolates but also resistant isolates will promote the proposal of effective alternative treatments. To our limited knowledge, this is the first report trying to recover the potential mechanism of erythromycin in the MRBp.\u003c/p\u003e \u003cp\u003eErythromycin, an essential and common macrolide antibiotic, exerts its antibacterial activity by inhibiting the protein synthesis of target bacteria\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Meanwhile, erythromycin also has an anti-inflammatory function, regulating airway secretion and immune regulation-related antimicrobial effects\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As for pertussis with MRBp, it is reported macrolides, including erythromycin, still have the potential to clear the MRBp from the nasopharynx (NP). In this study, we selected a representational isolate of MRBp, BP19147, as the target research bacteria. We tried to reveal the adaptive characteristics and proteomics (especially virulence factor-related proteins) of MRBp under the pressure of different concentrations of erythromycin.\u003c/p\u003e \u003cp\u003eIt was obvious that a concentration-dependent inhibitory effect of erythromycin on the growth curve, biofilm formation ability, and autoaggregation ability in BP19147. Our previous research found that, unlike the predominant \u003cem\u003eptxP1\u003c/em\u003e/\u003cem\u003efhaB3\u003c/em\u003e-MRBp in China, the predominant strain in Europe and America is \u003cem\u003eptxP3\u003c/em\u003e/\u003cem\u003efhaB1\u003c/em\u003e-MSBp (macrolide-sensitive \u003cem\u003eBordetella pertussis\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the strain \u003cem\u003eptxP1\u003c/em\u003e/\u003cem\u003efhaB3\u003c/em\u003e-MRBp selected in this study with hyperbiofilm forming ability is still inhibited by sub-MIC erythromycin. There are also consistent results in other studies, such as macrolide can inhibit the synthesis of alginate (the main component of the biofilm) and reduce the number of live bacteria of antibiotic-sensitive pseudomonas aeruginosa in the biofilm\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Therefore, sub-MIC erythromycin may inhibit the adaptability of MRBp.\u003c/p\u003e \u003cp\u003eThe two components of the BvgAS system, the BvgA and BvgS, critical virulence regulatory systems of Bp, showed no significant differences between the groups at the protein level through the RT-qPCR and proteome in this study. However, there were differences in the enrichment factors between the KEGG pathway enriched groups, which were concentration-dependent. The reason may be that sub-MIC erythromycin will not affect BvgA and BvgS but will inhibit the phosphorylation level of the system. Related studies have also pointed out that when Bp is under unsuitable conditions (such as in MgSO4, nicotinic, or low-temperature environments), Bvg (+) will transform into Bvg (-), and the phosphorylation level of the BvgAS system shows low levels\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Under Bvg (-), the expression of virulence-repressed genes (vrgs) is up-regulated, and the level of toxin secretion is significantly reduced\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe T3SS, the toxic efflux pump of Bp, has an inject effector function and modulates the pathway to enhance colonization. Some studies suggest that T3SS, as a needle-like, is used to inject cytotoxic effector into host cells and is not activated. However, other studies have isolated T3SS protein in clinical isolates and laboratory environments\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The results of this study indicate that erythromycin inhibits the proteins of the T3SS efflux pump of BP19147. This may reduce the toxicity and colonization ability of Bp. A similar effect of macrolides was also reported in \u003cem\u003eEscherichia coli\u003c/em\u003e O157\u003csup\u003e12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRegarding the specific toxin of Bp, Ptx, FHA, and pertactin do not show statistical differences in proteomics. Other exotoxins, Dnt and Cya, are inhibited by erythromycin. In fact, we observed that the hemolysis ability of BP19147 is also inhibited by erythromycin in the BG plate (data not shown). A recent study reported that macrolides can reduce the hemolysis of Escherichia coli\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Tcfa is also inhibited, suggesting that the colonization ability of Bp may be inhibited by erythromycin. A recent study reported that azithromycin therapy can significantly reduce the colonization ability of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e in patients with severe chronic obstructive pulmonary disease\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The DMT and RND related to the antibiotic are affected by erythromycin, and the protein expression is up-regulated.\u003c/p\u003e \u003cp\u003eExcept for these toxin proteins, any KEGG pathways (such as quorum sensing and biofilm formation) also have essential regulation functions, while pathogenic bacteria exert pathogenicity\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Moreover, the BvgAS system is also a critical virulence regulation system in Bp. Some studies have pointed out that antibiotics can inhibit the quorum-sensing effect of bacteria, and the downstream effect is usually related to virulence-related characteristics and biofilm formation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Our data indicate that the enrichment factor of the three virulence-related pathways (quorum sensing, the BvgAS system, and biofilm formation) are affected by erythromycin and shows a positive gradient trend. Except for the virulence-related pathway, the sub-MIC erythromycin inhibits the metabolic pathways(mapko01100) of Bp. Recent evidence suggests that biofilm cells tend to complete the TCA, and planktonic cells push forward the GAC in Bp. The two factors may increase cell survivability and lead to persistent infection\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, in our study, these two pathways both are inhibited. It may impact on the virulence of Bp. Some recent studies performed on the low-lever TAC and GAC will inhibit the virulence of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e and Salmonella\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA limitation of our research is that this study focuses on the mechanism of sub-MIC in MRBp in vitro. Lacking the clinical effect and possible mechanism of macrolides in MRBp in vivo, including the anti-inflammatory function and immune regulation function of erythromycin. To provide a theoretical basis for the treatment of clinical antibiotic-resistant bacterial infections, especially in MRBp, in the following research, we will focus on the changes in Bp bacterial load and blood drug concentration during erythromycin treatment, further monitoring various physiological and immune indicators of patients with MSBp and MRBp infection.