The Boom syndrome helicase (BLM) unwinds a variety of DNA structures such as Guanine (G)-quadruplex. Here we reveal a role of RNF111/Arkadia and its paralog ARKL1, as well as Promyelocytic Leukemia Nuclear Bodies (PML NBs), in the regulation of ubiquitination and control of BLM protein levels. RNF111 exhibits a non-canonical SUMO targeted E3 ligase (STUBL) activity targeting BLM ubiquitination in PML NBs. ARKL1 promotes RNF111 localization to PML NBs through SUMO-interacting motif (SIM) interaction with SUMOylated RNF111, which is regulated by casein kinase 2 (CK2) phosphorylation of ARKL1 at a serine residue near the ARKL1 SIM domain. Upregulated BLM in ARKL1 or RNF111-deficient cells leads to a decrease of G-quadruplex levels in the nucleus. These results demonstrate that a CK2- and RNF111-ARKL1-dependent regulation of BLM in PML NBs plays a critical role in controlling BLM protein levels for the regulation of G-quadruplex. The Bloom syndrome helicase (BLM) unwinds a variety of complex DNA structures including G quadruplex. Here the authors report RNF111-ARKL1-dependent ubiquitination of BLM in PML NBs, which limits BLM protein levels and maintains G quadruplex abundance in the nucleus. Among the many types of DNA structures, BLM has been shown particularly active in unwinding guanine (G)-quadruplex (G4) DNA in vitro5. G4 is formed in G-rich DNA region containing four stretches of three or more consecutive Gs that fold into a secondary structure8. Sequences that form G4 are widely present in the genome and G4 formation is linked with key biological processes ranging from transcription, translation, telomere homeostasis, genomic instability and cancer8. It has been indicated that BLM binds and unwinds G4 playing an important role in suppressing G4 levels in the cell10.The Bloom syndrome helicase (BLM) has multiple roles in many aspects of DNA metabolism, unwinding duplex DNA and a variety of complex DNA structures in DNA replication, recombination, transcription, and telomere maintenance13. PML NBs are membrane-less organelles forming a sphere of 0.1-1 um in diameter and are present in most mammalian cell nuclei15. BLM’s localization to PML NBs is dependent on both the SUMOylation of BLM and the two SUMO interacting motifs (SIMs)17. PML NBs have been implicated in a variety of biological processes such as senescence, apoptosis, and genome maintenance15. SUMOylation and SUMO-SIM interaction play essential roles during the formation and function of PML NBs18. The biological significance of PML NBs in the regulation of BLM function is still not clear.BLM primarily resides in Promyelocytic Leukemia Nuclear Bodies (PML NBs) in unperturbed cells23. RNF111  has been indicated as a STUBL since it possesses three successive SIMs at its N-terminal half of the protein and a RING domain at its C-terminus, capable of binding to SUMO2/3 chains for their ubiquitination in a SIM- and RING-dependent manner28. It has been shown that the SIMs of RNF111 are required for RNF111 localizing to PML NBs and binding to SUMOylated PML26. In addition, the E3 ligase activity of RNF111 is required for the ubiquitination and degradation of several regulators in the TGF-β signaling pathway33. The function of RNF111 and the regulation of RNF111-mediated degradation of SUMO-modified substrates remains to be further understood.SUMO-targeted E3 ubiquitin ligases (STUBLs) are a group of SIM-containing RING-type ubiquitin ligases that selectively recognize and ubiquitinate SUMO-modified proteins28. ARKL1 has been identified as a SUMO2 binding protein by GST-SUMO2 affinity capture and a yeast two-hybrid screen against SUMO35. It also has been shown that ARKL1 can localize to PML NBs, binds to casein kinase 2 (CK2) regulatory β subunit, and is involved in Epstein-Barr virus reactivation37. The function of ARKL1 remains largely uncharacterized.RNF111 has an N-terminal paralog Arkadia-like 1 , which shows similarity to the amino-terminal half of RNF111 and contains two SIMs that correspond to the SIM2 and SIM3 of RNF111In this study, we identify a CK2- and RNF111-ARKL1-dependent and PML-NB-involved regulatory mechanism controlling BLM ubiquitination and G4 resolution. RNF111 exhibits a noncanonical STUBL activity that requires SUMO-SIM-dependent interacion with ARKL1 as well as PML NBs localization for the ubiquitination and degradation of BLM. ARKL1 SIM domains bind to SUMOylated RNF111 at lysine 237 and 238 residues, promoting RNF111 localization to PML NBs. In addition, CK2-mediated phosphorylation of ARKL1 on a serine residue nearby the first SIM of ARKL1 promotes the SIM-SUMO interaction of ARKL1 and RNF111, facilitating RNF111 localization to PML NBs and ubiquitination of BLM. The PML NBs localization of RNF111 and BLM are critical for RNF111-dependent ubiquitination and protein degradation of BLM. Loss of RNF111 or ARKL1 or inhibition of CK2 leads to reduced G4 levels in the nucleus due to uncontrolled BLM protein levels. We proposes a model that BLM level in the cell is limited by RNF111-ARKL1-mediated ubiquitination and degradation of BLM in PML NBs under the regulation of CK2 activity for maintaining G4 levels.RNF111 knockout (KO) , but not a catalytic inactive mutant of RNF111 with two of the cysteine residues in the RING domain mutated to serine residues (RNF111CS), restored BLM ubiquitination and reduced elevated BLM levels in RNF111 KO cells  abolished the ubiquitination  Fig. . The eletor Fig. . Using ctor Fig. . These dtor Fig. . Furtherlls Fig. . In an iion Fig. . Togethe28. We investigated whether RNF111 interacts with BLM through its SIMs. By immunoprecipitating HA-tagged RNF111 and comparing WT and SIMs mutant of RNF111 (RNF111SIM*), we found that HA-tagged RNF111SIM* mutant exhibited similar degree of binding to BLM as RNF111WT did, indicating that the SIM domains of RNF111 are not required for RNF111 association with BLM  exhibited reduced interaction with RNF111 or BLM, indicating that the SIM domains of ARKL1 are critical for the ARKL1 interaction with RNF111 and BLM  in the K-X-E/D motifs or an inverted version of this sequence on a substratence Fig. . We reasnce Fig. . Furtherion Fig. . We furtion Fig. . Mutatioion Fig. . In addiion Fig. , its intion Fig. .Together, these results indicate that RNF111 is SUMOylated at K237/238 residues and that RNF111 interacts with ARKL1 in a SUMO-SIM-dependent manner.37. In U2OS cells expressing ARKL1-GFP and mcherry-PML, ARKL1-GFP is mainly in the nucleoplasm and forms a few foci that overlap with PML NBs marked by mcherry-PML  within 2 s . After photobleaching, the fluorescence of ARKL1-GFP in PML NBs immediately recovered with a half-time fluorescence recovery  that lacks the interaction with CK2β37. ARKL1 KO cells complemented with ARKL1∆S showed a reduced number of PLA foci when compared to that of ARKL1WT-complemented cells, indicative of a deficiency in RNF111 recruitment to PML NBs  antibody and inhibition of CK2 by TBB abolished the detection, supporting that ARKL1 is phosphorylated by CK2 and inhibition of CK2 decreases the phosphorylation  near the two SIM domains mutated to alanine (4A:S327/328/330/385A) and found that 4 A mutant abolished CK2 phosphorylation that can be recognized by anti- phosphor-CK2 substrate (pS/pTDXE) antibody  residue resides in a CK2 recognition motif nearby the first SIM domain of ARKL1 Fig.  and 6h. ody Fig. . Mutatioody Fig. . Sequenc∆S) or mutation of CK2 phosphorylation site (ARKL1S328A) reduced the binding of ARKL1 and RNF111 , however, still retains the interaction of ARKL1 and RNF111, consistent with the role of phosphorylation in promoting ARKL1-RNF111 interaction , a G4 stabilizer, significantly increased G4 levels that can be detected by an anti-G4 antibody, BG444.  fail to be ubiquitinated. It is noteworthy that both mutants are intact in their interaction with RNF111 in the in vitro co-immunoprecipitation experiment  mechanism is active, BLM is associated with the ALT-associated PML NBs (APBs) and plays a critical role in ALT58. The regulation of BLM through RNF111-ARKL1-dependent control may therefore also contributes to telomere maintenance and ALT.Due to the roles of BLM in multiple aspects of DNA processing such as DNA replication, repair, recombination, and telomere maintenance, the control of BLM protein level may also be critical in other aspects of the function of BLM in maintaining DNA metabolism homeostasis in cells. G4 structures are enriched in telomere since telomeric DNA consists of tandem copies of short G-rich repeats (5’-TTAGGG-3’). BLM also has been reported to promote telomere replication by resolving G4 structuresTogether, our study identifies a RNF111 and ARKL1 involved noncanonical STUBL activity for the ubiquitination and degradation of BLM and proposes a model that BLM level in the cell is limited by RNF111-ARKL1-mediated ubiquitination and degradation in PML NBs under the regulation of CK2 activity for maintaining G4 levels.U2OS cells  were grown in McCoy’s 5 A with L-glutamine medium  with 10% fetal bovine serum . 293 T , Hela , and BJ cells  were grown in Dulbecco’s modified Eagle’s medium (DMEM)  with 10% fetal bovine serum. For SiRNA transfection, cells were seeded one day before transfection. Lipofectamine™ RNAiMAX Transfection Reagent  was used for transfection based on manufacture’s protocol. For plasmid transfection, Lipofectamine™ 2000 Transfection Reagent  was used based on manufacture’s protocol. RNF111 KO U2OS was generated by CRISPR-Cas9 using Invitrogen TrueGuide™ sgRNA  and TrueCut™ Cas9 Protein v2 , Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent  was used for transfection according to manufacture’s protocol. ARKL1 KO U2OS and Hela cells were generated by CRISPR-Cas9 using pLentiCRISPR lentivirus carrying Cas9 and sgRNA targeting ARKL1 sequence 5’-ACAGAATGGCCGAGTCGCCA-3’. Cells were subjected to puromycin selection and single colonies were selected and expanded for a screen of ARKL1 clones. For Cycloheximide-chase assay, cells were treated with 100 μg/ml cycloheximide .59. pAID5.3-C was a gift from Masato Kanemaki (Addgene plasmid 145813)60. pCMV-Tol2 plasmid was a gift from Stephen Ekker (Addgene plasmid#31823)61. The primers used in this study for generating various mutants for BLM, ARKL1, and RNF111 are listed in Supplementary Table SiRNAs used in this study are listed in Supplementary Table 3VO4 and 20 mM N-ethylmaleimide. The lysate was sonicated in ice water and centrifuged at 21,000 × g for 10 min at 4 °C. The supernatant was collected and 5× SDS-PAGE sample loading buffer was added. Samples were denatured at 95 °C for 5 min. For immunoprecipitation, cells were harvest from plates by scrapers and immediately lysed by modified NETN lysis buffer . The lysate was sonicated in ice water and centrifuged at 21,000 × g for 10 min at 4 °C. The supernatant was collected and Flag/HA beads or antibodies conjugated with Protein A/G beads were added. After incubation at 4 °C overnight, beads were washed by modified NETN buffer for 4 × 15 min. Samples were eluted by 2× SDS-PAGE sample loading buffer at 95 °C for 5 min. For Immunoprecipitation to detect protein ubiquitination