Development of antibiotic resistance, a threat to global health, is driven by inappropriate antibiotic usage. Respiratory tract infections (RTIs) are frequently treated empirically with antibiotics, despite the fact that a majority of the infections are caused by viruses. The purpose of this study was to determine the prevalence of antibiotic treatment in hospitalized adults with viral RTIs, and to investigate factors influencing the antibiotic decision-making. We conducted a retrospective observational study of patients ≥ 18 years, hospitalized in 2015–2018 with viral RTIs. Microbiological data were taken from the laboratory information system and information on antibiotic treatment drawn from the hospital records. To investigate decisions for prescribing antibiotic treatment, we evaluated relevant factors such as laboratory and radiological results, in addition to clinical signs. In 951 cases without secondary bacterial RTIs , 720 (76%) were prescribed antibiotic treatment, most frequently beta-lactamase-sensitive penicillins, but cephalosporins were prescribed as first-line in 16% of the cases. The median length of treatment (LOT) in the patients treated with antibiotics was seven days. Patients treated with antibiotics had an average of two days longer hospital stay compared to patients with no such treatment, but no difference in mortality was found. Our study revealed that there is still a role for antimicrobial stewardship to further improve antibiotic use in patients admitted for viral RTIs in a country with relatively low antibiotic consumption. Clostridioides difficile infections, as well as toxic, allergic and other complications. Frequent and prolonged antimicrobial therapy is associated with higher rates of adverse events and promotes antibiotic resistance development [The development of antibiotic resistance is an increasing global health threat fueled by inappropriate antibiotic usage within and outside health care institutions. The prevalence of multidrug-resistant bacteria has been reported as increasing and spreading worldwide ,2,3. Accelopment ,7,8.Norway is a low-incidence country with regards to the consumption of antibiotics both in the community and in the hospital sector compared to the EU/EEA average. However, resistance to broad-spectrum antibiotics such as third-generation cephalosporins has been increasing over the past ten years . PenicilInappropriate antibiotic use is driven largely by lack of adherence to guidelines supporting sound antibiotic prescription. In addition, there are shortcomings of appropriate tools for decision-making in antibiotic treatment that is most often initiated by using an empiric regimen to control infections rapidly before results from microbiological investigations are available. Delays in laboratory testing and access to such services can be part of the problem.Streptococcus pneumonia, Haemophilus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae and Gram-negative bacilli, among others [The most common viral agents in respiratory tract infections (RTIs) are the influenza virus, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinovirus, parainfluenza virus, adenovirus and coronavirus ,12. Bactg others ,14,15.Respiratory infections are often treated empirically with antibiotics in primary health care and in hospital settings. However, more widespread use of molecular microbiological tests has revealed that most respiratory tract infections (RTIs) are caused by viruses, and do not require antibiotic treatment ,16. NeveIn Norway, the standard care for adults requiring hospital admission due to viral RTIs  includesThe aim of our study was to determine the prevalence of antibiotic treatment in hospitalized adults with confirmed viral RTIs. In addition, we also investigated factors influencing the antibiotic decision-making in this large cohort of viral RTI patients from a regional general hospital.The study population consisted of patients with viral RTIs hospitalized 1222 times in total during the inclusion period .A total of 12 cases of hospital-acquired infections were excluded, as well as 33 infection events (IE) with respiratory virus only detected in the readmission event. From the remaining 1177 IEs, 226 patients who received antibiotics due to bacterial infection were excluded.During 2015–2018, 932 patients were hospitalized on 951 occasions due to viral RTIs. Of these 951 IEs, 720 received antibiotic treatment with no sign of bacterial infection and 231 did not receive antibiotic treatment during hospitalization.p = 0.037 (Wilcoxon rank sum test). The distribution over age groups was 3.6% in 18–29 years, 3.3% in 30–39 years, 5.8% in 40–49 years, 9.5% in 50–59 years, 17.4% in 60–69 years, 28.9% in 70–79 years, 25.4% in 80–89 years and finally 6.2% in the age group of ≥ 90 years.Of the total IEs, 378 (39.7%) were hospitalized with influenza A, 231 (24.3%) with influenza B, 131 (13.8%) with respiratory syncytial virus (RSV), 122 (12.8%) with human metapneumovirus (hMPV), 81 (8.5%) with parainfluenza virus and 8 patients (0.8%) with adenovirus. In the 951 infection events included, no significant difference was found concerning gender when comparing patients who received antibiotic treatment to patients who did not receive such treatment during hospitalization . The medp = 0.037 (Wilcoxon rank sum test)  were categorized as immunocompromised, either due to treatment or underlying condition. In the cohort, 2.3% of the patients were organ transplanted. Patients with COPD, immunosuppression and who were organ transplanted showed a significant difference with an overrepresentation in the antibiotic-treated group, with p < 0.001, p = 0.049 and p = 0.029, respectively (Pearson’s chi-square test).In the group who received antibiotic treatment, the median Charlson score was 2 (range 0–9), while the median Charlson score in patients who did not receive such treatment was significantly lower with a median of 1 (range 0–8), um test) . Concernp < 0.001, logistic regression analysis).The proportion with National Early Warning Score (NEWS-score) ≥ 5 points was significantly higher in the patients receiving antibiotics compared to those not treated .Both C-reactive protein (CRP) level >60 mg/L and white blood cell (WBC) count ≥ 11.1 10p < 0.001, Wilcoxon rank sum test). Furthermore, in linear regression including age, gender and Charlson score, the antibiotic treatment had a significant coefficient of 2.24 , as antibiotic treatment showed an increase in LOS with on average 2.24 days.The length of the hospital stay (LOS) varied between 0 days (exceeding 5 h) and 27 days, with a median of 4 days. The median LOS was significantly longer in patients on antibiotic treatment compared with non-treated, 4 days vs. 2 days, respectively . Antibiotic treatment was given to 83% of the deceased patients, 18 patients (34.0%) needed Intensive Care Unit (ICU) treatment during their hospital stay, 17 (32.1%) needed non-invasive ventilation and 3 patients (5.7%) required mechanical ventilation. Out of the deceased patients, 22 (41.5%) suffered from influenza A, 12 from influenza B (22.6%), seven from RSV (13.2%), eight from hMPV (15.1%) and four from parainfluenza (7.5%). No significant difference linked to antibiotic treatment status was observed in mortality during the hospital stay  or 30 days after discharge . In addition, antibiotic treatment was not associated with higher mortality, when adjusted for age, gender and Charlson score .A total of 30 patients (3.2%) died during the hospital stay, and a further 23 patients (2.5%) died during the 30 days following discharge. The median age of the deceased was 81 years (range 57–103 years), 29 females (54.7%). Of these, two patients suffered from metastatic cancer. The median Charlson score was found to be 3 (range 0–8), significantly higher than surviving patients (median 2 (range 0–8), Virological investigation of respiratory samples detected influenza A in 39.7% of cases, influenza B in 24.3%, RSV in 13.8%, hMPV in 12.8%, parainfluenza in 8.5% and only 0.8% adenovirus of the events .The time to results, calculated from the sampling time to the final laboratory report on the respiratory virus panel, was a median of 1 day (ranging from 0.3 days up to 4 days). The analysis was not performed