Anti-Acinetobacter Baumannii single-chain variable fragments provide therapeutic efficacy in an immunocompromised mouse pneumonia model.

BACKGROUND: The emergence of carbapenem-resistant and extensively drug-resistant (XDR) Acinetobacter baumannii as well as inadequate effective antibiotics calls for an urgent effort to find new antibacterial agents. The therapeutic efficacy of two human scFvs, EB211 and EB279, showing growth inhibitory activity against A. baumannii in vitro, was investigated in immunocompromised mice with A. baumannii pneumonia. RESULTS: The data revealed that infected mice treated with EB211, EB279, and a combination of the two scFvs showed better survival, reduced bacterial load in the lungs, and no marked pathological abnormalities in the kidneys, liver, and lungs when compared to the control groups receiving normal saline or an irrelevant scFv. CONCLUSIONS: The results from this study suggest that the scFvs with direct growth inhibitory activity could offer promising results in the treatment of pneumonia caused by XDR A. baumannii.

Development of a human phage display-derived anti-PD-1 scFv antibody: an attractive tool for immune checkpoint therapy.

BACKGROUND: The PD-1 checkpoint pathway plays a major role in tumor immune evasion and the development of the tumor microenvironment. Clinical studies show that therapeutic antibodies blocking the PD-1 pathway can restore anti-tumor or anti-virus immune responses by the reinvigoration of exhausted T cells. Because of the promising results of anti-PD-1 monoclonal antibodies in cancer treatment, autoimmune disorders, and infectious diseases, the PD-1 has emerged as an encouraging target for different diseases. RESULTS: In the present study, we employed a human semi-synthetic phage library for isolation of some scFvs against the extracellular domain of PD-1 protein by panning process. After the panning, a novel anti-PD-1 scFv (SS107) was found that exhibited specific binding to PD-1 antigen and stimulated Jurkat T cells. The selected anti-PD-1 scFv could restore the production of IL-2 and IFN-γ by Jurkat T cells that were co-cultured with PD-L1 positive tumor cells. CONCLUSION: This anti-PD-1 scFv with high specificity and the ability to reactivate exhausted T cells has the potential to be developed as an anti-cancer agent or to be used in combination with other therapeutic approaches.

Identification of a novel fully human anti-toxic shock syndrome toxin (TSST)-1 single-chain variable fragment antibody averting TSST-1-induced mitogenesis and cytokine secretion.

BACKGROUND: Staphylococcal superantigens are virulence factors that help the pathogen escape the immune system and develop an infection. Toxic shock syndrome toxin (TSST)-1 is one of the most studied superantigens whose role in toxic shock syndrome and some particular disorders have been demonstrated. Inhibiting TSST-1 production with antibiotics and targeting TSST-1 with monoclonal antibodies might be one of the best strategies to prevent TSST-1-induced cytokines storm followed by lethality. RESULTS: A novel single-chain variable fragment (scFv), MS473, against TSST-1 was identified by selecting an scFv phage library on the TSST-1 protein. The MS473 scFv showed high affinity and specificity for TSST-1. Moreover, MS473 could significantly prevent TSST-1-induced mitogenicity (the IC50 value: 1.5 µM) and cytokine production. CONCLUSION: Using traditional antibiotics with an anti-TSST-1 scFv as a safe and effective agent leads to deleting the infection source and preventing the detrimental effects of the toxin disseminated into the whole body.

BK polyomavirus in Iranian patients with papillary thyroid cancer: Is it a major future challenge?

BK polyomavirus (BKPyV) is a well-known cause of nephropathy in renal transplant recipients. It has recently received much attention from researchers as a major predisposing factor for various cancers. This study aimed to investigate how BKPyV affected the advancement of papillary thyroid carcinoma (PTC). A total of 1057 samples were tested for BKPyV DNA and RNA, comprising 645 paraffin-embedded PTC biopsy samples (PEBS), 412 fresh biopsy samples (FBS), and 1057 adjacent noncancerous samples. The BKPyV DNA was found in 511 (48.3%) of the specimens, including 347 (84.2%) FBS and 164 (25.4%) PEBS. The mean BKPyV copy number was significantly lower in patients with PEBS (0.5 × 10-4 ± 0.1 × 10-4 copies/cell) than in FBS (1.3 × 10-1 ± 0.2 × 10-1 copies/cell) and non-PTC normal samples (0.3 × 10-5 ± 0.04 × 10-5 copies/cell). The PEBS had lower LT-Ag RNA expression than FBS, and no VP1 gene transcript expression was detected. In conclusion, although our findings indicated the presence of BKPyV in some Iranian PTC patients, more research is needed to corroborate these findings.