or SUMOylation, cells were treated with 10 µM MG132  for 4 hrs and lysed by modified NETN lysis buffer with 0.4% SDS. For SUMOylation, similar results were obtained when cells were not treated with MG132. The lysate was sonicated in ice water and centrifuged at 21,000 × g for 10 min at 4 °C. The supernatant was collected and SDS was diluted to 0.1% by modified NETN buffer. Flag/HA beads or antibodies conjugated with Protein A/G beads were added. After incubation at 4 °C overnight, beads were washed by NETN buffer for 4 × 15 min. Samples were eluted by 2× SDS-PAGE sample loading buffer at 95 °C for 5 min.For cell lysis, cells were harvest from plates by scrapers and immediately lysed by RIPA lysis buffer with 1× protease inhibitor cocktail, 1 mM PMSF, 50 mM β-Glycerophosphate, 5 mM NaF, 1 mM NaWT-Flag or RNF111CS-Flag expressed in 293 T cells was immunoprecipitated with Flag beads and eluted with 200 ng/µL Flag peptides . Eluted RNF111WT-Flag was then applied to Zeba™ Spin Desalting Columns  for further purification. GFP-BLM was purified from 293 T cells expressing GFP-BLM using GFP antibody-couple protein A/G beads. The in vitro purification reaction was performed following the instruction of Enzo Ubiquitinylation kit . Briefly, purified GFP-BLM and RNF111-Flag were incubated with 1x ubiquitination buffer  with 2.5uM ubiquitin , 100 nM ubiquitin Activating Enzyme E1 , 2.5 μM ubiquitin-conjugating enzyme E2 UbcH5c , 5 mM Mg2+-ATP , 1 mM DTT, 20 U/mL pyrophosphatase  at 37 °C with rotation for 2 h. Reaction was quenched by 2x non-reducing gel loading buffer.RNF1112, 300 mM Sucrose, 0.5% Triton-X) at 4 °C for 6 min, followed by PBS washing for 4 × 5 min. After permeabilization, cells were incubated with primary antibodies in immunofluorescence buffer  and 0.2% Triton-X in PBS) at 4 °C overnight, followed by 4 × 5 min PBS washing. After incubation with dye-conjugated secondary antibodies (1:1000 dilution) for 1 h at 37 °C and PBS washing for 4 × 5 min, cells were mounted using ProLong™ Gold Antifade Mountant with DAPI . Images were collected by Nikon A1-Confocal using ×60 objective and analyzed by NIH Elements AR software (AR 5.21.03 64 bit).Cells grown on coverslips were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at room temperature for 10 min, followed by PBS washing for 4 × 5 min. Cells were then permeabilized with permeabilization buffer . A baseline of 20 pre-bleach images was initially collected, followed by a bleaching sequence using 25% of our current laser power in the 488 nm laser line and a small circular region of interest (1.3 μm). A total of 180 image frames was recorded for post-bleach recovery continuously until signal reached a steady state. Movies were analyzed using NIS-Elements, and curve fitting was done with GraphPad Prism software. For curve fitting, a single exponential function (f(t) = α(1-e-kT), where T1/2  is ln 0.5/(-k), and α is the mobile fraction was used. Raw recovery curves were corrected for background and photofading. The lowest fluorescence signal and the time-point after bleaching were scaled to 0, and curves were normalized to 1 based on the reference signal before bleaching.FRAP acquisition was performed with A1-Nikon confocal located at the department of Genetics MD Anderson (BSRB Microscopy facility) equipped with LUNV laser launch and DU-G GaASP detector unit and a Nikon Plan-Apo 60X oil 1.4 NA objective. The entire experiments were performed under incubation  according to the manufacturer’s protocol. Briefly, coverslips were blocked with Duolink® blocking solution at 37 °C for 60 min and incubated with primary antibodies in Duolink antibody diluent at 4 °C overnight, followed by wash buffer A  washing for 2 ×5 min at room temperature. Ligation was performed with ligase in ligation buffer at 37 °C for 60 min, followed by wash buffer A washing for 2 ×5 min at room temperature. Amplification was performed with polymerase in amplification buffer at 37 °C for 100 min, followed by wash buffer B washing for 2 ×10 min at room temperature. After washing with 0.01x Wash Buffer B for 1 min, coverslips were mounted using ProLong™ Gold Antifade Mountant with DAPI. Images were collected by Nikon A1-Confocal using ×60 objective and analyzed by NIH Elements AR software (AR 5.21.03 64 bit).Statistical analysis was performed with GraphPad Prism 9 ). One-way ANOVA with Sidak’s correction, two-way ANOVA, or two-tailed unpaired t-test was used for statistical analyses as indicated. P < 0.05 was considered statistically significant. All experiments were performed with at least three biological repeats except indicated in the figure legends. No statistical method was used to predetermine sample sizes. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Western blots and microscopic imaging are representative of at least three biological repeats unless specified in the legends.Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Rates of antibiotic-resistant bacteria have increased worldwide over recent years, but the Italian Institute of Health reported a disruption to this trend in 2021 compared with 2020. Children are often recipients of unnecessary antibiotic prescriptions, especially for respiratory tract infections (RTIs). During the initial phase of the COVID-19 pandemic, common RTIs substantially decreased, so it is conceivable that antibiotic prescriptions also reduced during this time. To test this hypothesis, we retrospectively collected data on all visits to a pediatric primary care clinic in Northern Italy from February 20, 2020 to June 2, 2020 and compared data with the same period in 2019. We evaluated the antibiotic prescription rate according to the diagnosis at discharge. While the total number of visits significantly decreased (1335 in 2020 vs. 4899 in 2019), there was only a slight reduction in the antibiotic prescription rate . However, this corresponded to a 73.8% decrease in the total number of antibiotic prescriptions, with antibiotics for RTI accounting for 69% of the total reduction. It is possible that, at the larger scale, reduced antibiotic prescription in pediatrics during the COVID-19 pandemic resulted in a slight reduction in antimicrobial resistance. Acinetobacter [Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp).There are growing concerns about the threat from antibiotic-resistant bacteria. The Centers for Disease Control and Prevention (CDC) estimates that there was a 15% increase in drug-resistant nosocomial infection rates in 2020, the first year of the COVID-19 pandemic, compared with the previous year, especially with regard to rates of carbapenem-resistant Enterobacterales and carbapenem-resistant Children are often recipients of unnecessary antibiotics, especially for respiratory tract infections (RTIs), in most cases because these are caused by self-limiting viral conditions. Antibiotics are inappropriately prescribed for RTIs in children due to a lack of readily available tests to assess the viral origin of most RTIs coupled with pediatricians and family doctors seeking to limit parental anxiety about their children. During the first phase of the COVID-19 pandemic, when health measures for SARS-CoV-2 containment were stricter, there was a significant decrease in common RTIs and almost completely suppressed circulation of respiratory viruses other than SARS-CoV-2. It is conceivable that antibiotic prescriptions were similarly reduced. This trend, described in pediatric primary care settings in many countries –6, was aRegardless of the underlying motivations, it is possible that, at a larger scale, the reduction in antibiotic prescriptions in pediatrics during the COVID-19 pandemic may have led to a slight reduction in antimicrobial resistance. If confirmed in larger reports, these data suggest that further modifying antibiotic prescription attitudes to reduce prescriptions would likely result in a global reduction in antimicrobial resistance, even over the short term.In the future, given that it is not going to be possible to reduce the burden of RTIs in children, further education of clinicians on proper antibiotic prescription needs to be implemented and, if possible, guided by point-of-care tests able to discriminate viral from bacterial infections. In this regard, the recent increase in cases of scarlet fever and invasive group A streptococcus (GAS) in the pediatric age group in Europe , 9, prob


Suboptimal antibiotics use and the development of antibiotic resistance is a universal calamity. The theoretical model of therapeutic efficacy correlates quality use of antibiotics with healthcare practitioners’ understanding of antibiotic use and resistance. Keeping this phenomenon in mind, we aimed to evaluate hospital pharmacists’ understanding of antibiotic use and resistance at a public healthcare institute in Quetta city, Pakistan. This was a qualitative study that employed a semi-structured interview guide for data extraction. The phenomenology-based approach commissioned in-depth, face-to-face interviews with hospital pharmacists stationed at the surgical unit of Sandeman Provincial Hospital, Quetta. The interviews were audio taped followed by transcribed verbatim and were then analyzed for thematic contents by the standard content analysis framework. Although the saturation was reached after the 10th interview, we conducted two additional interviews for definite validation. Content analysis revealed five major themes: (1) Defining antibiotics, quality use of antibiotics and resistance, (2) antibiotic use: awareness and concern, (3) antimicrobial resistance: awareness and concern, (4) responding to antibiotic use and resistance, and (5) barriers to quality use of antibiotics and prevention of antibiotic resistance. The knowledge of quality use of antibiotics and resistance was promising, and the respondents were eager to address the drastic situation. The respondents were aware of the critical situation and provided valuable insights that can offer valued input while promoting the quality use of antibiotics in a developing country. The current study managed to identify an adequate understanding of antibiotic use and resistance among hospital pharmacists. Additionally, prospective concerns and possible predictors of antibiotic resistance were also highlighted. The current findings must be disseminated to the policymakers and prescribers to take prompt restorative actions to address antibiotic use and the development of antibiotic resistance in a developing country like Pakistan. The golden era of antibiotics regrettably did not last long as mankind was faced with the development of bacterial resistance against antibiotics . AlthougImproving the quality use of antibiotics in hospitals and other healthcare settings in addition to limiting use in agriculture is a crucial area to safeguard antibiotics for future generations. In line with what is being discussed, the World Health Organization highlights the significant role of healthcare professionals in limiting the frequency of antibiotic resistance . HealthcAmong healthcare professionals, hospital pharmacists occupy a conspicuous position in reducing the