Staphylococcus aureus (S. aureus) is a widely distributed opportunistic pathogen capable of causing a variety of serious infections, with its antibiotic resistance becoming increasingly prevalent. As natural bactericidal agents, bacteriophages (phages) have emerged as promising therapeutic alternatives to antibiotics. In this study, we isolated a lytic phage P108 capable of targeting methicillin-resistant S. aureus (MRSA) strains. Phage P108 features an icosahedral head with a diameter of approximately 84.7 nm and a contractile tail measuring about 221.5 nm in length. It has a latent period of 20 min and completes lysis within 60 min. Whole-genome sequencing revealed a linear dsDNA genome of 140,807 bp, encoding 226 putative proteins and 3 tRNAs. Four novel structural protein-coding genes were identified. Phylogenetic analysis demonstrated that P108 represents a member of the Herelleviridae family, Kayvirus genus. P108 is capable of lysing 79.2% (95/120) of clinical isolates of S. aureus and demonstrates broad-spectrum lytic activity against MRSA (84.8%, 39/46). Furthermore, phage P108 exhibits high stability, potent in vitro bactericidal activity, and effective bacterial biofilm removal, outperforming vancomycin in overall efficacy. These findings highlight its potential for antibacterial applications and support its development as a novel therapeutic strategy against drug-resistant S. aureus infections.

Multidrug-resistant (MDR) pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) pose a substantial challenge to global public health, particularly because of chronic and persistent infections associated with bacterial biofilms, which call for safe and innovative therapeutic strategies. Here, we present a novel antibiofilm system inspired by the preferential uptake properties of isogenous bacterial membrane vesicles (MVs). This system employs vancomycin (VAN) for bacterial killing, while MVs act as delivery vehicles to increase VAN penetration into biofilms. VAN@ΔagrMVs demonstrated sustained drug release and improved VAN accessibility within biofilms. Treatment with VAN@ΔagrMVs considerably reduced the number of planktonic MRSA strain USA300 cells and effectively eradicated MRSA biofilms in vitro. RNA sequencing revealed substantial alterations in genes associated with bacterial cell wall biosynthesis, global regulators, virulence factors, and biofilm formation. Treatment with VAN@ΔagrMVs substantially reduced the MRSA burden within biofilms in vivo. Safety evaluation demonstrated the avirulent properties of the VAN@ΔagrMVs, highlighting its potential for clinical application. Overall, this study offers a promising alternative for MRSA biofilm eradication, providing a viable strategy to combat chronic infections caused by MDR biofilm-forming pathogens.

Active lysogeny is a newly characterized mechanism that the dynamic integration and excision of prophages serve as molecular switches to coordinately regulate bacterial gene expression without generating progeny virions. The Sa3int family phages, the most prevalent prophages in Staphylococcus aureus, specifically integrate into the β-toxin-coding gene hlb. While infection conditions favor the loss of Sa3int phages and the emergence of Hlb-producing variants, highlighting their potential for active lysogeny, the environmental cues and underlying mechanisms controlling the peculiar life cycle of Sa3int phages remain largely unexplored. In this study, we identified a Sa3int phage, designated ΦSa3XN, from the methicillin-resistant S. aureus strain XN108. The active lysogeny feature of ΦSa3XN was analyzed by combinational PCR, plaque assay, transmission electron microscopy, and DNase protection assay. Additionally, glucose-induced active lysogeny of ΦSa3XN and its impact on S. aureus virulence were evaluated via reporter assay, electrophoretic mobility shift assay, hemolytic assay, and mouse infection models. ΦSa3XN acts as a genuine molecular switch, capable of excision without producing progeny phages. Glucose serves as an environmental cue that triggers ΦSa3XN excision and reinstates hlb expression, wherein the catabolite control protein A (CcpA) directly binds to the promoter region of cI and suppresses the expression of CI repressor, thus switching the phage life cycle. Moreover, glucose-induced active lysogeny of ΦSa3XN significantly enhances bacterial hemolytic activity, exacerbating skin inflammation and subcutaneous abscess formation in hyperglycemic mice. This study illustrates a novel example of active lysogeny for Sa3int phages and elucidates a glucose-responsive CcpA pathway that regulates ΦSa3XN excision to augment S. aureus virulence, advancing our understanding of the sophisticated interactions between S. aureus and phages.