during weekends and holidays.For 21 samples (2.2%), the turn-around time ranged from 5 to 17 days, with median of 7 days, due to delay in transport to the laboratory or technical problems. Of these 21 cases, 18 (86.0%) received antibiotic treatment; among these 18 cases, 5 had already discontinued antibiotics before the test result was returned. In four other cases, the antibacterial treatment was discontinued the same day or the day following the test result.During the hospital stay, 720 (75.7%) of the 951 cases were prescribed antibiotics. Antibiotic prescription ranged from 71.0% in the influenza B-positive group to 83.2% in the RSV group.In total, 15.0% of patients received antiviral treatment with oseltamivir, with no significant difference between the patients in the antibiotic-treated group and those who did not receive antibiotic treatment.Of the 720 cases treated with antibiotics, 383 (53.2%) received only one antibiotic agent. Of the 337 cases receiving two or more agents, 32.1% were treated with two agents, 11.5% with three agents, 1.8% with four agents, 1.3% with five agents and 0.1% with six agents.It is apparent from this table that most cases were prescribed with various types of penicillins, the most prevalent types ordained were beta-lactamase sensitive penicillins (46.7%) and extended-spectrum penicillins (42.8%), such as ampicillin and amoxicillin. Cephalosporins were prescribed in 23.1% of the IEs, and in 114 cases (15.8%), cephalosporins were prescribed as first-line treatment. Gentamicin was prescribed in 18.8%, but only in combination with penicillin.p = 0.003, Wilcoxon rank sum test), and more comorbidities with a higher Charlson score . They were more frequently in need of ICU treatment , and had a higher in-hospital mortality rate, 18 vs. 9 .Of the 720 patients who received antibiotic treatment, 291 patients (40.4%) received an antibiotic agent from the WHO Watch category . The patp < 0.001). In addition, patients who received a Watch agent had on average a 1.67 days longer hospital stay compared with patients receiving Access agents, adjusted for age, gender and comorbidity .Considering length of treatment (LOT), adjusted for age, gender and comorbidity, receiving a Watch agent prolonged the LOT with 1.4 days compared to an Access agent  ranged from 0.5 to 36 days with a median duration of 7 days.In the total cohort, 212 (30.5%) had a LOT of 0–5 days, while in 484 (69.5%) of the infection events treatment for six days or longer was prescribed.Days of therapy  ranged from 0.5 to 40 days, with median 8 days. Data on LOT and DOT were missing in 24 cases (3.3%).In the minority of patients, 107 (15.8%), treatment was discontinued quickly (within 1 day) after confirmation of a viral RTI. In 439 cases (64.7%), antibiotic treatment was terminated after five days or more after the confirmation of a viral RTI, ranging up to 35 days. In 18 cases, the treatment was stopped before the confirmation of a viral RTI, and in 24 cases, the date for the end of treatment was not found in the patient records.In the patient group hospitalized with influenza A, the antibiotic treatment was terminated within 1 day after viral RTI confirmation in 14.0% of the cases. For patients with influenza B and adenovirus, the antibiotic termination rate was found to be 12.8% and 12.5%, respectively, RSV 10.4%, parainfluenza 8.9% and for patients with hMPV 6.1%.p = 0.013, logistic regression analysis).Compared to influenza A, the only virus found to be statistically different in termination rate was hMPV, with OR of 0.35 .As described in p < 0.001, Wilcoxon rank sum test).In the group treated with antibiotics (n = 720), the median evaluation score was two (0–5), and 65.8% of the patients scored ≥ 2 points. In the group of patients not treated with antibiotics, the median score was 1 (0–4), and only 24.7% of these patients scored ≥ 2 points. The difference in the evaluation score median was found statistically significant (p < 0.001).Using regression analysis adjusted for age and gender, we found a significantly higher use of antibiotic treatment among patients with an evaluation score of two or more, with OR of 5.80  and CRP level >60 mg/L . Receiver Operating Characteristic (ROC) curve analysis of the evaluation score showed an Area Under the Curve (AUC) of 0.755.p = 0.150, Wilcoxon rank sum test). Further, we found that patients in need of a stay in the intensive care unit (ICU) had a significantly higher evaluation score median of 3 (range 0–5) compared to patients not in need of the ICU with a median evaluation score 2 (range 0–5)) , and only 4.4% of these received no antibiotic treatment.Both immunocompromised patients and patients without such a risk factor had the same median evaluation score of two (range 0–5), with no significant difference  for the evaluation score, each additional point in the evaluation score leads to an average increase of 0.9 days longer hospital stay (LOS). When further adjusting for antibiotic treatment, each additional point increase in evaluation score added 0.7 days to total LOS.Considering the length of stay (LOS), adjusted for age, gender and Charlson score, we found a coefficient of 0.90  is narrow-spectrum antibiotics, i.e., benzylpenicillin. For severe CAP and non-complicated sepsis, benzylpenicillin or ampicillin in combination with gentamicin is recommended . Our stuIn 2017, WHO developed the AWaRe classification of antibiotics as a tool to support antibiotic stewardship efforts at local, national and global levels . In AWaRAccording to the national antibiotic guidelines for primary care in Norway , and alsOn the other hand, the detection of a viral respiratory infection does not exclude a bacterial respiratory infection, as secondary bacterial infection can arise during the course of illness ,24 and aWe also found that patients in need of treatment in the ICU department during their hospital stay had significantly higher evaluation scores compared to patients not needing ICU admission. This may reflect that the variables included in the evaluation score is a suitable predictor for severity of the disease.In our study, 362 patients (37%) had radiological findings (chest CT or chest X-ray) characteristic of pneumonia, and regression analysis showed a highly significant OR of initiation of antibiotic treatment, in addition to CRP level > 60 mg/L. Radiographic results may be of uncertain value when differentiating between viral and bacterial etiology. Studies have shown that viral pneumonia also presents with similar radiological findings as certain types of bacterial pneumonia ,29. EvenIn addition, in our study we found that in only 27 cases (2.8%) were neither chest X-ray nor CT scans performed during the hospital stay. A recently published multicenter study aimed to investigate the influence of X-ray results on antibiotic prescription in the pediatric population . They coThe CRP level is another deciding factor, which in many cases may be of uncertain value in distinguishing between bacterial and viral etiology. The sensitivity and specificity of the CRP test in bacterial pneumonia is relatively low, and CRP values above the normal range can be due to other illnesses . AdditioIn many situations, antibiotic initiation happens early in the course of infection based solely on a clinical evaluation, which in most cases cannot distinguish between bacterial and viral RTIs ,14,37. CA study from Spain concluded that not only did RSV patients more frequently receive antibiotic treatment when compared to influenza patents, the antibiotic withdrawal at the time of diagnosis was lower among these patients . In our The British Thoracic Society recommends seven days of appropriate antibiotic therapy for patients with low- or moderate-severity community-acquired pneumonia . The IDSThe median turn-around time for laboratory results on respiratory viruses was only one working day in this study. A short time-lag to viral diagnosis did not lead to antibiotic de-escalation for the majority of our patients, which may be due to the more time-consuming bacterial diagnostics, and clinicians’ hesitancy to discontinue treatment prior to culture results. Access to rapid laboratory testing of a suitable panel of both viral and bacterial respiratory agents, according to the