The role of miRNA-377 as a tumor suppressor in lung cancer by negative regulation of genes belonging to ErbB signaling pathway.

BACKGROUND: The ErbB signaling pathway plays important role in the pathogenesis of lung cancer. We explored the role of miRNA-377 as a tumor suppressor in NSCLC through silencing of some genes in the ErbB pathway. METHODS AND RESULTS: The targeting effect of miRNA-377 on EGFR, MAPK1, ABL2, and PAK2 was evaluated. The expression levels of these genes and miRNA-377 were surveyed in NSCLC and normal human tissues, Calu-6, and A549 cells. Real-time PCR was used to figure out whether miRNA-377 could decrease the target genes mRNAs in transfected lung cancer cell lines. The effects of miRNA-377 on apoptosis cell and proliferation were analyzed. We showed that miRNA-377 targets EGFR, MAPK1, and PAK2 mRNAs in in-silico and luciferase reporter assay. The expression of miRNA-377 was significantly downregulated in human NSCLC tissues, Calu-6 and A549 cells compared to their controls. We observed a negative correlation between EGFR, MAPK1, PAK2, and miRNA-377 expression in human NSCLC tissues. A significant reduction in EGFR, MAPK1, and PAK2 mRNA levels was detected, following miRNA-377 transfection in Calu-6 and A549 cells. The higher levels of miRNA-377 in Calu-6, and A549 cells induced apoptosis and reduced proliferation, significantly. CONCLUSIONS: All these data reveal that miRNA-377 functions as a tumor suppressor in NSCLC and may serve as a potential therapeutic target for the treatment of NSCLC.

Comparative study of interruption of signaling pathways in lung epithelial cell by two different Mycobacterium tuberculosis lineages.

Alveolar epithelial cell (AEC) provides a replication niche for Mycobacterium tuberculosis. Based on the role of AEC in M. tuberculosis pathogenesis and existence of genetic diversity within this bacterium, we investigated interactions between AEC II and two different M. tuberculosis lineages. We have compared the transcriptome and cytokines/chemokines levels of A549 infected by M. tuberculosis lineage three and four using qRT-PCR and ELISA arrays, respectively. We showed different M.  tuberculosis strains induced changes in different effectors that involved in TLRs and NF-κB signaling pathways. We observed different reaction of the studied lineages specifically in pathogenesis, immune evasion mechanism, IL-12/IFN-γ axis, and autophagy. Similar behavior was detected in regarding to apoptosis, necroptosis, anti-inflammatory responses, and canonical inflammasome. Our findings contribute to elucidate more details in pathogenesis, immune evasion strategies, novel target and druggable pathway for therapeutic intervention, and host directed therapy in tuberculosis infection. Also, different M.  tuberculosis lineages-dependent host-pathogen interactions suggested using only one strain for this kind of research will be controversial.

High Prevalence of Bedaquiline Resistance in Treatment-Naive Tuberculosis Patients and Verapamil Effectiveness.

In the regions where bedaquiline (BDQ) is introduced into the regimen, analysis of MIC and screening for preexisting resistance mutations could be crucial. The high prevalence of isolates with high BDQ MICs without prior exposure to BDQ was worrisome. It was also concluded that efflux pumps play a pivotal role in intrinsic BDQ resistance; therefore, the potential of verapamil as an adjunctive therapy to combat BDQ resistance should be investigated.

Host genetic factors and clinical parameters influencing the occult hepatitis C virus infection in patients on chronic hemodialysis: Is it still a controversial infection?