Perinephric abscess, a common complication after kidney transplantation, often results from drug-resistant bacterial infections and is notoriously difficult to treat. Phage therapy has emerged as a promising alternative for such resistant infections. Here, we present two cases of perinephric abscesses in kidney transplant recipients (KTRs) treated with phage therapy. Our findings highlight the need for personalized treatment plans and timely intervention with phage therapy to improve patient outcomes. Future research should focus on overcoming barriers like biofilms to make this treatment more effective. Ultimately, phage therapy could lead to better survival rates and improved quality of life for transplant patients facing severe infections. This study is a step forward in the fight against superbugs, offering a potential alternative when antibiotics fail.

Vibrio vulnificus is a highly pathogenic marine bacterium that can cause life-threatening conditions such as septicemia and necrotizing fasciitis; in medically fragile individuals, mortality rates can exceed 50%. Its virulence factors, including the MARTX toxin and hemolysin VvhA, function to disrupt host cells, evade immune responses, and facilitate bacterial dissemination. Climate warming has dramatically expanded the geographic range of V. vulnificus, doubling infection risks in high-latitude northern waters, while microplastic pollution and extreme weather events further enhance its ecological adaptability. High-risk populations, such as patients with liver disease, immunocompromised individuals, and coastal workers, face elevated mortality due to iron metabolism disorders or frequent exposure. Traditional diagnostic pathogen culture methods are time-consuming and lack sensitivity, whereas technologies such as CRISPR-Cas12a enable quick detection with a sensitivity of 1-10 CFU/mL, facilitating timely intervention. The standard treatment for V. vulnificus infections relies on doxycycline combined with cefotaxime; however, rising antibiotic resistance, poses a significant challenge. This challenge underscores the need to develop alternative strategies, such as virulence-targeted therapies and immunomodulatory approaches .Multivalent vaccines such as RtxA1/VvhA fusion antigens offer broad-spectrum protection, while nanoparticle delivery systems and mucosal vaccines like Lactobacillus-based oral vaccines may enhance immune responses. Future efforts must integrate a "monitoring-blocking-treatment" framework, combining satellite-based early-warning systems, CRISPR rapid detection, and climate-medicine modeling. International collaboration is essential to implement vaccination programs for high-risk groups and enforce seafood safety protocols. Addressing the public health threat of V. vulnificus in the era of climate change demands interdisciplinary innovation and global governance.

Introduction: Co-infection with Pneumocystis jirovecii (PJP) and Mycobacterium tuberculosis complex (MTBC) is rare in HIV-seronegative patients. Because it is associated with unknown morbidity and a high mortality rate especially in patients with immunosuppression. But concurrent infections pose a diagnostic challenge owing to similar clinical features. Data suggests a high prevalence of such concurrent infections in developing countries but limited diagnostic modalities especially in resource constraint setup limits accurate diagnosis. This study reports a rare case of PJP co-infection with MTBC in a relatively immunocompetent patient which presented with uncommon radiological findings. Case presentation: A 84-year-old man (former smoker, 30 pack years) with a newly discovered unclassified interstital lung disease (ILD) and a history of hypertension presented with dyspnea, fever, and dry cough for one month to the hospital. He was treated with glucocorticoid for one week briefly and had been under treatment of antibiotics during his one-month disease course before this admission. HIV testing and immunoglobulin levels were normal in the hospital course. A lung computed tomography (CT) scan revealed mostly emphysema appearance but a few regions look like cysts and ground glass opacity diffused infiltration (Figure 1 A). Antibiotics and corticosteroids were administrated at the beginning. However, the patient experienced recurrent fever and a new epilepsy with the reduction of the glucocorticoid dosage. Sputum mNGS detected Mycobacterium tuberculosis complex and Pneumocystis jiroveci confirmed the diagnosis of co-infection with PJP and MTBC. Standard antituberculosis agents and sulfamethoxazole/trimethoprim and corticosteroids were administrated. After one month follow-up, the patient was in good condition despite of ILD and emphysema in his lung (Figure 1 B). Disscussion: Patients with interval fever on long-term antibiotics and glucocorticoid therapy in areas with a high incidence of Mycobacterium tuberculosis may be co-infected with Pneumocystis jirovecii. When opportunistic infections are suspected, diagnostic procedures including invasive ones should be performed as soon as possible and appropriate interventions need to be carried out promptly. Immunodeficiency patients are susceptible to opportunistic infections. mNGS is valuable for diagnosis and treatment. Although the image of MTBC and PJP infections lack specificity, they exhibit distinctive features.