epidemiological situation, is important. Fast and pertinent microbiological testing is crucial to help clinicians make the right treatment decision for the patients’ benefit . Rapid dOur study has some limitations. This study is a single-center study, so generalization of our findings must be carefully considered. We did not use any questionnaires for clinicians to gather information on reasons for continuing antibiotics after diagnosis of a viral RTI. Observational studies have a potential for bias due to using data collected retrospectively. As this study was designed to investigate patients presenting with respiratory symptoms, the antibiotic agents mainly used to treat urinary tract infections  were excluded from analyses. Recognizing that some antibiotic agents, such as cephalosporins, are used to treat cases with suspicion of concomitant RTIs and UTIs, we may have overrated the number of patients given antibiotics to treat RTIs.In addition, the study includes a few patients who may have been admitted with neutropenic fever. In these patients, empiric antibiotic treatment is often initiated. Even in the absence of bacterial growth, a broad-spectrum antibiotic treatment will, as a rule, be given in its full course. In addition, empiric antibiotic treatment is often necessary at the onset of fever in patients with underlying illnesses or drug-induced immunosuppression.The possibility of a bacterial infection despite negative cultures cannot be excluded. In addition, the lack of microbiological evidence of a bacterial infection may reflect insufficient microbiological sampling.Procalcitonin could have been an important factor in the decision-making regarding antibiotic initiation, but unfortunately this test was not available as a routine test in the hospital in the study period. However, the data on microbiological results and antibiotic treatment gave the basis for a comprehensive evaluation of a cohort of patients admitted to a large hospital serving the entire population in the region.Østfold Hospital Trust is a 500-bed, secondary acute care hospital with a catchment area of approximately 320,000 inhabitants. We conducted a retrospective observational single-center study, and included patients admitted to the hospital with viral RTIs during the years 2015–2018 . The patWe excluded cases with a finding of two viral agents, and in addition cases of enterovirus infections, because of the uncertainty of the clinical relevance of this virus agent in respiratory samples .Hospitalized patients who presented symptoms of respiratory tract infection >48 h from admission were considered hospital-acquired infections (HAI) and were excluded, as well as the IEs with respiratory virus only detected in the readmission event. A bacterial infection was diagnosed if microbiological examination revealed growth of relevant pathogenic microbes in respiratory specimen or blood culture, or by legionella/pneumococcus urine antigen or Mycoplasma/Chlamydia pneumonia PCR positive tests. These patients were excluded, leaving the final study population of hospitalized patients in two groups; antibiotic treated with no sign of bacterial infection and patients who did not receive antibiotic treatment during hospitalization.Concomitant medical conditions were registered and standardized using the Charlson comorbidity index, an assessment tool designed specifically to predict long-term mortality. The Charlson comorbidity index includes 19 medical comorbid conditions, with a weighted score of either 1, 2, 3 or 6 assigned to each condition, based on the adjusted relative risk associated with each comorbidity to predict 1-year mortality ,48,49.Information on immunosuppression was registered according to hospital records. A patient was categorized as immunosuppressed if treated with immunosuppressive medicines (such as methotrexate or rituximab), organ transplanted, suffering from disorders of the immune system, received cancer chemotherapy 1 month or less prior to admission or treated with systemic corticosteroids 14 days or less prior to admission.NEWS  is a tool to quickly detect changes in the patient’s vital signs and clinical condition to prevent deterioration. Therefore, we evaluated the disease severity by reviewing the first NEWS value registered at admission, with a special analysis of the number of cases with ≥5 points, which is considered the critical threshold value for urgent medical response .We retrospectively scored the patients by using CRB-65, which is a score system tool to estimate the severity and risk of death of community-acquired pneumonia .Respiratory samples were analyzed for respiratory viruses by using Seegene Allplex Respiratory Panel 1 and 2 assays  as previously reported ,46. The Clostridioides difficile infection were excluded. In addition, we excluded cloxacillin/dicloxacilllin treatment when administered together with a microbiological finding of staphylococcus in soft tissue, blood culture or urine.Data on the administration of antimicrobial drugs during hospitalization were collected from the electronic hospital records and analyzed using a purpose-built structured database. If the date for the end of antibiotic treatment was not found in the hospital record, these patients were excluded in the calculation of DOT and LOT. Antibiotic agents specifically aimed for urinary tract infections , and treatment of Chest X-ray/Chest CT compatible with or susceptible of pneumonia (1 point);CRP level > 60 mg/L at admission (1 point);Oxygen saturation < 90% at admission (1 point);Respiratory rate > 20/min at admission (1 point);Heart rate > 100 at admission (1 point).Due to the lack of a validated tool to evaluate antibiotic prescription retrospectively, an evaluation score was designed as a helping tool to better understand the decision-making in this cohort. We included clinical-, biochemical- and radiological factors that clinicians seem to emphasize when deciding whether to prescribe antibiotics, to investigate the relevance and indication of the antibiotic treatment. The infection events were scored as follows:The diagnosis of pneumonia by chest X-ray or CT was made by a medical specialist in radiography at the time of the examination during the hospital stay.Ethical approval was obtained from the Regional Committee for Medical and Health Research Ethics (REK ref. 2017/1917 A) and the hospital Privacy Appeal Board (public 17/05444).For statistical analyses, we used SPSS .Categorical variables are presented as frequencies and percentages; continuous variables are described as mean, median, standard deviation and range as appropriate.Statistical analysis was performed on categorical variables by using Pearson’s chi-square test, and Fisher’s exact test for variables with expected count n ≤ 5, for continuous variables Wilcoxon rank sum test was performed. We adjusted for patient age and gender in all regression analyses. Length of stay analysis is presented as Kaplan–Meier plot.ROC curve analysis was performed on the evaluation-score.Normality examination was performed with Shapiro–Wilk test.p-values less than 0.05 were considered statistically significant.Our study revealed that antimicrobial stewardship and rational antibiotic prescription could be improved even in a country with relatively low antibiotic usage. Doctors were reluctant to stop antibiotic treatment in cases with viral infection, even after receiving negative bacterial culture results, as most cases continued with a full treatment course.In addition, length of stay was significantly higher in the group of patients on antibiotic treatment compared to those not treated, showing that patients may benefit from more stringent antibiotic stewardship by a shorter hospital stay. Additionally, the fact that mortality was similar in both groups supports that not giving antibiotics in these cases is safe and reassuring for physicians.Together, these results provide important insights into medical decisions governing the antibiotic administration and can be useful for improvement in the management of viral RTIs.Access to relevant microbiological tests and rapid communication of results are crucial for reducing the antibiotic prescription. Development of better tools for antibiotic stewardship needs to be further investigated.