rates of antibiotic resistance . Our claParallel to the published literature, the role of the hospital pharmacist in disease management and clinical decision-making is shadowed in a developing country like Pakistan. There is a paucity of data on hospital pharmacists’ integration into the healthcare system. Moreover, the capability and proficiency of a hospital pharmacist are also questioned by other healthcare professionals. Based on this dearth of information, we aimed to evaluate how hospital pharmacists view the quality use of antibiotics and antibiotic resistance practicing at a local healthcare facility in Quetta city, Pakistan. Conducting this study had twofold reasons: to generate data that can be used as a potential reference for further studies, and to highlight what hospital pharmacists can offer while dealing with antibiotic resistance.The demographics are given in Most of the participants were male (75%), with age ranging from 25 to 35 years (80%). Half of the pharmacists had a Doctor of Pharmacy degree and had 11–20 years of experience.The thematic content analysis revealed five themes and eight subthemes which are shown in As expected, all pharmacists had a clear understanding of antibiotics, the quality use of antibiotics, and the development of antibiotic resistance. Additionally, the pharmacists mentioned routinely updating their knowledge about antibiotic use and resistance through updated information sources . This is encouraging because evidence-based information in pharmacy practice incorporates pharmacists’ clinical expertise with the most accessible evidence. The availability of evidence-based information also helps in justifying the medication-related needs of the healthcare system, prescribing practices, and patients’ predilections ,19.“Antibiotics; Fleming’ gift for mankind are static and cidal in nature. These are the drugs of choice against primary and secondary bacterial infections.” (Pharmacist 1)In parallel, pharmacists had decent knowledge about antibiotic resistance. It was obvious that pharmacists understood the phenomenon that can eventually help in the delivery of pharmaceutical care .“The over-use and misuse of antibiotics, bacterial mutations, and substantial use of antibiotics in agriculture and among animals (like poultry and livestock) result in developing antibiotic resistance.” (Pharmacist 3)Respondents of the current study acknowledged frequent use of antibiotics at the setting site. Furthermore, as the influx of inpatients is high compared to outpatients, the use of parenteral antibiotics was commonly reported. Among the commonly prescribed antibiotics were Ceftriaxone , Ciprofloxacin (fluoroquinolones), Vancomycin (glycopeptides), Meropenem (carbapenem), and Piperacillin-Tazobactam (β-lactam/beta-lactamase inhibitors).“The physicians prescribe oral antibiotics to the outpatients ; however, Meropenem and Vancomycin are frequently used (inpatient) when compared to other antibiotics. It is estimated that every third or maybe fourth prescription contains these two drugs.” (Pharmacist 6)The absence of an effective surveillance system and poorly regulated legislature results in the free availability of antibiotics without prescription. Community pharmacies  lack authorized personnel (community pharmacists) and are operated by laymen with no prior knowledge, qualification, or training in running a community pharmacy. Most of the pharmacists had information about antimicrobial dispensing rules according to Pakistan’s Drug Act 1976 and the Drug Regulatory Authority of Pakistan Act 2012. However, few know the drug laws (schedule G and D) that elaborate the use and dispensing of antibiotics at the communal level.“From brands to generics, everything is freely available at the pharmacies. Everybody knows about it including policymakers and officials of the inspection teams. Till today, no one took serious action against the free availability and public sale of antibiotics.” (Pharmacist 7)The limited availability of antibiotics in the hospital and a high influx of patients requiring antibiotics was reported as a significant concern by all pharmacists. This is reasonable as the healthcare facilities are limited  and the prescribers have no or limited choice to prescribe antibiotics based on the availability in the central pharmacy.“If I can recall, there are not more than 10 antibiotics available in the hospital. Therefore, antibiotic selection is based on availability and not on therapeutic needs. Rationally, yes this is malpractice, but what other choice do we have?” (Pharmacist 10)The free availability of antibiotics, antibiotic sharing, and nonprofessional referrals of antibiotic use in the community was the primary concern of the pharmacists. These factors were linked to economic and therapeutic loss to society. Moreover, this free availability and frequent use of antibiotics were rated as important factors in developing antibiotic resistance. In comparison, antibiotic use in the community was ranked as a substantial problem when compared to antibiotic use in hospitals.“Just name the antibiotic, the quantity and there you have it. There are zero concepts of the recommended dosage, treatment duration, and actual need for antibiotics. Community pharmacies are encouraging antibiotic resistance, and this is increasing day by day.” (Pharmacist 8)The respondents agreed that antibiotic resistance is a significant issue and has increased after the pandemic. They were also of the opinion that the irrational use of antibiotics at the communal level is promoting antibiotic resistance and needs imperative attention.“Irrational prescribing, self-medication, using leftovers, all results in antibiotic resistance. Other reasons are also reported in the literature, but we have to admit that the issue is serious and needs prompt actions.” (Pharmacist 4)“Last week a six-month child was subjected to cultural sensitivity. The kid was resistant to eleven tested antibiotics.” (Pharmacist 5). The development of antibiotic resistance was taken seriously by all respondents that are resulting in suffering, deaths, and economic loss. Pharmacists also reported that antibiotic resistance at their practicing site is also emerging as treatment failure is often reported with the use of first or second-line antibiotics.“I observed that compared to last year, antibiotics  are least effective. The physicians are now routinely prescribing Meropenem, Vancomycin, and Tazobactam. We must wake up because this is a serious concern and as I see it, there is no solution in near future too.” (Pharmacist 1)As evident from the above conversation, our respondents were versed in antibiotic use and resistance. Consequently, we wanted to extract pharmacists’ viewpoints on how they respond to antibiotic use and the development of antibiotic resistance in their practice settings. Although all respondents agreed that they play a pivotal role in medicine management, mixed views were observed when the response to antibiotic use was investigated.“Although I follow need and evidence-based medication , I must keep an eye on the generic availability in our stock.” (Pharmacist 9)Six pharmacists claimed that they often try to convince the patients about generic substitution to save costs as well as about the importance of completing the complete therapy. Most of the patients come from a meager income background; they have no idea about the quality use of antibiotics.“I normally guide the patients about the importance of antibiotics, the hazards of antibiotic resistance, and the financial and social repercussions. I hope that a medically educated patient can help in halting the development of antibiotic resistance.” (Pharmacist 11)Three of the interviewees agreed that we could not control antibiotic resistance alone without implementing guidelines, laws, and legislation for antibiotic use. The interviewed hospital pharmacists also pointed out a few strategies to control the emergence of antibiotic resistance by involving all key stakeholders of the healthcare system.“Alone, we cannot reduce antibiotic resistance. It is emerging at a high pace and a collective approach is needed to overcome this problem. The policymakers should target a mass population as well as an individualized strategy that must focus on community members and healthcare professionals to safeguard the use of antibiotics.” (Pharmacist 9)It is now acknowledged that antibiotic resistance can be reduced by prescribing antibiotics rationally based on established guidelines, antibiotic susceptibility testing, and clinical response. In parallel, the surveillance of antibiotic availability in healthcare settings, restrictive self-medication, and over-prescription is also needed. However, antibiotic resistance can only be controlled and minimized by the determined efforts of all healthcare professionals . Several barriers were identified by the study respondents while addressing the quality use of antibiotics and the development of antibiotic resistance and will be discussed consequently.Self-medication, using leftover antibiotics, referral of antibiotics (friends and families), expecting an antibiotic during the consultation, and demanding an antibiotic by themselves were identified as the key barriers to the quality use of antibiotics and antibiotic resistance. Patient education and counseling were last reported at the healthcare institutes and that was rated as a major factor in developing the false ideology about antibiotics as mentioned above.“Our patients demand antibiotics and will go to different stores to get one. The physician is considered incompetent if an antibiotic is not prescribed. This mindset is shaping as a key barrier to quality use of antibiotics in our society.” (Pharmacist 3)Another barrier to rational use of antibiotics was related to the urgency of being cured. We must agree that when faced with a disease, expecting a quick recovery is obvious. However, this tendency does not allow misuse of antibiotics considering that it will pace up the recovery time. Nevertheless, patients are in the habit of using antibiotics for a fast pace of recovery, and this is shaping as a factor in developing antibiotic resistance.“While being questioned (by physicians or nurses), using an antibiotic before coming to the hospital is usually reported by the patients. The reason is always the same (it cures everything). This is an issue that we are facing almost daily. Don’t you think this is causing antibiotic resistance?” (Pharmacist 4)Most of the respondents also emphasized certain institution-related deficiencies and limitations as barriers to the quality use of antibiotics and antibiotic resistance. The most reported barrier was the limited number of antibiotics available at the central pharmacy. Medicines are supplied annually based on hospital demand and because purchasing budgets are low, medicines are not adequately available. The availability is attained by reducing the quantity and types of medicines . However, this limits the choice of the prescribers, and they must prescribe what is available at the hospital.