AIM: The presence of occult hepatitis C virus (HCV) infection (OCI) is still controversial, however, this infection cannot be ignored. Therefore, the current study aimed at assessing the OCI frequency in patients on chronic hemodialysis (CHD) and also evaluating the association between OCI incidence with clinical parameters and interferon lambda 3/4 (IFNL3/4) gene polymorphisms. METHODS: A total of 515 patients on CHD and HCV negative markers were selected. Plus- and minus-stranded HCV-RNA was tested in peripheral blood mononuclear cell samples by reverse transcription-polymerase chain reaction and then genotyped using the restriction fragment length polymorphism method. RESULTS: The frequency of OCI was 11.3% in patients on CHD. Among 58 patients with OCI, 25.8%, 62.1%, and 12.1% were infected with HCV-1a, HCV-1b, and HCV-3a, respectively. The mean alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase levels were 31.9 ± 24.8, 32.3 ± 19.1, and 171.6 ± 88.9, respectively. None of the patients with OCI had a history of liver disease. Multivariate logistic regression analysis indicated that cholesterol, triglyceride, low-density lipoprotein, 25-hydroxyvitamin D, platelets, duration of hemodialysis, HCV subtypes, IFNL3 rs12979860 TT, IFNL3 rs8099917 GG, IFNL3 rs12980275 GG, and IFNL4 ss469415590 ΔG/ΔG genotypes were associated with OCI. CONCLUSION: There was a moderate prevalence of OCI in Iranian patients on CHD. The current study findings indicated that this infection was associated with clinical parameters and unfavorable genotypes of IFNL3 single nucleotide polymorphisms and IFNL4 ss469415590. Further studies are required to determine the correlation between OCI incidence with clinical parameters and host genetic factors.

Low viral load of Merkel cell polyomavirus in Iranian patients with head and neck squamous cell carcinoma: Is it clinically important?

Recent studies show that the human Merkel cell polyomavirus (MCPyV) may be involved in causing cancer. The objective of this study was to assess the impact of MCPyV on the development of head and neck squamous cell carcinoma (HNSCC). In total, 50 paraffin-embedded HNSCC biopsy samples and 50 adjacent non-cancerous samples were evaluated for the presence of MCPyV DNA and RNA. Among patients, the five most frequent histopathologic sites were the tongue (22.0%), lip (16.0%), submandibular (14.0%), cheek (14.0%), and throat (14.0%). MCPyV DNA was positive in eight (16.0%) samples. The median MCPyV LT-Ag copy number in the eight positive samples and in one non-cancerous sample was 4.8 × 10-3 and 2.6 × 10-5 copies/cell, respectively. Quantification of MCPyV LT-Ag revealed increased expression in stage III (5.6 × 10-3 copies/cell) than in the other stages. The MCPyV DNA load in different stages of HNSCC was also statistically significant (P = 0.027). The viral load was low, suggesting that only a fraction of cancerous cells is infected. This result provides evidence confirming the presence of MCPyV in a subset of Iranian patients with HNSCCs, but further studies needed to confirm our findings.

Pros and cons of direct genotyping on tuberculosis clinical samples.

OBJECTIVE: Prompt genotyping of Mycobacterium tuberculosis (M. tuberculosis) is crucial for improving molecular epidemiological investigation of tuberculosis (TB). METHODS: We performed a retrospective study to evaluate the use of 24 loci MIRU-VNTR (mycobacterial interspersed repetitive unit-variable number of tandem-repeat) directly on 135 clinical samples from 84 TB patients. RESULTS: There was a direct correlation between genotyping on clinical samples by MIRU-VNTR and bacterial load (P = 0.001). VNTR loci were amplified successfully for 41.5% of the clinical samples (19-24 loci), 32.6% (13-18 loci), 23.7% (7-12 loci) and 2.2% (1-6 loci). Loci of 2401, 577, 2996 and 154 had the highest power to show the mixed strains infection in clinical samples. CONCLUSIONS: Direct MIRU-VNTR is partially successful in complete genotyping of M. tuberculosis strains. On the other hand, detection of polyclonal infection is undoubtedly reliable based on the direct MIRU-VNTR.

Bias in detection of Mycobacterium tuberculosis polyclonal infection: Use clinical samples or cultures?

The application of MIRU-VNTR has unveiled that infection by Mycobacterium tuberculosis can be polyclonal. Our comparative study demonstrated that based on the studied samples (clinical specimen or culture) detection of polyclonal M. tuberculosis infection can be significantly different.

A comparative study of various methods for detection of IL28B rs12979860 in chronic hepatitis C.