The cucurbit[n]uril supramolecular hydrogels are driven by weak intermolecular interactions, of which exhibit good stimuli responsiveness and excellent self-healing properties. According to the composition of the gelling factor, supramolecular hydrogels comprise Q[n]-cross-linked small molecules and Q[n]-cross-linked polymers. According to different driving forces, hydrogels are driven by the outer-surface interaction, the host–guest inclusion interaction, and the host–guest exclusion interaction. Host–guest interactions are widely used in the construction of self-healing hydrogels, which can spontaneously recover after being damaged, thereby prolonging their service life. The smart Q[n]s-based supramolecular hydrogel composed is a kind of adjustable and low-toxicity soft material. By designing the structure of the hydrogel or modifying the fluorescent properties, etc., it can be widely used in biomedicine. In this review, we mainly focus on the preparation of Q[n]-based hydrogels and their biomedical applications including cell encapsulation for biocatalysis, biosensors for high sensitivity, 3D printing for potential tissue engineering, drug release for sustained delivery, and interfacial adhesion for self-healing materials. In addition, we also presented the current challenges and prospects in this field. Self-assembly is ubiquitous. Molecular self-assembly mainly diffuses molecules to form nanostructures through a series of non-covalent interactions, such as hydrogen bonding, the hydrophobic interaction, π-π stacking interaction, electrostatic interaction, and van der Waals forces ,2. It caCucurbit[n]uril (Q[n]) is a macrocyclic compound constructed by methylene-bridged urea units ,7. It diIn this paper, the construction and application of Q[n]-based supramolecular hydrogels are widely summarized. According to the composition of the gelator, Q[n]-based supramolecular hydrogels can be formed through the self-assembly of a low molecular gelator and via the non-covalent crosslinking of polymer chains modified with functional groups. The research on hydrogels constructed by small molecules mainly focused on the selection and design of gelators, the process of self-assembly, and the diversity of structures. The research on supramolecular hydrogels constructed by polymers mainly focused on the construction method of supramolecular networks, the performance of gels and their practical application values. The role of Q[n]s in supramolecular hydrogels provides a theoretical and practical basis for subsequent research and practical applications.Q[n]-cross-linked polymer-based supramolecular hydrogels are the main building blocks of Q[n]-based hydrogels. Polymers can be constructed from natural macromolecules and synthetic monomers. Natural polymers exhibit good biocompatibility and biodegradability and can be widely used in the biological field. The monomers of synthetic polymers can be designed and customized based on demand. Q[n]-cross-linked polymer hydrogels are based on non-covalent crosslinks between cucurbit[n]uril and guest molecules. Due to the physical properties of non-covalently cross-linked hydrogels, most prepared hydrogel materials exhibit excellent responsiveness, self-healing ability, and have broad field prospects in the field of biomaterial science . Unlike Li et al. prepared a novel supramolecular gel via a hydrogen-bonding interaction between the carbonyls of Q[7] and the amino groups of polyacrylamide (PAAm). As shown in Supramolecular hydrogels are generally formed through the host–guest inclusion between Q[n]s and the side chain groups of polymer chains ,26,27,28Scherman et al. reported several studies on the preparation of supramolecular hydrogels through the host–guest interactions between Q[8] and specific guest groups ,32,33,34Q[8] can simultaneously bind with two guests in its portal to form a ternary complex. Webber et al.  proposedThe surface of Q[n] is not easy to modify, thus limiting their derivatization and application. However, many studies have studied the surface modification of Q[n]s and achieved some results. Kim and co-workers developed an allyloxylated Q[6] monomer, which was surface-activated to modify Q[6] at the end of the polymer chain. As shown in Small molecule hydrogels have attracted great interest from numerous researchers due to their unique structural properties, designability, and potential biological applications ,41,42. VThe Q[n] outer surface exhibits a positive electrostatic potential and displays electrostatic interactions with other electronegative elements, including the ion–dipole interaction, dipole interaction, etc. Therefore, we propose the outer-surface interaction of Q[n]s. In 2007, Kim et al.  first reDue to the different cavity sizes of Q[n]s, all guest molecules could easily enter Q[n]s with larger ports, including Q[7] and Q[8]. The study of Q[n]s-based supramolecular hydrogels showed that most hydrogels were prepared through the host–guest inclusion interaction. However, owing to their smaller sizes, Q[5] and Q[6], which are often excluded, have not received attention in the field of hydrogels. Recently, these hydrogels were constructed through the formation of a host–guest exclusion interaction based on the hydrogen bond and ion–dipole interaction between the guest and carbonyl oxygen atom of the Q[5] or Q[6] port.10Q[5]) and para-phenylenediamine (p-PDA) [10Q[5] has potential applications in the development of more practical and marketable Q[n]-based hydrogels.In 2021, we found that a small molecular supramolecular hydrogel could be formed by the host–guest exclusion interaction between decamethylcucurbit[5]uril (Me (p-PDA) . DiffereIn addition, as shown in Supramolecular hydrogels formed by the inclusion interaction between Q[n] and organic small molecules mainly used Q[n] with larger cavities, such as Q[7] and Q[8]. Q[n] has a wider range of binding to guest molecules, which can encapsulate larger guest molecules such as amantadine, ferrocene, and bipyridine.In 2017, Scherman and co-workers reported the formation of supramolecular hydrogels using simple molecules and Q[8] ternary complexes . As showSimilarly, Qu et al. designed an achiral guest 4,4′-bipyridin-1-ium chloride (BPY+) salt derivative and Q[8] to construct a ternary complex by a 2:1 host–guest interaction in an aqueous solution. The flexible chain of the monomer provides moderate water solubility, and the increased solubility in solutions driven by the host–guest interaction between Q[8] and BPY+ groups leads to the spontaneous assembly and formation of helical nanofibers. By varying the guest concentration, the helical nanofibers were transformed into pH-responsive hydrogels, thereby tuning the morphology of the material .As cross-linked three-dimensional network structure materials, conventional hydrogels can absorb and retain a large amount of water . The intCell encapsulation can prevent biological macromolecules, such as enzymes and cells, escaping from the microcapsules, while small molecule substances and nutrients in the medium can freely egress and ingress, thereby achieving the purpose of catalysis or cultivation. It is an important challenge to develop effective crosslinking and biofunctionalization strategies under physiological conditions without compromising the function of cell encapsulation . The intHydrogels are very promising