Streptococcus mutans is recognized as the primary etiological agent of dental caries, one of the most prevalent infectious diseases globally. Its remarkable acid tolerance enables survival and proliferation in the low-pH biofilm microenvironment, establishing S. mutans as the dominant species in dental plaque and a key contributor to cariogenesis. Although numerous studies have identified genes linked to acid tolerance mechanisms, the full set of essential acid tolerance genes within its genome remains incompletely characterized, largely due to the lack of systematic, genome-scale investigations. To address this knowledge gap, we constructed a genome-wide pooled CRISPR interference (CRISPRi) library targeting 95% of the predicted S. mutans genes and employed next-generation sequencing to identify acid tolerance determinants systematically. Our screen revealed 95 acid tolerance-associated genes, a subset of which were functionally validated through gene knockout studies. Functional enrichment analysis demonstrated significant associations with metabolic pathways (including cofactor biosynthesis and amino/nucleotide sugar metabolism), tRNA modification, and transcriptional regulation. Protein-protein interaction (PPI) network analysis identified critical interactors (ComYC, SMU\_1979c, DeoC, AcpP, NadD, and SMU\_1988c) and two functionally cohesive modules. These findings provide novel mechanistic insights into the acid adaptation strategies of S. mutans and highlight potential therapeutic targets for caries prevention.

Membrane vesicles (MVs) are produced by species across all domains of life and have diverse physiological functions as well as promising applications. While the mechanisms for vesiculation in Gram-negative bacteria are well-established, the genetic determinants and regulatory factors responsible for MV biogenesis in Gram-positive bacteria remain largely unknown. Here, we demonstrate that a Q225P substitution in the alternative sigma factor B (SigB) triggers MV production in Staphylococcus aureus strain Newman by hindering the specific binding of SigB to the asp23 promoter, thereby repressing expression of alkaline shock protein 23 (Asp23). Isogenic deletion of asp23 also promotes MV formation in Newman, confirming the critical roles played by sigB and asp23 in modulating S. aureus vesiculation. While bacterial growth and cytoplasmic membrane fluidity are not impaired, mutation of asp23 weakens the cell wall and enhances autolysis, consistent with decreased expression of alpha-type psm and lrgAB that modulate murein hydrolase activity. TEM and proteomic analysis show that Newman and asp23 deletion mutant generate MVs with nearly identical morphology and composition, but virulence-associated factors are significantly enriched in MVs from the asp23 mutant. Overall, this study reveals novel genetic determinants underlying S. aureus vesiculation and advances the understanding of the physiology of MV biogenesis in S. aureus.

This study examines the total viable counts (TVC) of air, contact surfaces, raw materials, and semi-finished products throughout the production leisure dried tofu (LD-tofu) at three different companies. High-throughput sequencing was used to analyse the microbial diversity of fermented soybean whey (FSW), marinade, and LD-tofu to evaluate the effect of occupational environment on the microbial diversity of LD-tofu. Results indicated that the average TVC of marinade and LD-tofu was 3.79 and 1.83 lg CFU/g, respectively, which were within clean levels. The workshop air (4.14 - 5.27 lg CFU/m3), contact surfaces (4.61 - 7.01 lg CFU/m2), FSW (5.88 lg CFU/g), and tofu (4.89 lg CFU/g) were within mild to severe contamination levels. The pressing wooden frames were within severe contamination levels, as they are difficult to clean and may have residual soybean residue. Pearson correlation analysis showed that Lachnospiraceae, Moraxellaceae, and Bacillaceae were closely related to the TVC of FSW, while Vibrionaceae, Pseudomonadaceae, and Burkholderiaceae were closely related to the TVC of LD-tofu and marinade. This study provides scientific basis for improving the microbial control of the production environment, equipment, and raw materials for LD-tofu production, as well as the theoretical basis for the storage and safety control of soybean products.

Generating strong adhesion by engineered proteins has the potential for high technical applications. Current studies of adhesive proteins are primarily limited to marine organisms, e.g., mussel adhesive proteins. Here, we present a modular engineering strategy to generate a type of exotic protein adhesives with super strong adhesion behaviors. In the protein complexes, the lanmodulin (LanM) underwent α-helical conformational transition induced by lanthanides, thereby enhancing the stacking density and molecular interactions of adhesive protein. The resulting adhesives exhibited outstanding lap-shear strength of ≈31.7 MPa, surpassing many supramolecular and polymer adhesives. The extreme temperature (−196 to 200 °C) resistance capacity and underwater adhesion performance can significantly broaden their practical application scenarios. Ex vivo and in vivo experiments further demonstrated the persistent adhesion performance for surgical sealing and healing applications.