“We work in a public hospital where 70–80% of medicine is provided by the hospital to the inpatients. However, we have financial limitations, and availability of antibiotics from all therapeutic classes is not possible.” (Pharmacist 12)Another barrier to the quality use of antibiotics was related to the limited capacity of cultural sensitivity. By practice, the developed guidelines advocate an initiation of empirical therapy followed by a culture sensitivity test. However, this was last performed at the study site, and without sensitivity reports being available, the practice promoted antibiotic resistance.“We have limited the capacity of performing a culture sensitivity test. Because most of our patients belong to the below-average income group, ordering a sensitivity test is unaffordable for the patients. In such scenarios, we have no choice but to continue using the same antibiotics.” (Pharmacist 10)For decades, healthcare and social scientists have been trying to minimize the burden of antibiotic resistance. Among those, numerous interventions and measures have been taken that too have reported their effectivity and effectuality ,22,23,24Pharmacists’ positive perception towards the quality use of antibiotics and antibiotic resistance was an encouraging finding of the current study. However, mixed observations were reported when the results of the current study were cross-compared with the published literature. Al-Tanni et al. in their study concluded that although pharmacists’ knowledge of the quality use of antibiotics was satisfactory, the perception towards the spread of resistance was unsatisfactory . SimilarThe results of this qualitative study revealed that the use of broad-spectrum antibiotics is widespread at the study site. Our respondents were aware and anxious about this imprudent use of antibiotics, and this is similar to the findings of Tarrant et al. . The autAnother key finding of the current study was the limited availability of antibiotics in the hospital and hence a reduced choice for the prescribers while dealing with infectious diseases. In addition, poor culture sensitivity testing was highlighted. Unfortunately, the evidence-based data are lacking from Baluchistan, and we do not have an actual picture of such limitations. However, our personal observation goes parallel to what is reported by the respondents as there is a lack or limited number of antibiotics at the hospital. Within this context, the healthcare system of Pakistan is faced with severe financial limitations and is unable to cater to the needs of the patients . HealthcFinally, patient-reported factors were rated as a predictor of antibiotic resistance by the study respondents. Among these, self-medications, personal referrals, and demanding an antibiotic were the major variables. These findings are not new, and the literature does support the claims of our study respondents. Nepal and Bhatta in their systematic review highlighted the high prevalence of self-medication of antibiotics in the WHO Southeast Asian Region and this hence was the leading cause of antibiotic resistance . SimilarBecause of the scarcity of information, a qualitative study design  was the ultimate choice. Being amenable, qualitative methods can extract attitudes, experiences, and intentions that are often missed in a quantitative phase ,45. BecaThe research was conducted at the Surgical Department of Sandeman Provincial Hospital, Quetta (SPHQ). Centrally located, SPHQ is a tertiary care hospital and provides generalized healthcare facilities. Additionally, being a public institute, SPHQ is a choice for most of the population .Hospital pharmacists stationed and practicing at the surgical unit and consenting to participate in the study were approached for data collection. Based on our objective, it was apparent to adopt the purposive sampling method . PharmacWe constructed a semi-structured interview guide after an extensive literature review ,53,54,55The guide was constructed in the English language and was subjected to face and content validity through a panel of experts (senior pharmacists). Once the validity was ensured, the guide was piloted with four pharmacists to ensure that the topics to be discussed were at the level that respondents would comprehend with ease. The preliminary data and conclusion confirmed that the discussion topics were enough and appropriately phrased to answer research questions and minimize validity and reliability threats. As the validity and reliability of the discussion guide were ensured, it was made available for the main study. Data and participants of the pilot study were not included in the final analysis.ZI  and FS  conducted the interviews. FS was also involved in carrying out field notes during the interviews.Interviews were conducted at the pharmacist’s office in the surgical unit. All participants were briefed about the study objectives before the interviews. A debriefing session was again conducted at the end of the discussion. The interviews started with an ice-breaking session. Probing questions were asked in between conversations to clarify the meanings of responses and to gain insight into the topic being discussed.The phenomenology-based approach commissioned in-depth, face-to-face interviews. All interviews were audio-taped followed by transcribed verbatim and were then analyzed for thematic contents by the standard content analysis framework. Each interview lasted for approximately half an hour. To draw in-depth views, the freedom to express additional reviews and comments was given. Interviews were conducted until thematic saturation was reached ,60. No r® was used for coding and analysis through iterations [The research team analyzed the recordings (verbatim) and later arranged an informal gathering where pharmacist were presented with the finalized interview scripts . They weerations  and incoThe current study managed to identify an adequate understanding of antibiotic use and resistance among hospital pharmacists. Additionally, prospective concerns (limited availability of antibiotics) and possible predictors of antibiotic resistance  were also highlighted factors contributing factors were also identified. As corrective measures, respondents of the current study focused on strict legislation to overcome the free availability and sales of antibiotics at the community level, ensuring the implementation of guidelines for the prescribers and copious availability of antibiotics and sensitivity testing facilities at the healthcare institutes. Therefore, we urge that these findings must be disseminated to policymakers and prescribers to take restorative action as soon as possible. Antibiotic resistance is a global threat, and we need a holistic approach to tackle this issue for generations while combating infectious diseases with precision.Qualitative methods have their limitations. Although we ensured saturation, the convenience sampling approach does not offer views of the participants that are not interviewed. Likewise, generalizability is always an issue with qualitative methods. However, with rich data extraction, we are confident that the study has provided baseline information for prospective studies. Therefore, we are recommending an in-depth and detailed study (quantitative) focusing on a large cohort of hospital pharmacists to ensure the validity of the current findings.


This paper presents an experimental study on the compressive performance of longitudinal steel-fiber-reinforced polymer composite bars (SFCBs) in concrete cylinders confined by different type of fiber-reinforced polymer (FRP) composites. Three types of concrete cylinders reinforced with (or without) longitudinal SFCBs and different transverse FRP confinements were tested under monotonic compression. The results showed that the post-yield stiffness of SFCBs is higher when confined with high elastic modulus carbon fiber-reinforced polymer (CFRP) composite than with low elastic modulus basalt fiber-reinforced polymer (BFRP) composite. Decreasing confinement spacing did not significantly improve the compressive strength of SFCBs in concrete cylinders. The compressive failure strain of SFCBs could possibly reach 88% of its tensile peak strain in concrete cylinders confined by CFRP sheets, which is significantly higher than the value (around 50%) in previous studies. Existing design equations, which applied a strength reduction factor or a maximum compressive strain of concrete to consider the compressive contributions of SFCBs in concrete members, underestimate the load-carrying capacity of SFCB-reinforced concrete cylinders. The design equation that considers the actual compressive stress of SFCBs gives the most accurate prediction; however, its applicability and accuracy need to be verified with more experimental data. Modern performance-based seismic design (PBSD) requires structures to be repairable after certain levels of earthquake ,2,3. HowHowever, a defect of FRP composites is that their compressive behavior is usually considered to be lower than their tensile strength . This liThis paper presents an experimental study on the compressive performance of longitudinal SFCBs in concrete cylinders confined with different type of FRP composites. Three types of concrete cylinders reinforced with (or without) longitudinal SFCBs and different types of transverse FRP confinements were designed and tested under monotonic compression. The failure process of the test cylinders was discussed, as well as their stress–strain curves. The compressive strength and possible failure strain of SFCBs were thoroughly analyzed and discussed. Three types of existing design equations  were evaluated with the test results in this paper.The experiment investigated the effect of different FRP composites (used as confinements) on the compressive performance of longitudinal steel-fiber-reinforced concrete (SFCB) cylinders. Three types of concrete cylinders were designed: (1) plain concrete cylinders (C); (2) concrete cylinders reinforced with longitudinal SFCBs and transverse FRP composites, i.e., SFCB and CFRP sheet-reinforced cylinders (SF-CFs) and SFCB and CFRP strip-reinforced cylinders (SF-CDs); and (3) concrete cylinders confined with only transverse FRP composite, i.e., CFRP sheet-confined cylinders (CFs) and CFRP strip-confined cylinder (CDs). The CF and CD cylinders were used as control cylinders for the SF-CF and SF-CD cylinders, respectively. The concrete cylinder was 300 mm high and had a cross-sectional diameter of 150 mm. The thickness of the concrete cover was 15 mm. For comparison, two previously tested concrete cylinders  were alslρ is the longitudinal reinforcement ratio; lE is the transverse confinement stiffness as in Equation (1), in which fE is the elastic modulus of FRP composites; fρ is the transverse reinforcement ratio as in Equation (2), in which ft is the thickness of the CFRP sheet/strip (=0.167 mm); fb is the widths of the CFRP sheet (=300 mm) and CFRP strip (=36 mm); D is the diameter of the concrete column (=150 mm); fs is the spacing of the CFRP strip (=18 mm); ss1A is the cross-sectional area of the BFRP spiral (=50.2 