for fabricating transductive biosensors because of their inherent cellular similarities . SupramoHydrogel inks are particularly useful in biomedical applications to reproduce features of the cellular environment, and can form structures used to replace, enhance, and mimic tissue . HydrogeOwing to its 3D network structure, the hydrogel that consists of hydrophilic polymer chains can encapsulate drug molecules to form potential drug delivery systems ,57. The In recent years, numerous studies have been conducted on bioadhesives as soft and wet materials used in medicine and engineering, including soft electronics , biomediThis paper mainly discusses the recent preparation methods of Q[n]-based supramolecular hydrogels and their applications in the field of biomedicine. The unique structure of Q[n] makes it have stable properties, excellent molecular recognition ability, and strong binding ability with guest molecules, which leads to an easy non-covalent bond interaction. As specific stereochemical building blocks in the system, supramolecular hydrogel can be found not only by the self-induced outer-surface interaction, but also by the host–guest inclusion or exclusion interaction. Utilizing the interaction between Q[n] and the guest, combined with the properties of different functional guests, the researchers prepared self-healing, adhesive, stimuli-responsive or biocompatible hydrogels. The low toxicity of Q[n] and the biocompatibility of some hydrogels that can be used as an extracellular matrix make it prone to be applied in biomedical fields, such as cell encapsulation, biosensors, 3D printing, drug releases, and interfacial adhesion. However, the application of supramolecular hydrogels still faces many practical challenges due to the refractory and long-term toxicity of various chemical groups. In order to enrich the application, the development of biosafety, degradable hydrogels, and high mechanical strength is the main focus of current research. With relatively strong biocompatibility of the self-assembled system, supramolecular hydrogels are expected to be a good application for bioscience and medicine. On the other hand, the supramolecular bonding strength is controllable by designing suitable guests, and the regulation of mechanical strength with self-healing in the Q[n]-based hydrogel materials should be realized with supramolecular functionalization. Therefore, the development of the application of Q[n]-cross-linked guest hydrogels is also one of the solutions to the current problems. The Q[n]-based supramolecular hydrogel still has great potential application, providing unlimited possibilities for the research of cucurbit[n]uril chemistry.


Background: For many years it has been studied how eye movements influence reading andlearning ability. The objective of this study is to determine the relationships between thedifferent publications and authors. As well as to identify the different areas of research ocularmovement.; Methods: Web of Science was the database for the search of publicationsfor the period 1900 to May 2021, using the terms: “Eye movement" AND “Academicachiev*”. The analysis of the publication was performed using the CitNetExplorer,VOSviewer and CiteSpace software.; Results: 4391 publications and 11033 citation networkswere found. The year with the most publications is 2018, a total of 318 publicationsand 10 citation networks. The most cited publication was "Saccade target selection and objectrecognition: evidence for a common attentional mechanism." published by Deubel et al.in 1999, with a citation index of 214. Using the Clustering function, nine groups were foundthat cover the main research areas in this field: neurological, age, perceptual attention, visualdisturbances, sports, driving, sleep, vision therapy and academic performance.; Conclusion:Even being a multidisciplinary field of study, the topic with the most publications to date isthe visual search procedure at the neurological level. Ocular motility has been studied since the 20th century ; therefoDuring reading processes, the eyes move through the text making aseries of saccadic movements with different ranges and directions aswell as different fixations with variable durations. These movementstend to go in a forward direction, that is to say, the eyes fixate onone word before moving onto another. However, in order to fixate on aprevious word, or to move to the next line of the text, a backwardsmovement is performed (regression). Saccadic movements are the fastestmovement that the human body is capable of performing, with an averagespeed of 100° to 800° per second and a frequency of 100,000 saccades perday .The necessary skills, which are most influenced by vision, to achievea good academic performance are: a fluent reading, precision and a goodunderstanding of the text. These skills depend on eye movements ; 17; 24.Eye movements have many functions in a range of everyday tasks, giventhat these work in coordination with body and head movements. In socialinteractions, eye movements provide us with information (i.e.: bylooking at someone´s face or following their look) and indicate that weare looking at someone in particular. In other words, eye movementsserve an additional purpose as a communicative signal. On the otherhand, for example while driving, the interaction between head and eyemovements is complex, therefore meaning that saccadic movements, smoothpursuit vergence eye movements and the vestibulo-ocular reflex arerequired ; 64; 35.Citation network analysis is a powerful tool, which allows us toanalyse, classify and deepen the scientific literature on a specificsubject. Moreover, through this type of analysis is it possible todetermine the most cited article and create groups that allow for linksbetween articles and authors to be established. This therefore meansthat it is a great tool for broadening our knowledge on a specific fieldof interest .This study presents an analysis of citation networks. In which therelationship between authors and publications is analysed. As well as anin-depth analysis of the research areas with the greatest interestwithin the research field of the influence of eye movements on academicperformance.The search of publications was carried out in the Web of Science(WOS) database, using the following search terms: "Eyemovement" AND “Academic achiev*”. These terms were used inaccordance with the aim of this study, how eye movements influenceacademic performance.As the search results had articles in common, the Boolean operator“NOT” was used, as well as the truncation symbol “*”, which was used tosearch for the singular and plural forms of the terms. Therefore, in thesecond search we used the following terms: (“Eye movement*” AND“Academic performance” NOT “Academic achiev*”), in the third search weused the following terms: (“Eye movement*” AND “Binocular vision” NOT“Academic performance” NOT “Academic achiev*”), and in the fourthsearch, the following terms: . The selected time interval to carry out the search was from1900 to May 2021.CitNetExplorersoftware. CitNetExplorer software  has been used for the analysis ofpublications and the creation of citation networks.Once the Web of Science bibliography is downloaded by exporting plaintext files, it is loaded into the Using the Citation Score attribute, the quantitative analysis wasperformed, in order