The subtilisin-like protease-1 (SspA-1) plays an important role in the pathogenesis of a highly virulent strain of Streptococcus suis 2. However, the mechanism of SspA-1-triggered excessive inflammatory response is still unknown. In this study, we demonstrated that activation of type I IFN signaling is required for SspA-1-induced excessive proinflammatory cytokine production. Further experiments showed that the TLR2 endosomal pathway mediates SspA-1-induced type I IFN signaling and the inflammatory response. Finally, we mapped the major signaling components of the related pathway and found that the TIR adaptor proteins Mal, TRAM, and MyD88 and the downstream activation of IRF1 and IRF7 were involved in this pathway. These results explain the molecular mechanism by which SspA-1 triggers an excessive inflammatory response and reveal a novel effect of type I IFN in S. suis 2 infection, possibly providing further insights into the pathogenesis of this highly virulent S. suis 2 strain.

Argonaute proteins (Agos) bind short nucleic acids as guides and are directed by them to recognize target complementary nucleic acids. Prokaryotic Agos (pAgos) are extremely diverse, with potential functions in microbial defense. The functions and mechanisms of a group of full-length yet inactive pAgos, long-B pAgos, remain enigmatic. Here, we show that most long-B pAgos constitute cell suicide systems together with their various associated proteins, including nucleases, Sir2-domain-containing proteins and trans-membrane proteins, respectively. Among them, the long-B pAgo-nuclease system utilizes an RNA-programmed and target-recognition-activated collateral DNA cleavage activity to sense invaders and kill the infected cells. This results in depletion of the invading plasmid from the cell population. Together, our data indicate that the long-B pAgo systems induce cell death with various effector proteins after recognition of invading nucleic acids, corresponding to an immune response via abortive infection.

Medical EducationVolume 57, Issue 11 p. 1122-1122 REALLY GOOD STUFF Medical licensing examination-based reform of medical microbiology teaching Xiaodong Shen, Xiaodong ShenSearch for more papers by this authorYuhan Wang, Yuhan WangSearch for more papers by this authorDan Zhao, Dan ZhaoSearch for more papers by this authorMing Li, Corresponding Author Ming Li [email protected] Correspondence Ming Li, Department of Microbiology, College of Basic Medical Sciences, Army Medical University, No. 30 Gaotanyan Centre Street, Shapingba District, Chongqing 400038, China. Email: [email protected]Search for more papers by this author Xiaodong Shen, Xiaodong ShenSearch for more papers by this authorYuhan Wang, Yuhan WangSearch for more papers by this authorDan Zhao, Dan ZhaoSearch for more papers by this authorMing Li, Corresponding Author Ming Li [email protected] Correspondence Ming Li, Department of Microbiology, College of Basic Medical Sciences, Army Medical University, No. 30 Gaotanyan Centre Street, Shapingba District, Chongqing 400038, China. Email: [email protected]Search for more papers by this author First published: 13 September 2023 https://doi.org/10.1111/medu.15206 Funding information: This project is funded by the Educational Research Project of Army Medical University (NO. 2022A02). Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. REFERENCE 1Wang X. Experiences, challenges, and prospects of National Medical Licensing Examination in China. BMC Med Educ. 2022; 22(1): 349. doi:10.1186/s12909-022-03385-9 Volume57, Issue11November 2023Pages 1122-1122 ReferencesRelatedInformation

Both Gram-positive and Gram-negative bacteria release nano-sized lipid bilayered particles, known as membrane vesicles (MVs), into external environments. Although MVs play a variety of roles in bacterial physiology and pathogenesis, the mechanisms underlying MV formation in Gram-positive microorganisms such as Staphylococcus aureus remain obscure. Bacterial MV production can be induced in response to stress conditions, and the alternative sigma factor B (SigB) functions as a central regulator of the stress response in Gram-positive bacteria. In a previous study, we demonstrated that the SigB(Q225P) substitution mutation in S. aureus promotes biofilm formation. Here, we report that the SigB(Q225P) mutation also increases MV production in this important pathogen. LacZ reporter assays and electrophoretic mobility shift assays showed that the Q225P substitution reduces SigB binding to the promoter region of the thermonuclease gene (nuc), resulting in a significant reduction in Nuc expression. Deletion of nuc markedly enhances S. aureus MV generation, possibly due to the accumulation of nucleic acids. These results are not only important for understanding MV biogenesis in S. aureus, but also useful for the development of a S. aureus MV-based platform for MV application.