mm2); cord is the diameter of the concrete core of the cylinder (=120 mm); s is the spacing of the BFRP spiral (=23 mm); and lf is the transverse confinement strength as in Equation (3), in which ff is the tensile strength of FRP composites. The negative sign in Equation (3) indicates that it is in a state of passive constraint, which is opposite of the direction of lateral expansion.The design details of all cylinders are shown in For the SF-CF, SF-CD, and SF-BS cylinders, four longitudinal SFCBs were embedded in each cylinder, as shown in D refers to the diameter; eqD refers to the equivalent diameter of the SFCB, which is calculated based on the principle of equivalent flexural stiffness  of SFCBs tested in different conditions is shown in A comparison of the compressive peak stress ratio [i.e., ratio of compressive peak stress to tensile strength  could be achieved in the test. However, due to the lack of sufficient experimental data, the actual compressive peak stress of the SFCB confined in high-stiffness FRP composites, as well as the factors affecting it, remain unknown.The design method for the load-carrying capacity of SFCB-reinforced concrete cylinders is crucial to realizing a precise and cost-effective design of SFCB-reinforced concrete structures. However, it has not been studied before. Since the elastic failure mode (determined by the outer fiber layer failure) of a SFCB is similar to that of FRP reinforcement in concrete cylinders, the design equations for predicting the load-carrying capacity of FRP-reinforced concrete cylinders can be used to evaluate the load-carrying capacity of SFCB-reinforced concrete cylinders.There are generally three types of design equations for evaluating the load-carrying capacity of FRP-reinforced concrete cylinders, considering the variable compressive contributions of the longitudinal reinforcements for the concrete cylinders:Method I: Considering the contribution of longitudinal reinforcement with a strength-reduction factor.preP is the predicted load-carrying capacity of the reinforced concrete cylinder; α1 is the capacity reduction coefficient of the column; cf′ is the compressive strength of the confined concrete; gA is the cross-sectional area of the column; and sfα is the strength reduction coefficient of the longitudinal reinforcement.Tobbi et al.  set the Afifi et al.  conducteMethod II: Considering the contribution of longitudinal reinforcement with a maximum compressive strain.Mohamed et al. , Hadi etMethod III: Considering the contribution of longitudinal reinforcement with its actual compressive strength in concrete cylinders obtained through tests.ccf′ is the compressive strength of concrete with only transverse reinforcement.Tang et al.  proposedcf′ used in this paper is not the compressive strength of plain concrete, but the compressive stress of the CF/CD/BS cylinders that corresponds to the failure strain of SFCBs in the SF-CF/SF-CD/SF-BS cylinders, respectively. This is done to offset the confinement concrete effect. The predicted-to-experimental ratios of different design equations are shown in expP is the load-carrying capacity of reinforced concrete cylinders from experiments.Note that the value of The predicted-to-experimental ratios of Methods I and II (0.80~0.96) are generally lower than 1, which indicates that Methods I and II underestimated the load-carrying capacity of SFCB-reinforced concrete cylinders. This is potentially due to the fact that the combination of longitudinal SFCB and transverse FRP composite restraint exerts stronger confinement on concrete in SF-CF/SF-CD/SF-BS cylinders than in the case of only transverse FRP composite restraint in CF/CD/BS cylinders. The actual compressive contribution of concrete for SF-CF/SF-CD/SF-BS cylinders may be larger than that of the corresponding CF/CD/BS cylinders. Therefore, the compressive contribution of concrete in SF-CF/SF-CD/SF-BS cylinders may be underestimated when the compressive strength of concrete in CF/CD/BS cylinders is predicted. This conclusion can be further supported by comparing the predicted-to-experimental ratios of SF-CF cylinders to those of SF-CD cylinders. The predicted-to-experimental ratios of SF-CF cylinders vary from 0.80 to 0.88, which are generally lower than those of SF-CD cylinders, which vary from 0.89 to 0.94. This is because the relatively strong confinement effect for the concrete in SF-CF cylinders generates more errors in the estimation of the compressive contribution of concrete than those in SF-CD cylinders.The average predicted-to-experimental ratio of the two equations in Method I is 0.90, which is higher than the average predicted-to-experimental ratio of 0.84 for the four equations in Method II. This finding is consistent with the results of previous studies  on FRP-rMethod III provides the most accurate prediction of the load-carrying capacity of SFCB-reinforced concrete cylinders, with an average predicted-to-experimental ratio of 0.96. This is likely due to the fact that Method III considers the actual compressive strength of SFCBs in concrete cylinders. However, this method requires the compressive strength of SFCBs to be obtained in advance. Therefore, further studies are required on the compressive behavior of different SFCBs embedded in concrete cylinders to improve its applicability and accuracy.The post-yield stiffness of the confined SFCBs developed to become relatively higher when it was confined with a relatively high elastic modulus CFRP composite than when it was confined with a relatively low elastic modulus of BFRP composite.The compressive failure strain of the SFCB in the SF-CF cylinder could have reached 88% of its tensile peak strain, which indicates that a relatively high utilization of the material strength may be achieved for the SFCB restrained with relatively high elastic modulus confinements.The design equations that consider the compressive contribution of SFCBs in concrete cylinders with a strength reduction factor of the SFCB (in Method I) or a maximum compressive strain of concrete (in Method II) generally underestimated the load-carrying capacity of SFCB-reinforced concrete cylinders. In addition, this underestimation appears more significant for the cylinder with a relatively strong confinement.The approaching method, which applies an actual compressive strength of the SFCB for considering its compressive contribution in concrete cylinders (in Method III), gave the most accurate prediction for the load-carrying capacity of SFCB-reinforced concrete cylinders. However, its applicability and accuracy need to be verified with more experimental data in the future.The compressive performance of SFCBs confined by different type of FRP composites in concrete cylinders were experimentally studied in this paper. The following conclusions can be drawn:


In terms of prophylactic and therapeutic antibiotic rational use, respectively, we evaluated implementation conditions, the correlation between implementation and corresponding disease progress, and the cost-effectiveness of China’s antibiotic stewardship. For perioperative prophylactic antibiotic use, antibiotic stewardship was found to have been well-implemented, cost-effective, and reduced the incidence of surgical site infection. However, concerning therapeutic use and antibiotic-resistant bacterial infection prophylaxis, the complexity of influencing factors and the contradiction between stewardship implementation and clinical demand needs to be further evaluated.Antibiotic stewardship has been prioritized by governments and health care organizations in recent years as antibiotic resistance is markedly increasing. A tertiary hospital in Guangzhou, China was chosen as a study example to undertake an implementation and effectiveness evaluation of China’s antibiotic stewardship to improve and promote antimicrobial stewardship nationwide. The general surgery department of the study hospital was utilized to examine surgical site infection, and samples from across the hospital were used to identify bloodstream infection. Data was analyzed using descriptive analysis, the Mann–Kendall trend test, logit model and panel data model, and  Bacterial infection is a global issue, increasingly drawing the attention of healthcare workers, administrations, and organizations . During Governments and health organizations worldwide have developed stewardship guidelines for the standardization of antibiotic use and to prevent drug-resistant bacterial infections. High-income countries’ antibiotic stewardship, mainly focus on: 1) education for healthcare providers concerning microbiology and antibiotics; (2) promotion of antibiotic stewardship in all health care settings; (3) education in antibiotic stewardship for the general public and healthcare providers; 4) establishment of surveillance systems and databases for antibiotic use and antibiotic resistance; and 5) promotion of further research in antibiotic resistance and stewardship [ establis promotio educatioIn China, the policy of improving the management of the clinical application of antibiotics was released by the General Office of the Bureau of Medical Administration and Hospital Authority in 2008. Since then, clinical antibiotic use in China has entered a new era of stewardship management. As opposed to antibiotic stewardship in most other countries, in China, national antibiotic stewardship is mandatory at all levels of the healthcare system. Antibiotic stewardship in China mainly involves three aspects: (1) guidelines and supervision regarding the use of antibiotics based on the rate of drug resistance; (2) regulation of prophylactic antibiotic use during the perioperative period; and (3) use of a drug grading system for the clinical use of antibiotics .In the first aspect, the Ministry of Health of the People’s Republic of China has published several versions of the Guiding Principles for Clinical Application of Antibiotics and has developed monitoring systems for the rational use of antibiotics at all levels of the healthcare system.The second aspect, the rational use of perioperative prophylactic antibiotics, concerns surgical procedures with clean incisions and clean-contaminated incisions. For clean incisions, antibiotics cannot be used during the perioperative period for prophylactic purposes, otherwise, it would be considered irrational antibiotic use. For clean-contaminated incisions, rational use of antibiotics can be separated into drug appropriateness and time appropriateness. Drug appropriateness refers to surgeons use of prophylactic antibiotics according to the common bacteria that cause particular site infections. For time appropriateness, preoperative antibiotic administration should begin 0.5–1 h before the start of surgery. Any antibiotics used during the perioperative period for prophylactic purposes beyond this regulation would be considered irrational use. Administration of postoperative antibiotics should be stopped within 24–48 h after surgery ,11.For the third aspect, the antibiotic grading system contains three grades. First is the unrestricted grade, which includes antibiotics that