to quantify the internal connections of the Web ofsciences database and other external databases .i denotes the cluster to which node i isassigned, δ denotes afunction that equals 1 if ci = cj and 0 otherwise, and γ denotes aresolution parameter that determines the level of detail of theclustering. The higher the value of γ, the larger the number of clustersthat will be obtained. This formula allows to obtain the connectionsbetween the publicationsFirst, the clustering function has been used, which is based on theformula developed by VanEck in 2012 , wherecSecondly, the "core Publications" function has been used,which consists of identifying the publications that are considered to beat the core of a citation network. Only publications with 4 or morecitations were considered.H Index(number and level of scientific production of authors and institutions).Degree . Centrality .For the use of The CiteSpace software (5.6.R2), the bibliographydownloaded from the Web of Science is also used. First, the period ofyears to be analyzed is selected. Next, it is selected based on what youwant to analyse, the country, the institutions, authors, keyword, etc.;and thus obtain the following parameters: VOSviewer software allows the visualization and creation ofbibliometric networks. Therefore, it was used for creating the graphs.To obtain the graphs, the CitNetexplorer software groups are downloadedin the Pakej format. Next, this file is loaded in Vosviewer in theCreate- Create a map based on network data sectionOn the other hand, the Web of Science and Scimago Journal &Country Rank databases have been used to obtain bibliometric data andthe impact of the journals.The first articles on eye movements were published in 1976. Theperiod of study was from 1900 to May 2021. Through the WOS search, 4391publications and 11033 citation networks were found.As shown in After having analysed the 20 most cited articles, 10 of them concernthe visual search procedure in primates on a neurological level ;45; 26,Of all publications, 84.8% were articles, 6.1% were proceedingspapers, 5.9% were reviews, and the remaining 3.2% were meetingabstracts, book chapter or editorial materials. With regards to thelanguage of the publications, 99% were in English, 0.8% were in Germanand the remaining 0.2% were in Russian. As shown in The research area on eye movements is multidisciplinary. It is worthnoting the field of Psychology (34.7%) and Neuroscience (34.2%) .The institutions with the highest number of publications were UniThe most commonly used keywords were “Eye-movements”, “Attention”,“Performance”, “Saccadic Eye movements” and “Perception”.Using the Clustering function, 13 groups were found, nine of whichhad a significant number of publications. The remaining four groups onlymade up 1.2% of the publications, which did not have enough publicationsto obtain connections between authors and publications.Group 1 includes 692 publications and 2184 citations. The most-citedwork is Itti & Koch  in VisioThe scientific references in this group analyse the visual searchprocedure at the neurological level .Group 2 comprises 536 publications and 1500 citations. The most-cited work isMunoz et al.  in ExperGroup 3 includes 384 publications and 1209 citations. The most-citedwork is Deubel & Schneider  in VisioGroup 4 comprises 264 publications and 910 citations. The most-cited workis Zihl  in NeuroThe scientific references in this group examine eye movements in patientswith visual disturbances .Group 5 includes 253 publications and 809 citations. The most-citedwork is Savelsbergh et al.  in JournThis study analyses the skills of anticipation and visual search inexpert and novice soccer goalkeepers. Expert goalkeepers are generallymore accurate. That is, they use a more efficient search strategyinvolving fewer fixation. No differences in visual search behaviour areobserved between successful and unsuccessful penalties.The scientific references in this group discuss the importance of oculomotormovements in sports .Group 6 comprises 249 publications and 474 citations. The most-citedwork is Konstantopoulos, Chapman & Crundall  in AccidThe scientific references in this group assess the importance of eyemovements in driving .Group 7 includes 232 publications and 477 citations. The most-citedwork is Shadlen & Newsome  in JournThe scientific references in this group discuss the importance ofcortical areas in eye movements, as well as changes in eye movementsduring sleep .Group 8 comprises 188 publications and 444 citations. The most-citedwork is Alvarez et al. . In other words, the field ofeye movement research is multidisciplinary.Major databases such as WOS or Scopus allow for the creation ofcitation networks. However, it is not possible to perform asystematic review of all of the existing scientific literaturepublished on a topic, given that this does not provide a generaloverview of the connections between the citations of a group ofpublications. For this reason, the CitNetExplorer software was used,as, besides creating citation networks, it also offers a moredetailed analysis of the scientific literature than the WOS orScopus databases The general objective of the present study was to analyse theexisting scientific literature on ocular motility. The source ofpublications was the WOS database, which only accepts prestigiousinternational journals after a rigorous selection process, and whosesearch range begins in the year 1900.Therefore, once the bibliography had been downloaded from the WOSdatabase, the CitNetExplorer software allowed us to collect and analyseevery available piece of literature on ocular motility from 1900 to May2021. In addition, it was also possible to obtain the connectionsbetween the fields of study and the different research groups byanalysing the citation networks. The "Clustering" functionallowed us to obtain the results and group the publications according tothe links between the citations. The "Core publications"function was used to select the most cited publications . Those publications with 4or more citations were selected according to other studies . All theVision Research, with an impact factor of 2.61, was the journal withthe highest number of publications (213) on ocular motility. In turn,the journal with the highest impact factor was The Journal ofNeuroscience with 5.67. In any case, it is important to consider thatthe impact factor is a critical index of the journal’s importance but itis not an absolute index. The main difference between a critical indexand an absolute index is that the latter is based on the impact of theresearch results and the authors’ physical and intellectualcontributions .However, the Journal of Eye Movement Research, since 2016, is thejournal that has most increased its publications on eye movements andthe impact they can have on academic performance ; 59; 40.The country with the highest publication rate was the United States(33.9%), followed by England (14.9%) and Germany (13.1%). Our resultsagree with the study by Hernandez-Torre & Yuh-Shan . ThissuAmong the first publications in this field of research, the articlesby Schall , with a In Ralph, Hager & Christine , the autFrom 2013 to date, the curve of publications on eye movements hasbeen steadily increasing, perhaps due to the increasing interest in theimportance of vision on academic performance in recent years, andtherefore