Interleukin-28B (IL28B) single-nucleotide polymorphisms (SNPs) constitute important host-related factors influencing the response rate to Hepatitis C virus (HCV) standard antiviral therapy. In the last few years, several new technologies for SNP detection have been developed. However, the sensitivity and specificity of various methods are different and needs evaluation. Five different methods (resolution melting curve [RMC], polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP], PCR-sequencing analysis, amplification refractory mutation system [ARMS], and zip nucleic acid probe-based real-time PCR [ZNA]) were developed for genotyping rs12979860 associated with IL28B. In this study, limit of detection (LD), costs and turnaround time of these methods were compared in 350 subjects. As for IL28B rs12979860 polymorphisms, 348/350 (99.4%) samples were consistent among the five methods, while results for 2/350 (0.57%) samples were concordant by ZNAs and PCR-sequencing, and discordant by other methods. Without considering the cost of DNA extraction, the price of each reaction for ARMS-PCR, RMC, PCR-RFLP, ZNA and PCR-sequencing were respectively: US$3.10, US$5.0, US$5.50, US$8.50 and US$17.0. RMC was the fastest method, while the ZNA method was easy to use, reliable and effective. Lower LD was determined to be 50-60 copies/µL for the PCR-RFLP, RMC and ARMS-PCR assays; whilst ZNA assay was able to detect 2-3 copies/µL. In conclusion, in the current study, all four methods are suitable for IL28B rs12979860 genotyping, but the ZNA assay can be a reliable tool. Due to its lower LD for SNP identification, this method is better than others for detecting this type of polymorphism.

High prevalence of Mycobacterium tuberculosis mixed infection in the capital of moderate tuberculosis incidence country.

OBJECTIVE: Recent studies using molecular epidemiological techniques have demonstrated mixed infection with multiple strains of Mycobacterium tuberculosis especially in countries with high tuberculosis (TB) burden. We aimed to determine the prevalence of mixed infection among patients with TB in the capital of Iran as a country with moderate incidence rate. METHODS: Samples were collected randomly from January 2011 to December 2013 in Tehran, capital of Iran. A total of 75 M. tuberculosis isolates were genotyped by 24 loci mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-VNTR) for screening the mixed infection. RESULTS: Twenty patients (20/75) were identified with mixed infection, and the estimated rate of mixed infection was 26.6%. Thirteen out of the 24 loci were able to detect the mixed infection in our study. CONCLUSIONS: Mixed infections occur at high prevalence among studied Iranian TB patients. Further research is inevitable to evaluate the association of mixed infection and disease progression and treatment.

Inhibitory effect of miR-377 on the proliferative and invasive behaviors of prostate cancer cells through the modulation of MYC mRNA via its interaction with BCL-2/Bax, PTEN, and CDK4.

The MYC gene is a regulatory and proto-oncogenic gene that is overexpressed in the majority of prostate cancers (PCa). Numerous studies have indicated that aberrant expression of microRNAs is involved in the initiation and progression of prostate cancer. In this investigation, we assessed the impact of miR-377 on MYC through luciferase assay. Real-time PCR was employed to determine whether miR-377 could reduce the levels of MYC mRNA in transfected PCa cell lines (PC-3 and DU145) and change in the mRNA levels of BCL-2/Bax, PTEN, and CDK4 as a consequence of MYC downregulation. Moreover, we analyzed the effects of miR-377 on apoptosis, proliferation, cell cycle, and wound healing. Our findings demonstrate that miR-377 effectively targets MYC mRNA, as confirmed by luciferase assay and Real-time PCR. We observed a significant reduction in BCL-2 and CDK4 expression, along with an increase in Bax and PTEN, in prostate cancer cell lines upon MYC suppression. Additionally, elevated levels of miR-377 in PCa cell lines induced apoptosis, inhibited proliferation and migration, and arrested the cell cycle. Taken together, these results unveil the inhibitory role of miR-377 in MYC function within PCa, thereby suggesting its potential as a therapeutic target for the treatment of this malignancy.

Targeting long non-coding RNA MALAT1 reverses cancerous phenotypes of breast cancer cells through microRNA-561-3p/TOP2A axis.