are cheap with good efficacy and fewer side effects, such as cefuroxime zinc and ceftriaxone. Unrestricted grade antibiotics can be prescribed by all-grade doctors. Second is the restricted grade, which contains antibiotics that have good efficacy but are expensive or have limitations regarding side effects or drug resistance, such as cefoperazone–sulbactam and piperacillin–tazobactam that can be prescribed by doctors more senior than resident doctors. Third are special-grade antibiotics, which have good efficacy but are expensive and work for special drug-resistant bacteria, and require usage restrictions for avoiding drug resistance production, such as imipenem, meropenem, and vancomycin, which can only be prescribed under the directions of both chief doctors and antibiotic experts .This system of mandatory antimicrobial stewardship has been followed in China for more than 10 years and has been revised several times during this period. However, comprehensive evaluation is lacking to improve and promote antimicrobial stewardship. In comparison, evaluation reports in high-income countries mostly focus on the implementation of antimicrobial stewardship, its influencing factors, and the evaluation of cost and effectiveness ,13,14. IThe rate of rational use perioperative prophylactic antibiotics in the general surgery department of FAH, SYSU increased each year, from 32.95% in 2013 to 60.19% in 2018. The increasing trend in the first three years was rapid and then slowed in subsequent years .E. coli (corresponding CRO-R-ECO in our sample), third-generation cephalosporin-resistant Klebsiella pneumoniae (corresponding CRO-R-KPN), MRSA, and VREA decreased significantly. The detection rate of carbapenem-resistant Klebsiella pneumoniae  increased significantly. The detection rates of carbapenem-resistant E. coli  and CR-ABA (corresponding IMP-R-ABA) demonstrated no significant changes.We assessed trends in the detection rate of multiple antibiotic-resistant bacteria among patients with bloodstream infection at FAH, SYSU from 2013 to 2018. A comparison with nationally reported trends in detection rates for multiple antibiotic-resistant bacteria between 2014 and 2018 is shown in Trends in drug use intensity for each grade of antibiotics from 2013 to 2018 at FAH, SYSU are shown in From 2013 to 2018, the rate of surgical site infection in the general surgery department of FAH, SYSU decreased with an increased rate of rational perioperative prophylactic antibiotic use .After screening all variables with a logit model independently, we included the main study point of perioperative prophylactic antibiotic rational use, and smoking, alcohol consumption, and hospitalization duration in the logit model and panel model analysis as explanatory variables. In logit model analysis of the entire general surgery department, we found that in patients with rational prophylactic antibiotic use, the rate of surgical site infection was lower, and was 0.383 times that of patients with irrational prophylactic antibiotic use. We found that smoking and hospitalization duration were positively correlated with surgical site infection, whereas alcohol consumption and rational use of perioperative prophylactic antibiotics were negatively correlated with surgical site infection.We also conducted a subgroup analysis according to surgical site. For clean-incision surgical sites, such as in surgeries of the thyroid or mammary gland, we found that rational use of perioperative prophylactic antibiotics was a main influencing factor that protected against surgical site infection. However, for surgical sites with clean-contaminated incisions, such as in surgeries of the upper and lower gastrointestinal tract, liver, and biliary system, the duration of hospitalization was the main influencing factor; longer hospitalization duration was related to higher rate of surgical site infection. As for panel model analysis, when considering the impact of time and surgical site, hospitalization duration was the only significant influencing factor.From 2013 to 2018, the detection rate for multiple antibiotic-resistant bacteria in patients with bloodstream infection at FAH, SYSU decreased with a decreased ratio of antibiotic use duration to hospitalization duration. No significant correlation was found between the detection rate and drug use intensity of the corresponding antibiotics .In the logit model and the panel model analysis, the main study points include: total hospital drug use intensity; the ratio of antibiotic use duration/hospitalization duration; patient demographic characteristics; hospital department; and common influencing factors of bloodstream infection, such as neutrophil deficiency and vascular catheter use were included as variables in screening. Different variables were included in the analysis for different bacteria. In the logit model analysis, among all factors, the ratio of antibiotic use duration/hospitalization duration was the most common factor positively correlated with most bacteria investigated in this study. Regarding total hospital drug use intensity for the corresponding antibiotics, we found no relationship with the rate of detection of multiple antibiotic-resistant bacteria. Further panel model analysis confirmed this result.t-tests, patients with irrational antibiotic use had greater total and single day hospitalization costs, hospitalization medicine costs, and hospitalization antibiotics costs. On average, with rational antibiotic use per patient, CNY 578.33, 480.72, and 131.72 could be saved for the costs of single day hospitalization, medicines during hospitalization, and antibiotics during hospitalization, respectively, compared to one patient treated with irrational antibiotic use.The results of the cost comparison between perioperative prophylactic antibiotic rational use and irrational use are shown in t-tests demonstrate that patients with an antibiotic use duration/hospitalization duration ratio ≤0.9 had lower total and single day hospitalization costs, hospitalization medicine costs, and hospitalization antibiotics costs per patient. Furthermore, in patients with an antibiotic use duration/hospitalization duration ratio ≤0.9, CNY 338.12–4203.06, CNY 380.63–1993.10, and CNY 329.25–1085.32 were saved in costs of single day hospitalization, and medicines and antibiotics given during hospitalization, respectively ; extended-spectrum β-lactamase-producing gram-negative bacteria (ESBLs), including ceftriaxone-resistant Escherichia coli (CRO-R-ECO) and ceftriaxone-resistant Klebsiella pneumoniae (CRO-R-KPN); vancomycin-resistant enterococci, including vancomycin-resistant Enterococcus faecium (VREM) and vancomycin-resistant Enterococcus faecalis (VREA); carbapenem-resistant Enterobacteriaceae, including imipenem-resistant E. coli (IMP-R-ECO) and imipenem-resistant Klebsiella pneumoniae (IMP-R-KPN); and carbapenem-resistant Acinetobacter baumannii (CR-ABA), including imipenem-resistant Acinetobacter baumannii (IMP-R-ABA). [This study was a retrospective observational sample evaluation focused on FAH, SYSU during the period 2013–2018. This evaluation comprised three parts: (1) evaluation of antibiotic stewardship implementation; (2) evaluation of the relationship between antibiotic stewardship implementation and disease progress; and (3) evaluation of cost-effectiveness. According to the main contents of China’s antibiotic stewardship, we focused on two aspects in each of the above parts of the study: rational use of prophylactic antibiotics during the perioperative period and antibiotic rational use in the treatment of infectious disease. Considering the integrity of medical records in FAH, SYSU, surgical site infection in the general surgery department was chosen as the sample for evaluation of perioperative prophylactic antibiotic use. As a type of hospital-acquired infection in sterile tissue, we chose bloodstream infection to investigate the rational use of antibiotics for infectious disease treatment. Among the seven most common multidrug-resistant bacteria in China, we included methicillin-resistant -R-ABA). After exclusion of patients with missing data, study inclusion patients are shown in As mentioned, this study comprised three parts. First, in the evaluation of antibiotic stewardship implementation, we investigated the rational use of perioperative prophylactic antibiotics, the monitoring of multiple antibiotic-resistant bacteria, and the use of common antibiotics. In examining the relationship between stewardship implementation and disease progress, we focused on perioperative prophylactic antibiotic rational use and surgical site infection, as well as clinical use of antibiotics and bloodstream infection owing to multiple antibiotic-resistant bacteria. Finally, we conducted a cost-effectiveness evaluation in terms of perioperative prophylactic antibiotic rational use, as well as the rational use of antibiotics in patients with a bloodstream infection.Most data were collected from the electronic clinical database in the information data center of FAH, SYSU. We included the clinical data of patients admitted into the general surgery department, hospitalized patients with a bloodstream infection caused by multiple antibiotic-resistant bacteria or the corresponding antibiotic-sensitive bacteria, and data of antibiotic-use intensity  in each hospital department at each antibiotic grade of FAH, SYSU between 2013 and 2018. Detection data of antibiotic-resistant bacteria were collected from the monitoring system in the microbiology unit of the medical laboratory department.t-tests. Data integration and data analysis were performed using Stata SE 15.1 for Mac .After formatting the original data, all data were sorted into binary, categorical, and continuous variables. Among these variables, in the evaluation of perioperative prophylactic antibiotic rational use, we chose the main study point, perioperative prophylactic antibiotic use, as well as patients’ demographic characteristics, the common influencing factors for surgical site infection, such as smoking, alcohol consumption, and duration of hospitalization, as the screening explanatory variables. As for the rational use of antibiotics in the treatment of the patients with bloodstream infection, we chose the main study point, total hospital drug use intensity and the ratio of antibiotics use duration/hospitalization duration, as well as patients’ demographic characteristics, department in which patient is hospitalized, and common influencing factors of bloodstream infection, such as neutrophil deficiency and vascular catheter use, as the screening explanatory variables. For data analysis, descriptive analysis and Mann–Kendall trend tests were used to evaluate antibiotic stewardship implementation. We used a logit model and a panel data model to evaluate the relationship between stewardship implementation and disease progress. In this area, we carried out subgroup analysis in a logit model analysis of relativity of the perioperative prophylactic antibiotic use. The subgroup was categorized by different surgical sites, such as surgeries of the thyroid gland, mammary gland, the upper and lower gastrointestinal tract, liver, and biliary system. We conducted the cost-effectiveness analysis using Our findings indicate that at FAH, SYSU, antibiotic stewardship for perioperative prophylactic antibiotic use was effectively implemented, reduced the incidence of surgical site infection, and lowered costs. However, for therapeutic antibiotic use and infection prophylaxis for antibiotic-resistant bacteria, the complexity of influencing factors and contradiction between stewardship implementation and clinical demand should be further evaluated. Our evaluation can serve as a reference for other healthcare organizations in future large-scale studies of antimicrobial stewardship implementation, effectiveness, and assessment.