\u003c/p\u003e \u003cp\u003eTo sum, our study found that some proteins, including colonization, hemolysis, and other functional proteins of MRBp, are inhibited by erythromycin with concentration-dependent. Moreover, the expression level of the BvgAS in the virulence regulation system and the T3SS virulence efflux pump is also inhibited. Our results demonstrate that erythromycin has inhibitory effects on the toxicity of MRBp, and further clinical case-control studies need to be conducted to verify.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the sub-MIC erythromycin inhibits the growth ability, biofilm formation ability and any toxins of MRBp. Moreover, the inhibiting ability has a concentration-dependent effect. Thus, the sub-MIC of erythromycin may reduce the virulence of MRBp. It will provide a theoretical basis for the rational use of macrolide for MRBp infection and help the development of new antibiotics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the [National Natural Science Foundation of China] under Grant [number 8217081223], and [Shaanxi Health and Family Planning Commission] under Grant [number 2021E002].\u003c/p\u003e\n\u003cp\u003eEthical approval\u003c/p\u003e\n\u003cp\u003eNot required.\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eAuthor Contribution\u003c/p\u003e\n\u003cp\u003eKaichong Jiang and Yuan Luan: Contributed to the Investigation and Writing - Original Draft. Wei Wang: Contributed to the isolate of clinical strains. Da Xue and Shuyue Tang: Contributed to the Formal analysis. Xiaokang Peng: Contributed to the Funding acquisition. Xiaoguai Liu: Contributed to the study design. Zengguo Wang: Contributed to the study design, Project administration and Funding acquisition.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eThe datasets generated during this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTiwari, T., Murphy, T. V., Moran, J. \u0026amp; National Immunization Program, C. D. C. Recommended antimicrobial agents for the treatment and postexposure prophylaxis of pertussis: 2005 CDC Guidelines. \u003cem\u003eMMWR Recomm Rep\u003c/em\u003e \u003cstrong\u003e54\u003c/strong\u003e, 1-16 (2005).\u003c/li\u003e\n \u003cli\u003eMi, Y. 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P.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Acetyl-CoA-mediated activation of Mycobacterium tuberculosis isocitrate lyase 2. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 4639, doi:10.1038/s41467-019-12614-7 (2019).\u003c/li\u003e\n \u003cli\u003eCohen, H.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The ancestral stringent response potentiator, DksA has been adapted throughout Salmonella evolution to orchestrate the expression of metabolic, motility, and virulence pathways. \u003cem\u003eGut Microbes\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1997294, doi:10.1080/19490976.2021.1997294 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bordetella pertussis, Resistance, Erythromycin, Virulence Factors, Proteomics","lastPublishedDoi":"10.21203/rs.3.rs-3933379/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3933379/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe macrolide-resistant \u003cem\u003eBordetella pertussis\u003c/em\u003e (MRBp) has appeared in Asian and even been prevalent in China. Causing of the antibiotic sensitivity test is not carried out in the clinic application, macrolide is still the first choice of antibiotic when MRBp infection. The macrolide therapy for pertussis needs to be revised. Macrolide is always shown a positive effect on other macrolide-resistant bacterium infenction in clinical application. However, the mechanism of macrolide on MRBp is unclear.This study conducted a representative isolate BP19147 (\u003cem\u003eptxP1\u003c/em\u003e/\u003cem\u003efhaB3\u003c/em\u003e-MRBp) under a series of sub-inhibitory concentrations of erythromycin. We measured the growth curve, biofilm formation and autoaggregation assay under SS broth. The relative genes expression was detected by RT-qPCR. The proteomics was detected by label-fee DIA. The MR isolate BP19147 is inhibited by sub-MIC of erythromycin and has a concentration-dependent effect. From the proteomics results, the Ptx, FHA, and pertactin do not show a statistical difference (\u003cem\u003ep\u003c/em\u003e \u0026gt;0.05). Other virulence factors (including Dnt, Cya, and et al) show the statistical difference (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05). In the KEGG enrichment, the BvgAS system, biofilm formation, and some adaptive systems are inhibited by erythromycin. The sub-MIC of erythromycin may reduce the virulence of MRBp, which will provide a theoretical basis for the rational use of erythromycin for MRBp infection and help the development of new antibiotics.\u003c/p\u003e","manuscriptTitle":"The effect of erythromycin in Macrolide-Resistant Bordetella pertussis: Inhibitory on Growth, Toxin Expression, and Virulence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 14:08:53","doi":"10.21203/rs.3.rs-3933379/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2555bc12-cae7-4e14-bb46-c229669d781e","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29015389,"name":"Biological sciences/Microbiology"},{"id":29015390,"name":"Health sciences/Diseases/Infectious diseases"}],"tags":[],"updatedAt":"2024-03-13T12:42:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-28 14:08:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3933379","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3933379","identity":"rs-3933379","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}