the relevant role played by eye movements ; 67; 31.The year with the highest number of "key year" publicationson eye movements was 2018. In this year, the study by Stewart &Schütz , which eConsequently, eye movements are present in all areas of daily life.Therefore, in recent years there has been an increasing amount ofresearch conducted into the ways in which eye movements vary with ageand how they influence memory ; 11; 56.Another topic of interest, although to a lesser extent, is theimportance of eye movements in sports vision ;63. It In conclusion, by analyzing citation networks it has been possible toextend knowledge about eye movements. Nine groups were identified inthis study , therefore meaning that the field of research on eyemovements is multidisciplinary. The topic with the most publications todate is the visual search procedure at the neurological level. However,research topics on the importance of eye movements with age and sportsvision have gained growing interest in recent years.http://biblio.unibe.ch/portale/elibrary/BOP/jemr/ethics.htmland that there is no conflict of interest regarding the publication ofthis paper.The author(s) declare(s) that the contents of the article are inagreement with the ethics described in


Negligible leaching of PFOS after sealing with Product B was observed ( < 5 × 10−9 mg/m2). Results from modeled rainfall scenarios suggest PFAS leachability is reduced from sealed versus unsealed AFFF-impacted concrete surfaces.Per- and polyfluoroalkyl substances (PFAS) within concrete pads impacted by historical firefighting training using aqueous film-forming foam (AFFF) may be potential secondary sources of PFAS due to surficial leaching. This study aimed to (i) characterize the effectiveness of two commercially available sealants (Product A and Product B) in mitigating leaching of five PFAS  from concrete surfaces at the laboratory-scale, and (ii) develop a model to forecast cumulative leaching of the same five PFAS over 20 years from sealed and unsealed concrete surfaces. Laboratory trials demonstrated that both sealants reduced the surficial leaching of the five PFAS studied, and Product B demonstrated a comparatively greater reduction in surface leaching than Product A as measured against unsealed controls. The cumulative PFOS leaching from an unsealed concrete surface is estimated by the model to be about 400 mg/m  The use of aqueous film-forming foams (AFFF) containing per- and polyfluoroalkyl substances (PFAS) at some military bases, airports, and industrial facilities for firefighting and training purposes has impacted hardscape surfaces  . These iA current limitation with respect to PFAS leaching from hardscape is that long-term loss of PFAS under dynamic conditions  has not been fully considered, despite surficial leaching of PFAS during a few runoff events being evaluated . Hence, In this study, short-term laboratory-scale experiments were conducted to assess the effectiveness of sealants to reduce surficial leaching of PFAS into runoff during simulated rainfall events. Further, a deterministic model was developed, evaluated, and applied to forecast surficial leaching of PFAS from sealed and unsealed AFFF-impacted concrete pads over a 20-year period.p < 0.01) between the leaching results from Product A-sealed cores and those of unsealed cores reported by p < 0.01). Further, there are substantially higher PFAS concentrations in leachate from the unsealed cores than Product B-sealed cores ( > 100 times). The maximum concentration of PFOS in runoff water from Product B-sealed cores was < 0.2 µg/L. There was an apparent decrease in the maximum PFOS concentration after each rainfall event that eventually reduced to below the limit of detection. PFAS leaching from Product B-sealed cores was significantly reduced (p < 0.01) with respect to unsealed controls under the experimental conditions tested. Other PFAS , perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), and 6:2 fluorotelomer sulfonate (6:2 FTS) demonstrated similar leaching trends with respect to unsealed and sealed cores.The PFAS leaching results in water samples collected from concrete cores during the rainfall simulations are shown in Given different mechanisms of leaching mitigation between Product A  and Product B , and the results summarized in PFAS impacts associated with the historical AFFF use can represent decades of different types of AFFF and application methods . CorrespC1585 found initial and secondary rates of absorption were both 0.0 × 10−4 mm/√s for a Product B-sealed core compared to 1.7 × 10−3 mm/√s and 4.7 × 10−4 mm/√s of initial and secondary absorption rates for an unsealed core, respectively (Table S3). Conversely, the inorganic sealant (Product A) did not reduce the rate of absorption of water into AFFF-impacted concrete matrix. Product A facilitates a crystalline-based chemical reaction within the porosity of the concrete, attempting to stabilize PFAS. Initial and secondary rates of absorption were 2.8 × 10−3 mm/√s and 9.2 × 10−4 mm/√s, respectively, on a Product A-sealed core using sorption tests (ASTM C1585) (Table S3).The rate of water absorption was evaluated for sealed and unsealed cores. The organic sealant (Product B) reduced the rate of water absorption into the AFFF-impacted concrete matrix, which would otherwise facilitate surficial leaching and subsequent runoff of PFAS. Sorption tests performed following ASTM hrunoff) started immediately at the beginning of the modeled rainfall scenario because the concrete reached saturation after 2–4 min and the infiltration (hinfiltration) rate was low. Evaporation (hevaporation) and logging (hlogging) had no impact on the runoff (hrunoff) during the simulated rainfall event (60 min). Seemingly the rainfall intensity of 1 mm/min used in the experiments was sufficient to achieve constant runoff and negate the potential "wicking" effect attributable to evaporation . Runoff ) are shown in des of the unsealed core (53 L/kg) is two times less than the Product A-sealed core (120 L/kg), suggesting that the former can leach ∼2-fold more PFAS than the latter over the experimental period. Further, Product B-sealed cores showed a minor leaching and EKdes decreased after each rainfall event. EKdes of the Product B-sealed core could not be comparably modeled to the unsealed and Product A-sealed cores as many data points of the leaching profiles were at or below the LODs for the five PFAS. However, for comparative purposes, EKdes for PFOS was estimated for the first two rainfall simulations of Product B-sealed cores to be 32,000 L/kg =  0.014) and 110,000 L/kg =  0.014), respectively.The values of EKAfter fitting the model to the experimental results, the model was evaluated for validity using data from replicated rainfall simulations from the same concrete cores. Most NSE values of the evaluation are > 0.70, which indicate reasonable predictive performance of the model . The comThe model was used to forecast PFAS runoff from the concrete pad characterized in It is noteworthy that the forecasted median PFOS leaching concentration from the Product A-sealed pad was lower than the unsealed pad for years 1–8. For other PFAS, the median leaching concentration in the Product A-sealed pad is less than the unsealed pad for years 2–5. This suggests that the use of the Product A sealant on AFFF-impacted concrete