Non-coding RNAs, including Inc-RNA and miRNA, have been reported to regulate gene expression and are associated with cancer progression. MicroRNA-561-3p (miR-561-3p), as a tumor suppressor, has been reported to play a role in preventing cancer cell progression, and MALAT1 (Lnc-RNA) have also been demonstrated to promote malignancy in various cancers, such as breast cancer (BC). In this study, we aimed to determine the correlation between miR-561-3p and MALAT1 and their roles in breast cancer progression. The expression of MALAT1, mir-561-3p, and topoisomerase alpha 2 (TOP2A) as a target of miR-561-3p was determined in BC clinical samples and cell lines via qRT-PCR. The binding site between MALAT1, miR-561-3p, and TOP2A was investigated by performing the dual luciferase reporter assay. MALAT1 was knocked down by siRNA, and cell proliferation, apoptotic assays, and cell cycle arrest were evaluated. MALAT1 and TOP2A were significantly upregulated, while mir-561-3p expression was downregulated in BC samples and cell lines. MALAT1 knockdown significantly increased miR-561-3p expression, which was meaningfully inverted by co-transfection with the miR 561-3p inhibitor. Furthermore, the knockdown of MALAT1 by siRNA inhibited proliferation, induced apoptosis, and arrested the cell cycle at the G1 phase in BC cells. Notably, the mechanistic investigation revealed that MALAT1 predominantly acted as a competing endogenous RNA in BC by regulating the miR-561-3p/TOP2A axis. Based on our results, MALAT1 upregulation in BC may function as a tumor promoter in BC via directly sponging miRNA 561-3p, and MALAT1 knockdown serves a vital antitumor role in BC cell progression through the miR-561-3p/TOP2A axis.

Development of polyclonal heavy chain antibodies targeting programmed death ligand-1.

Programmed death ligand-1 (PD-L1, CD274 and B7-H1) has been described as a ligand for immune inhibitory receptor programmed death protein 1 (PD-1). With binding to PD-1 on activated T cells, PD-L1 can prevent T cell responses via motivating apoptosis. Consequently, it causes cancers immune evasion and helps the tumor growth; hence, PD-L1 is regarded as a therapeutic target for malignant cancers. The anti-PD-L1 monoclonal antibody targeting PD-1/PD-L1 immune checkpoint has attained remarkable outcomes in clinical application and has turned to one of the most prevalent anti-cancer drugs. The present study aimed to develop polyclonal heavy chain antibodies targeting PD-L1via Camelus dromedarius immunization. The extra-cellular domain of human PD-L1 (hPD-L1) protein was cloned, expressed, and purified. Afterwards, this recombinant protein was utilized as an antigen for camel immunization to acquire polyclonal camelid sera versus this protein. Our outcomes showed that hPD-L1 protein was effectively expressed in the prokaryotic system. The antibody-based techniques, such as enzyme-linked immunosorbent assay, western blotting, and flow cytometry displayed that the hPD-L1 protein was detected by generated polyclonal antibody. Due to the advantages of multi-epitope-binding ability, our study exhibited that camelid antibody is effective to be applied significantly for detection of PD-L1 protein in essential antibody-based studies.

Identification of two neutralizing human single-chain variable fragment antibodies targeting Staphylococcus aureus alpha-hemolysin.

Objectives: The inability of the host immune system to defeat Staphylococcus aureus is due to various secreted virulent factors such as leukocidins, superantigens, and hemolysins, which interrupt the function of immune components. Alpha-hemolysin is one of the most studied cytolysins due to its pronounced effect on developing staphylococcal infections. Alpha-hemolysin-neutralizing antibodies are among the best candidates for blocking the toxin activity and preventing S. aureus pathogenesis. Materials and Methods: A human single-chain variable fragment (scFv) phage display library was biopanned against alpha-hemolysin. The selected phage clones were assessed based on their binding ability to alpha-hemolysin. The binding specificity and affinity of two scFvs (designated SP192 and SP220) to alpha-hemolysin were determined by enzyme-linked immunosorbent assay. Furthermore, the neutralizing activity of SP192 and SP220 was examined by concurrent incubation of rabbit red blood cells (RBCs) with alpha-hemolysin and scFvs. Results: SP192 and SP220 showed significant binding to alpha-hemolysin compared with the control proteins, including bovine serum albumin, human adiponectin, and toxic shock syndrome toxin-1. Besides, both scFvs showed high-affinity binding to alpha-hemolysin in the nanomolar range (Kaff: 0.9 and 0.7 nM-1, respectively), leading to marked inhibition of alpha-hemolysin-mediated lysis of rabbit RBCs (73% and 84% inhibition; respectively). Conclusion: SP192 and SP220 scFvs can potentially be used as alpha-hemolysin-neutralizing agents in conjunction with conventional antibiotics to combat S. aureus infections.