The accessory gene regulator (agr) quorum-sensing system is an important global regulatory system of Staphylococcus aureus and contributes to its pathogenicity. The S. aureus agr system is divided into four agr groups based on the amino acid polymorphisms of AgrB, AgrD, and AgrC. The agr activation is group-specific, resulting in variations in agr activity and pathogenicity among the four agr groups. Strains with divergent agr system always have different phenotypes. In the present report, we, respectively, exchanged the agr system of a certain S. aureus with other three agr alleles and assessed the corresponding phenotypes of these congenic strains. Replacement of the agr system led to significant variations in hemolytic activity, protein expression, and virulence gene expression comparing with that of the parental strain. Interestingly, we found that the biological characteristics of these agr congenic strains in the same strain background were highly similar to each other, and the allele-dependent differences of the agr systems were weakened. These findings indicate that the allele-dependent agr predilections of S. aureus are determined by some factors in addition to the polymorphisms of AgrB, AgrD, and AgrC. Future studies may reveal the novel mechanism to improve our understanding of the agr network.

Burkholderia pseudomallei is the causal agent of melioidosis, a deadly tropical infectious disease that lacks a vaccine. On the basis of the attenuated Staphylococcus aureus RN4220-Δagr (RN), we engineered the RN4220-Δagr/pdhB-hcp1 strain (RN-Hcp1) to generate B. pseudomallei hemolysin-coregulated protein 1 (Hcp1)-loaded membrane vesicles (hcp1MVs). The immunization of BALB/c mice with hcp1MVs mixed with adjuvant by a three-dose regimen increased the serum specific IgG production. The serum levels of inflammatory factors, including TNF-α and IL-6, in hcp1MV-vaccinated mice were comparable with those in PBS-challenged mice. The partial adjuvant effect of staphylococcal MVs was observed with the elevation of specific antibody titer in hcp1MV-vaccinated mice relative to those that received the recombinant Hcp1 protein (rHcp1) or MVs derived from RN strain (ΔagrMVs). The hcp1MVs/adjuvant vaccine protected 70% of mice from lethal B. pseudomallei challenge. Immunization with hcp1MVs only protected 60% of mice, whereas vaccination with rHcp1 or ΔagrMVs conferred no protection. Moreover, mice that received hcp1MVs/adjuvant and hcp1MVs immunization had low serum TNF-α and IL-6 levels and no inflammatory infiltration in comparison with other groups. In addition, all surviving mice in hcp1MVs/adjuvant and hcp1MVs groups exhibited no culturable bacteria in their lungs, livers, and spleens five days postinfection. Overall, our data highlighted a new strategy for developing B. pseudomallei vaccine and showed that Hcp1-incorporated staphylococcal MV is a promising candidate for the prevention of acute melioidosis.

Introduction: Vancomycin-intermediate Staphylococcus aureus (VISA) is typically associated with a decline in virulence. We previously reported a WalK(S221P) mutation that plays an important role in mediating vancomycin resistance in VISA XN108. Whether this mutation is implicated in bacterial virulence remains unknown. Objectives: This study aimed to investigate the effect of WalK(S221P) mutation on the virulence of VISA and the underlying mechanism of this effect. Methods: The influence of WalK(S221P) mutation on VISA virulence and its underlying mechanism were explored using animal models, RNA-seq analysis, RT-qPCR, hemolytic assay, slide coagulase test, Western blot, β-galactosidase assay, and electrophoresis mobility shift assay (EMSA). Results: Compared with XN108, WalK(S221P)-reverted strain XN108-R exacerbated cutaneous infections with increased lesion size and extensive inflammatory infiltration in mouse models. The bacterial loads of S. aureus XN108-R in murine kidney increased compared with those of XN108. RNA-seq analysis showed upregulation of a set of virulence genes in XN108-R, which exhibited greater hemolytic and stronger coagulase activities compared with XN108. Introduction of WalK(S221P) to methicillin-resistant S. aureus USA300 and methicillin-susceptible strain Newman increased the vancomycin resistance of the mutants, which exhibited reduced hemolytic activities and decreased expression levels of many virulence factors compared with their progenitors. WalK(S221P) mutation weakened agr promoter-controlled β-galactosidase activity. EMSA results showed that WalK-phosphorylated WalR could directly bind to the agr promoter region, whereas WalK(S221P)-activated WalR reduced binding to the target promoter. Inactivation of agr in S. aureus did not affect their vancomycin susceptibility but mitigated the virulence alterations caused by WalK(S221P) mutation. Conclusion: The results of our study indicate that WalK(S221P) mutation can enhance vancomycin resistance in S. aureus of diverse genetic backgrounds. WalK(S221P)- bearing S. aureus strains exhibit reduced virulence. WalK(S221P) mutation may directly impair the activation of the agr system by WalR, thereby decreasing the expression of virulence factors in VISA.