Prominent infiltration of lymphocytes with distinct compositions were found in different organs of IgG4-RD patients. Transcription factors (TFs), including PRDM1/XBP1 and RUNX3, were upregulated in IgG4-RD, promoting the differentiation of plasmablasts and CTLs, respectively. Monocytes in IgG4-RD have stronger expression of genes related to cell adhesion and chemotaxis, which may give rise to profibrotic macrophages in lesions. The gene activation pattern in peripheral immune cells indicated activation of multiple interaction pathways between cell types, in part through chemokines or growth factors and their receptors. Specific upregulation of TFs and expansion of plasmablasts and CTLs may be involved in the pathogenesis of IgG4-RD, and each of these populations are candidate targets for therapeutic interventions in this disease.IgG4-related disease (IgG4-RD) is a systemic autoimmune disease with unclear pathogenesis. We performed single-cell RNA-seq and surface proteome analyses on 61,379 PBMCs from 9 treatment-naive IgG4-RD patients and 7 age- and sex-matched healthy controls. Integrative analyses were performed for altered gene expression in IgG4-RD, and flow cytometry and immunofluorescence were used for validation. We observed expansion of plasmablasts with enhanced protein processing and activation, which correlated with the number of involved organs in IgG4-RD. Increased proportions of CD4 Affecting approximately 0.28–1.08/100,000 individuals, IgG4-RD is a newly recognized rare disease (IgG4-related disease (IgG4-RD) is an insidiously progressive inflammatory and fibrosing condition characterized by tumefactive lesions, dense lymphoplasmacytic infiltrates, and abundant IgG4 disease . The dis+CD27+CD20–CD38hi plasmablasts are significantly clonally expanded, with extensive somatic mutation (+SLAMF7+ cytotoxic T lymphocytes (CTLs) and granzyme A–expressing CD8+ CTLs were the dominant infiltrating T cells, and circulating CD4+ CTLs decreased with improvement of symptoms , to characterize the cell composition, gene signature, developmental trajectories, and regulators of immune cells in peripheral blood (PB) from patients with IgG4-RD.https://doi.org/10.1172/jci.insight.167602DS1). After quality control and filtering, 61,379 cells, including 21,851 from HCs and 39,528 from patients, were obtained (+ monocytes (MNs) (LYZ+CD14+), CD16+ MNs (LYZ+FCGR3A+), DCs (CD1C+), CD4+ T cells (CD4+), CD8+ T cells (CD8A+), proliferating lymphocytes (MKI67+), natural killer (NK) cells (KLRF1+), B cells (CD19+), and platelets (PPBP+)  (obtained . Nine diobtained . These s (PPBP+) . Of notete trend , which ws) LYZ+CD+, CD16+ + plasma cells found in IgG4-RD lesions and their critical role in the pathogenesis, a more precise and detailed understanding of the features of B cells in circulation is essential. We identified 5 transcriptionally distinct B cell subsets, including naive B (MS4A1+IGHD+TCL1A+), memory B (MS4A1+CD27+), intermediate memory B (IGHD+CD27+), plasmablast (MZB1+), and dividing plasmablasts (MZB1+MKI67+) (Given the rise of IgG4+MKI67+) , A–C. No+MKI67+) , suggest+MKI67+) . Proport+MKI67+) . This fi+MKI67+) , and ind+ T cells in IgG4-RD (Gene Ontology (GO) analysis showed that genes involved in protein processing in endoplasmic reticulum (ER), ER stress response, and protein folding were significantly upregulated, suggesting enhanced protein synthesis during related processes in IgG4-RD . This ob IgG4-RD .IGHG4, IGHG1, and IGHE were most highly upregulated in IgG4-RD  and a proliferation-inducing ligand , are survival factors for B cells that also control B cell maturation (TNFRSF13B), BAFF receptor (BAFFR/TNFRSF13C), and B cell maturation antigen (BCMA/TNFRSF17) are closely related members of the TNF receptor superfamily and bind BAFF and APRIL , 3 subtypes of CD8+ T (CD3E+CD8A+), γδT (CD3E+CD4–CD8A–TRDC+), mucosal-associated invariant T (MAIT) cells (CD3E+SLC4A10+), proliferating lymphocytes (CD3E+MKI67+), double-negative T , and T/MN doublets , memory CD4+ T cell (GPR183+), effector memory CD4+ T cell (GPR183+KLRB1+), and regulatory T (Treg) cell (IL21RA+FOXP3+) subtypes, we defined 2 activated CD4+ T subtypes, CD4+ CTL (GZMA+ ZNF683+) and IFN-stimulated CD4+ T (IFI6+). The CD4+ CTL cluster was characterized by high expression of genes associated with cytotoxicity, including GNLY and GZMA (+ T cell included a naive CD8+ T subset (CCR7+ SELL+) and 2 effector CD8+ T subsets (CD8+ CTL-GZMK and CD8+ CTL-GNLY), which had high expression levels of GZMA and NKG7. In detail, CD8+ CTL-GZMK uniquely expressed GZMK, whereas CD8+ CTL-GNLY showed relatively high expression levels of GZMB, GZMH, and GNLY  cells was found to be specifically increased in IgG4-RD and was correlated with serum IgG4 levels and plasmablast counts (–CD4+PDCD1+CXCR5+CXCR3–CCR6–) than HCs did (8.3%) (P < 0.001), but not a significant elevation of Tfh cells (CD45RA–CD4+PDCD1+CXCR5+) (18.0%) compared with HCs (16.8%) (P > 0.05) (IL21 and CXCL13 mRNA in these circulating Tfh cells were very low in this data set (data not shown). Treg cytokines such as IL-10 and TGF-β are proposed to be involved in the class switching of plasma cells and fibrosis in IgG4-RD. We found that the proportion of Treg cells was comparable between IgG4-RD and HCs; however, the expression of FOXP3 was higher in Treg cells in IgG4-RD (Follicular helper T cells (Tfh) are a special subset of CD4t counts . Throught counts , IgG4-RD > 0.05) . However IgG4-RD , which i IgG4-RD . The RNAMoreover, GSEA showed that pathways related to the defense response to Gram-negative bacteria and regulation of B cell differentiation were upregulated in T cell genes in IgG4-RD , suggest+ T cells , nonclassical MNs , intermediate MNs , 2 DC subsets — DC (CD1C+) and pDC (LILRA4+) — and NK cells (NKG7+)  . We furt (NKG7+) . To expl (NKG7+) . However (NKG7+) . In cont (NKG7+) .RETN, SH3BGRL3, S100A9, EMP3, and CCL4 were significantly upregulated, while CD163 and some glycolysis-related genes were downregulated in IgG4-RD MNs compared with HCs (+CD163– cells among CD14+CD16– MNs was found to be associated with lung fibrosis (RETN) is a proinflammatory, profibrotic cytokine that induces fibroblast-myofibroblast differentiation (NAMPT) has opposite effects (RETN was upregulated in MNs (predominantly expressed in CMs) from IgG4-RD, and serum resistin was elevated in IgG4-RD patients  according to a standard protocol  within 4 hours (cell viability >93%). All granulocytes were depleted. PBMCs were labeled with 9 antibodies conjugated with polyadenylated and antibody-specific barcodes . The BD protocol . RNA expprotocol  and blooprotocol  from IgGPBMCs were stimulated with PMA  and ionomycin  in the presence of protein transport inhibitor cocktail (Thermo Fisher Scientific) for 6 hours. Cells were collected and stained with BV510-conjugated anti-CD3 , APC-Cy7–conjugated anti-CD4 , PerCP-Cy5.5–conjugated anti-CD8 , Alexa Fluor 647–conjugated anti-CD319 , and BV421-conjugated γδTCR antibody  for surface markers. Cells were fixed and permeabilized before staining with Alexa Fluor 488–conjugated anti-GNLY  and PE-conjugated anti-GZMA . After 3 washes, cells were measured using a BD flow cytometer. For MNs, cells were measured after staining with APC-Cy7–anti-CD14 , BV510–anti-CD16 , BV421–anti-HLA-DR , APC–anti-CCR1 , and PE–anti-CX3CR1 . All antibodies except anti-CCR1 and anti-CX3CR1 were purchased from BD Biosciences.Formalin-fixed lymph nodes, salivary glands, pancreas, and lacrimal glands biopsies from IgG4-RD patients were paraffin-embedded. Slides were deparaffinized in xylene and antigens were retrieved. Then, the sections were stained with anti-CD4 , anti-CD8 , anti-GNLY , and anti-γδTCR  according to the manufacturer’s protocol  and then were screened and analyzed with the Vectra Polaris Automated Quantitative Pathology Imaging system (PerkinElmer). For CCL5 and CX3CL1 staining, antibodies against CCL5  and CX3CL1  were used as primary antibodies, while Alexa Fluor 647– and 488–conjugated anti-IgG  were used as secondary antibodies. Salivary gland biopsies from patients with mucocele as controls. The images were captured with a Leica microscope.Cytokines in the sera were measured using ELISA kits from CUSABIO (CSB-E06884h) according to the manufacturer’s instructions.t test, while skewed data were analyzed by 2-tailed Mann-Whitney test or Kruskal-Wallis test. For multiple tests, Kruskal-Wallis test followed by post hoc Dunn’s multiple-comparison test was performed. Comparison of proportions of Tfh in combined data was performed by χ2 test. The relationship between IgG4 concentration and percentage plasmablasts/dividing plasmablasts among B cells was explored by linear regression. Pearson’s or Spearman’s correlation was performed to calculate the correlation coefficients of 2 factors according to normality of distribution. Data analyses were performed with GraphPad Prism software version 7.0. A P value of less than 0.05 was considered significant. DEGs between patients and HCs were analyzed with Wilcoxon’s rank-sum test implemented in Seurat. A permutation test for the CellphoneDB analysis was used to evaluate the significance of a ligand/receptor pair.Cell proportions are expressed as mean ± SD. Normality test was conducted to determine whether the data are normally distributed or skewed. Normally distributed data were analyzed by 2-tailed Student’s This study was approved by the ethics committee of the West China Hospital of Sichuan University (no. 2020-759) and written informed consent was obtained from each participant prior participation.https://ngdc.cncb.ac.cn/gsa-humanThe data that support the findings of this study are available within the article and its CL, DAF, CT, and Y Luo designed the study design, interpreted data, and wrote the manuscript. CL, PQ, CT, XJ, YH, XZ, QH, and YZ collected clinical data and samples. CL, PQ, and QZ isolated PBMCs and performed flow cytometry. CL, ZT, TW, and CC stained tissue sections and performed histological examination. CL and SL conducted bioinformatic analyses. CT, Y Luo, and Y Liu supervised the whole project. All authors contributed to the manuscript and approved the submitted version. CL, SL, and PQ contributed equally. CL was listed first because he organized the data for development of the manuscript in addition to planning and leading the experiments.