Mixed pulmonary infection with four isolates of nontuberculous mycobacteria: a case report of mycobacterium bacteremicum infection.

BACKGROUND: A mixed pulmonary infection of Mycobacterium bacteremicum and three different isolates of nontuberculous mycobacteria (NTM) is an unusual clinical manifestation and have not yet been indicated. In this case report, we reported four isolates of NTM using phenotypic and genotypic test of pulmonary sample in Tehran, Iran. CASE PRESENTATION: We report a case of severe pulmonary disease in a 19-year-old male patient with productive cough, shortness of breath, and low-grade fever for several weeks. The C-reactive protein (CRP) level (80.2 mg/L) and erythrocyte sedimentation rate (ESR) (95 mm/h) were high. The computed tomographic scan indicated bronchiectasis, nodular opacities, consolidation, and cavitary lesions on both sides. The result of purified protein derivative (PPD) test was equal to 15 mm. The sequences of hsp65, rpoB, and 16S rDNA genes indicated more than 99% homology to four isolates of nontuberculous mycobacteria (NTM), including Mycobacterium fortuitum, M. chelonae, M. mucogenicum, and M. bacteremicum. We found that all four strains were susceptible to amikacin, cefoxitin, ciprofloxacin, clarithromycin, imipenem, and linezolid. The patient was treated with ciprofloxacin, clarithromycin, and amikacin, along with Montelukast, for five months. CONCLUSION: We report a case of severe pulmonary infection by four isolates of NTM. After treatment, the patient reported complete resolution of the signs and a weight gain of 5 kg; also, the CRP and ESR were normal. Nine months after the infection diagnosis, a new CT scan revealed further improvements.

Detection of Mimivirus from respiratory samples in tuberculosis-suspected patients.

Acanthamoeba polyphaga mimivirus (APMV), a species of amoeba-infecting giant viruses, has recently emerged as human respiratory pathogens. This study aimed to evaluate the presence of Mimivirus in respiratory samples, collected from tuberculosis (TB)-suspected patients. The study was performed on 10,166 clinical respiratory samples from April 2013 to December 2017. Mimivirus was detected using a suicide nested-polymerase chain reaction (PCR) and real-time PCR methods. Of 10,166 TB-suspected patients, 4 (0.04%) were positive for Mimivirus, including Mimivirus-53, Mimivirus-186, Mimivirus-1291, and Mimivirus-1922. Three out of four patients, hospitalized in the intensive care unit (ICU), were mechanically ventilated. All patients had an underlying disease, and the virus was detected in both sputum and bronchoalveolar lavage samples. In conclusion, Mimivirus was isolated from TB-suspected patients in a comprehensive study. The present results, similar to previous reports, showed that Mimiviruses could be related to pneumonia. Further studies in different parts of the world are needed to additional investigate the clinical importance of Mimivirus infection.

Anti-Acinetobacter baumannii single-chain variable fragments show direct bactericidal activity.

Objectives: The high resistance rate of Acinetobacter baumannii and the limited number of available antibiotics have prompted a worldwide effort to develop effective antimicrobial agents. Accordingly, identifying single-chain variable fragment antibodies (scFvs), capable of exerting direct antibacterial activity in an immune system-independent manner, may be making immunocompromised patients more susceptible to A. baumannii infections. Materials and Methods: To isolate bactericidal scFvs targeting A. baumannii, we panned a large human scFv phage display library against whole-cell extensively drug-resistant (XDR) A. baumannii strains grown as biofilm or cultured with human blood or human peripheral blood mononuclear cells plus plasma. The binding of scFv-phages to A. baumannii was assessed by the dot-blot assay. Soluble scFvs, derived from the selected phages, were assessed based on their ability to bind and inhibit the growth of A. baumannii. Results: Five phage clones showed the highest reactivity toward A. baumannii. Among five soluble scFvs, derived from positive phage clones, two scFvs, EB211 and EB279, had high expression yields and displayed strong binding to A. baumannii compared with the controls. Moreover, XDR A. baumannii strains treated with positively-charged scFvs, including EB211, EB279, or a cocktail of EB211 and EB279 (200 µg/ml), displayed lower viability (approximately 50%, 78%, and 40% viability, respectively) compared with PBS-treated bacteria. Conclusion: These results suggest that combining last-resort antibiotics with bactericidal scFvs could provide promising outcomes in immunocompromised individuals with A. baumannii infections.