Staphylococcus aureus represents a notorious opportunistic pathogen causing various infections in biofilm nature, imposing remarkable therapeutic challenges worldwide. The catabolite control protein A (CcpA), a major regulator of carbon catabolite repression (CCR), has been recognized to modulate S. aureus biofilm formation, while the underlying mechanism remains to be fully elucidated. In this study, the reduced biofilm was firstly determined in the ccpA deletion mutant of S. aureus clinical isolate XN108 using both crystal violet staining and confocal laser scanning microscopy. RNA-seq analysis suggested that sak-encoding staphylokinase (Sak) was significantly upregulated in the mutant ∆ccpA, which was further confirmed by RT-qPCR. Consistently, the induced Sak production correlated the elevated promoter activity of sak and increased secretion in the supernatants, as demonstrated by Psak-lacZ reporter fusion expression and chromogenic detection, respectively. Notably, electrophoretic mobility shift assays showed that purified recombinant protein CcpA binds directly to the promoter region of sak, suggesting the direct negative control of sak expression by CcpA. Double isogenic deletion of ccpA and sak restored biofilm formation for mutant ∆ccpA, which could be diminished by trans-complemented sak. Furthermore, the exogenous addition of recombinant Sak inhibited biofilm formation for XN108 in a dose-dependent manner. Together, this study delineates a novel model of CcpA-controlled S. aureus biofilm through direct inhibition of sak expression, highlighting the multifaceted roles and multiple networks regulated by CcpA.

Staphylococcus aureus represents a major human pathogen that is frequently involved in polymicrobial infections. However, the prevalence and role of co-infectious microbes on the pathogenesis and fitness essentiality of S. aureus in vivo remain largely unknown. In this study, we firstly performed a retrospective surveillance of 760 clinical samples and revealed a notable predominance of co-infection with S. aureus and Acinetobacter baumannii. The high-density S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) further identified a core set of genes enriched in metabolism of inorganic ions, amino acids, and carbohydrates, which are essential for infection and tissue colonization of S. aureus in the murine systemic infection model. Notably, we revealed a differential requirement of fitness factors for S. aureus in tissue-specific (liver and kidney) and infection-type-specific manner (mono- and co-infection). Co-infection with A. baumannii dramatically altered the fitness requirements of S. aureus in vivo; 49% of the mono-infection fitness genes in S. aureus strain Newman were converted to non-essential, and the functionality of ATP-binding cassette (ABC) transporters was significantly elicited during co-infection. Furthermore, the number of genes essential during co-infection (503) outnumbers the genes essential during mono-infection (362). In addition, the roles of 3 infection-type-specific genes in S. aureus during mono-infection or co-infection with A. baumannii were validated with competitive experiments in vivo. Our data indicated a high incidence and clinical relevance of S. aureus and A. baumannii co-infection, and provided novel insights into establishing antimicrobial regimens to control co-infections. IMPORTANCE Polymicrobial infections are widespread in clinical settings, which potentially correlate with increased infection severity and poor clinical outcomes. Staphylococcus aureus is a formidable human pathogen that causes a variety of diseases in polymicrobial nature. Co-infection and interaction of S. aureus have been described with limited pathogens, mainly including Pseudomonas aeruginosa, Candida albicans, and influenza A virus. Thus far, the prevalence and role of co-infectious microbes on the pathogenesis and fitness essentiality of S. aureus in vivo remain largely unknown. Understanding the polymicrobial composition and interaction, from a community and genome-wide perspective, is thus crucial to shed light on S. aureus pathogenesis strategy. Here, our findings demonstrated, for the first time, that a high incidence rate and clinical relevance of co-infection was caused by S. aureus and Acinetobacter baumannii, illustrating the importance of polymicrobial nature in investigating S. aureus pathogenesis. The infection-type-specific genes likely serve as potential therapeutic targets to control S. aureus infections, either in mono- or co-infection situation, providing novel insights into the development of antimicrobial regimens to control co-infections.