Antimicrobial resistance (AMR) which has been ascribed to be due to community carriage of antibiotic-resistant bacteria is highly prevalent in the WHO South-East Asia region. One of the major reasons for this is the misuse of antibiotics in animal farming practices and at the community level, which threatens both human and animal health. However, this problem of antibiotic misuse in poultry farms and in respective farmers is not well studied in countries like Pakistan.We conducted a cross-sectional study in rural Punjab to explore the current practices of antibiotic use in poultry and poultry farmers, associated factors, their healthcare-seeking behavior and biosecurity practices.In the context of antibiotic use for poultry, 60% comprised of Colistin sulfate and Amoxicillin trihydrate whereas Colistin is considered as the last resort antibiotic. In addition, the significant consumption of antibiotics in poultry farms (60%) and poultry farmers (50%) was without prescription by either human health physicians or veterinarians. Most of the farms (85%) had no wastewater drainage system, which resulted in the direct shedding of poultry waste and antibiotic residue into the surrounding environment. The lack of farmers’ education, professional farm training and farming experience were the most significant factors associated with antibiotic use and knowledge of AMR.Our study findings show that it is necessary for an integrated AMR policy with the inclusion of all poultry farmers to be educated, a mass awareness program to be undertaken and that strict antibiotic usage guidelines be available to them. Such initiatives are also important to ensure food safety and farm biosecurity practices. As per WHO misuse of antibiotic referred to buy antibiotics for animal and human use without prescription, takin antibiotics for viral infections, e.g., cold, flu and using antibiotics for growth promotion on farms .Misuse of antibiotics in food-producing animal farming practices has become an inevitable challenge to the containment of global antimicrobial resistance (AMR) both in humans and in animals, particularly in low and middle-income countries (LMICs) . AMR hasAntibiotic use in food animals started in the 1940s when the use of tetracycline in animals resulted in improved growth . IntensiPakistan is among the top 10 countries that are producing food animals through modern farming practices and rely on antibiotics as growth promoters and for disease prevention . HoweverApart from the direct effect of antibiotic use on AMR development in humans and animals, an abundance of resistant pathogens in the environment and elevated environmental pressure of them, are also major transmission factors in such circumstances. AMR transmission to the environment occurs in different ways, e.g., dissemination of animal waste , uncontrolled grazing of animals, using organic fertilizer , and the fact that pharmaceutical companies and municipalities dump their waste and human waste in the environment –18. In mWhile the burden of AMR is high and difficult to quantify in LMIC settings, there are multiple challenges to mitigate against it . Adequatn = 40). The eligible participants were voluntarily agreed adult poultry farmers who provided their prior informed consent before data collection.We conducted a cross-sectional survey for poultry farms and poultry farmers in rural Punjab, Pakistan from January to March 2021. The Tehsil (sub-district) named Pindi Gheb from Attock district in Punjab was selected as our study area which is one of the more densely populated districts in Punjab with a large number of poultry farms . From TeData was collected using a validated and researcher administered questionnaire (which had been pre-tested in 4 non-study villages). Our questionnaire was adopted from a study conducted recently in North-western China regarding the use of antibiotics in poultry . For theAll statistical analyses were performed in an IBM SPSS Statistics version 28.0.1.1 (IBM Corp.) Descriptive statistic was used to analyze the characteristics of the study farms, demographics of farmers, and pattern of antibiotic use in poultry farms and farmers. Additionally, distributions of antibiotic misuse by the demographic characteristics and the education level of farmers were compared with the knowledge of farmers about antibiotics by cross-tabulation.p < 0.05.We also performed a chi-square test to check the association between the education level of participants and knowledge about antibiotics, AMR, and prohibited antibiotics in poultry. Statistical significance was considered at Our study variables include both dependent and independent variables. Dependent variable include purpose of antibiotics use while independent variables include education and professional farm training of farmers, knowledge of farmer about antibiotic resistance and prohibited antibiotics, physician prescription, veterinary doctor prescription, frequency of antibiotic, withdrawal time period follow up, method of disposing poultry waste.We performed the regression analysis  for these dependent and independent variables but there was no statistical significance association between variables.n = 25: 62.5%) had an experience of less than 15 years  completed their secondary education (10 years of education) and 10 (25.0%) had no formal education. Thirty-five participants (87.5%) never attended any professional farm training. Nineteen poultry farms (47.5%) included in this survey were medium scale broiler farms which had 2000–4,000 chickens per farm . The numn = 40: 100%), the major use of antibiotics as growth promoters , lack of compliance , and health care seeking from unqualified practitioners for antibiotics to a larger extent   reported the purchase of antibiotics from agents instead of pharmacy/drug stores. Agents act as a third party between the poultry farmers and feed/veterinary drug companies and supply feed and medicines to the poultry farms. Moreover, 45% (n = 18) of the respondents had received veterinary services from feed companies.Our current study reveals an extensive use of antibiotics in all farms  . All parn = 29, 72.5%) frequently used antibiotics. Half of the poultry farmers  did not follow withdrawal periods of the antibiotics. Interestingly, many of the farmers  used antibiotics for clinical conditions, which did not require antibiotics, such as flu, fungal infections, or malaise  used followed by Enrofloxacin, Tylosin and Doxycycline  respectively. Apart from antibiotics, other antimicrobials, e.g., antivirals (Amantadine HCl) and antifungal (Nystatin) were used by 25.0 and 2.5% of poultry farmers for the treatment of viral and fungal diseases.n = 6, 60.0% and n = 4, 80.0%), as compared to those having secondary level education  and above . Likewise, professionally trained farmers had not used antibiotics as growth promoters contrary to those having no professional farm training . The Chi-square test indicates the significant correlation between professional farm training and antibiotic use as a growth promoter (p = 0.05). While education level and antibiotic use as growth promoter had no significant correlation (p = 0.141).As illustrated in n = 5, 25.0%), had a rudimentary knowledge about antibiotic usage  had knowledge about prohibited antibiotics, and  about antibiotic usage. The majority  of the respondents having a higher secondary level of education or more, had some knowledge about antibiotic usage, while over half  had knowledge about prohibited antibiotics and  had knowledge about antibiotic resistance. There is a significant association between the education level of farmers and knowledge about antibiotic usage (p = 0.012) and prohibited antibiotics (p = 0.051).Again, the majority (90.0%) of the respondents who were not educated had no knowledge about antibiotic usage and prohibited antibiotics in poultry; and no farmer in this category had knowledge about antibiotic resistance . Farmersp < 0.05). Farmers having professional farm training (n = 5) have more knowledge about antibiotic usage  and prohibited antibiotics , while they had comparatively less knowledge about AMR . We have also observed that the number of years in farming has a direct relation to the knowledge about antibiotics. All farmers having more than 30 years in farming had enough knowledge, as compared to those having less experience in farming. Whereas more than 80% of the farmers had no idea about antibiotic usage, AMR, and prohibited antibiotics. Therefore, these variables have statistical significance (p < 0.05).Similarly, the correlation between professional farm training and knowledge of farmers about antibiotics was statistically significant  reported not having any wastewater drainage system in their farms; rather the poultry waste was being drained directly into adjacent open areas and agricultural land. Only 6 farms (15.0%) had proper drainage systems. Additionally, 24 (60.0%) farmers reported that they use poultry wastes as fertilizer, which is causing the further spread of AMR to the food system.n = 2, 5.0%), in the last 1–3 months, while  of the participants used antibiotics 6 months prior to the survey. Two-thirds  of the participants did not remember the last intake of antibiotics. About half  of the respondents reported self-medication of antibiotics without a physician’s prescription. Almost half of the participants  took previously used antibiotics without consulting a physician, while  used antibiotics after getting advice from relatives. One participant (2.5%) mentioned that he had no access to physicians, so he used antibiotics without prescription.Out of the 40 participants, more than one-third  used antibiotics within the last month preceding the survey (n = 30.75.0%), reported obtaining them from local pharmacies , from leftover antibiotics at home, and  obtained them from rural medical practitioners . Moreover (n = 28.70.0%) of the respondents used antibiotics for 1–3 days , used for 4–7 days, and  used for more than 7 days  of the participants mentioned use of antibiotics for treating flu/common cold , and about one-third  stated respiratory infections in general (where cough and chest pain were common symptoms)  .In the current study, we evaluated the practice of antibiotic use and healthcare-seeking behavior regarding poultry farming and farmers in rural areas of Punjab in Pakistan which is the first of this nature in the study area. While Health care seeking behavior is defined as any action taken by an individual who identifies themselves to be ill or having health-related issues for the purpose of finding an appropriate treatment .Our study findings confirm that the use of antibiotics in poultry is not well regulated in Pakistan, particularly in rural areas and it has the potential to enhance the emergence of drug resistant pathogens to develop AMR. Most of the participants used antibiotics as growth promoters without any consultation with trained veterinarians. This observation of the unregulated use of antibiotics in food-producing animals in Pakistan including improper dosage, wrong combination of antibiotics, misuse, and overuse is similar to other studies in similar settings , 24. In The majority of the participants in this study purchased antibiotics based on their previous experience and from local agents, which is a clear indication of a patron-client relationship and undue influence for unnecessary usage. Such resistance-provoking drug purchase behavior and practice is also evident in similar LMIC settings , 25–27. Another important concern of antibiotic use in poultry farming is the ‘Withdrawal period’. Any medicine or antibiotic consumed by humans or animals, has a withdrawal period when they become non-functional and eliminated from the body. The ‘Withdrawal period’ is particularly important for food animals such as poultry and cattle to ensure that no antibiotics have entered the human food chain. Unfortunately, nearly half of our participants were unaware of this term and so did not follow the recommended withdrawal period. This unhealthy practice increases the possibility of high levels of antibiotic residues in poultry meat with their detrimental health consequences. All these findings of violation of the withdrawal period for antibiotics have also been observed in other studies , 28.Antibiotic use in poultry and lack of proper biosecurity practices are major concerns in the environmental dissemination of antibiotic residues and resistant bacteria which in turn act as the mixing hub of human-animal superbugs. A majority of farms in the current study had no wastewater drainage system and wastewater was simply drained into nearby agricultural land or open sites near farms. This practice increases the chances of antibiotic contamination of agricultural land through raw and untreated wastewater . MoreoveIn terms of antibiotic use among poultry farmers in the study area, the easy accessibility of antibiotics from pharmacies/drug stores without doctor’s prescriptions is an important issue. One-third of the participants obtained antibiotics from pharmacies and self-medication is a common practice. People in LMIC settings have no idea about the risk of self-medication and they purchase antibiotics from drug stores without a physician’s prescription , 37, 38.Several studies have reported that patients understanding about illness and its treatment will increase their adherence to the medication , 44. In Knowledge about antibiotic resistance and antibiotic usage is a fundamental requirement to mitigate AMR at community level. A significant number of our study participants had no knowledge about these issues. Knowledge of the farmers about antibiotics was directly associated with their education level. Uneducated participants and those with a primary level of education had no or only a limited knowledge about antibiotic use, AMR, and the prohibited list of antibiotics in poultry as compared to those participants who had a secondary or higher level of education. These findings are consistent with other studies , 46, 47.While our study focuses on an imperative aspect of AMR in rural Pakistan, it has few limitations. The findings may not be generalized to the whole country as we collected data from a sub-district in Punjab. Yet, these results provide a descriptive picture of the overall situation of antibiotic use in rural Pakistan. Moreover, the findings of this study may also be affected by recall bias to some extent as participants had to remember the use of antibiotics and they have very minimal medicine-related knowledge. However, we tried to validate the findings by collecting and inspecting the antibiotic boxes from the farms and households.Our study highlights the risks of AMR due to non-professional farming practices and its hazards to humans, animals, and the environment. It furthermore emphasizes the need for education and professional farm training for the containment of AMR in resource-deficient settings. The current study also strongly supports the alignment of food safety policy with the current AMR mitigation plan. An integrated and sustainable national AMR and food safety policy needs to be adopted with the inclusion of farmers’ education, mass awareness, organic farming, and strict antibiotic usage guidelines.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was reviewed and approved by The Ethics Committee at Institute of Health and Society, UiO (Application no. 8849626), Norwegian Centre for Research Data (reference no. 726029) and the Bioethics Committee of faculty of biological sciences, Quaid-i-Azam University, Islamabad, Pakistan (reference no. #BEC-FBS-QAU2021-250). The participants provided their written informed consent to participate in this study.MA: conceptualization and project administration. MA and UH: methodology and writing – original draft preparation. UH and MA: formal analysis. AK: resources. UH: data curation. EM, IG, and AK: writing – review and editing. MA, AK, and EM: supervision. All authors have read and agreed to the published version of the manuscript.This research was funded by the Department of Community Medicine and Global Health, Institute of Health and Society, Faculty of Medicine, University of Oslo, Norway.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.