surfaces may slow the leaching of PFAS to runoff compared to unsealed concrete surfaces. A potential explanation for this is that Product A sealant formed a non-soluble crystalline precipitate within the concrete matrix likely reducing the ability for entrained water (and dissolved PFAS within the entrained water) to leach from the concrete.2), indicating near-complete (97%) PFOS leaching from the top 0.5 cm. Approximately 85% of PFOS mass from the Product A-sealed pad is forecasted to have been leached over the 20-year modeled rainfall scenario (∼340 mg/m2), and asymptotic conditions are not reached until after the study timeframe. The unsealed pad and Product A-sealed pad appear to leach approximately 50% of the PFOS after approximately 4 and 9 years, respectively. These are 2.5 to 6 times shorter than the 25 years estimated by −9 mg/m2). The leaching results from the other 4 PFAS studied closely resemble that of PFOS for both Product A- and Product B-sealed cores.The PFOS cumulative mass loss of the unsealed pad appears to reach asymptotic conditions after 15 years  that may vary if the rainfall intensity changes.Despite the findings summarized herein, the model remains limited in the following aspects. Firstly, this model does not incorporate various potential fate and transport mechanisms that may be occurring, such as the advection of potential moisture wicking and the potential for PFAS supramolecular structures and/or bilayer formation controlled dissolution . Owing tThe durability of the sealant to endure physical and chemical stresses on the sealed surface and natural concrete weathering also likely challenge the confidence of the modeling . RoutineFurther, the mechanics of actual rainfall were not included in study design, and incorporating the kinetic energy, droplet size, and runoff velocity of water in future efforts could be more representative of real-world conditions . The selWhile the modeling approach has merit, the variability of concrete chemistry/characteristics and erosion has not been fully represented. It might result in differences in absorption, diffusion, and leaching of PFAS. Significant changes in leaching of organic chemicals were reported for stamped concrete (an imprinted and textured concrete), which may be attributable to the increasing surface roughness and physical trapping . WhetherThe reduction of PFAS leaching from AFFF-impacted concrete surfaces due to the application of two different sealants suggests that sealants may serve as a mitigation strategy to manage PFAS leaching from concrete depending on site-specific conditions. Commercial concrete sealants have been used in many applications, and this study evaluates their ability to mitigate PFAS leaching. The use of concrete sealants to reduce PFAS leaching must also achieve site-specific requirements for the intended purpose of the concrete infrastructure . While Product B significantly reduced PFAS leachability from a concrete surface in comparison to an unsealed concrete surface, its organic composition may be incompatible with surfaces used for future fire training. Future work will evaluate the field-scale durability of multiple PFAS-relevant sealants to better understand the options available to mitigate potential PFAS leaching from concrete surfaces. The deterministic model described herein was used to forecast a 20-year comparison of the tested experimental conditions to support evaluation of the potential benefits of managing PFAS leaching from concrete with two sealants. Further, these results suggest that use of concrete sealants may be a viable management strategy to mitigate PFAS leaching from AFFF-impacted concrete.d = 120 mm; h = 10 cm) were collected from an AFFF-impacted concrete pad using a core drill (C1585 (Standard Test Methods for Measurement of Rate of Absorption of Water by Hydraulic-Cement Concretes)  .The PFAS mass concentration within the concrete was determined by drilling 4 to 5 holes around the circumference of the concrete core at different depths  to develop a profile . Concrete dust samples from the drilling were composited, extracted, and analyzed as described previously . Five PFRainfall simulation experiments were conducted to contain both wetting and drying periods, as described previously . RainfalA deterministic model for forecasting surficial leaching of PFAS in rainfall runoff from an AFFF-impacted concrete pad was developed based on the principle of mass balance and mass transfer. This modeling approach has been validated in a model for estimating pesticide runoff . The modhrunoff: runoff depth (mm), hrain: rainfall depth (mm), hinfiltration: water infiltration depth into the concrete pad (mm), hevaporation: water evaporation depth in the local meteorology conditions (mm), hlogging: water logging depth on the concrete surface (mm). Methods for estimating each component are presented in the SI.In a rainfall event, the water balance can be expressed by the following:MPFAS leaching) is calculated as follows:MPFAS leaching: mass of PFAS leaching (µg), S: surface area of the leaching concrete (m2), Cs: concentration of PFAS in concrete solid compartment (µg/kg), and effective Kdes (EKdes): where Kdes represents a desorption coefficient, EKdes represents a lumped coefficient expressing the migration of PFAS from the solid compartment to the liquid compartment (L/kg). A lumped coefficient is intentionally specified to acknowledge that the mechanisms controlling PFAS leaching from concrete are currently not well understood. A more detailed description of estimating EKdes from experimental data can be found in the SI .Mass of PFAS lost to runoff . For modeling purposes, only the top 0.5 cm of the concrete pad was considered, based on prior observations of PFAS mass primarily in surficial and upper portions of concrete matrix ]) ]) .i and Cm are the observed and simulation concentration from the concrete cores, respectively, The performance of the model is evaluated by two objective functions  to evaluate model uncertainty, which are expressed as below:hrain) for the days when rainfall occurred. The model was applied to forecast PFAS leaching in different scenarios: unsealed (control) and sealed (with Product A and Product B) concrete pads. The cumulative mass loss in 20 years is calculated by summing the mass loss of each rainfall event.The model was applied to forecast cumulative mass loss of PFAS due to rainfall runoff from a concrete pad over 20 years. Concrete pad parameters, such as the surface area of the concrete and PFAS concentrations in concrete, were retrieved from The input parameters for the model are shown in Phong H.N. Vo: Conceptualization, Methodology, Formal analysis, Investigation, Writing – original draft. Trent A. Key: Investigation, Formal analysis, Writing – review & editing. Tu Hoang Le: Methodology, Formal analysis, Investigation. Jeffrey T. McDonough: Methodology, Writing – review & editing. Scott Porman: Methodology, Writing – review & editing. Stephanie Fiorenza: Methodology, Writing – review & editing. Hong T.M. Nguyen: Methodology, Formal analysis. Vinh T.N. Dao: Writing – review & editing. Jochen F. Mueller: Methodology, Writing – review & editing. Phong K. Thai: Conceptualization, Methodology, Formal analysis, Investigation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Sepsis is the third leading cause of neonatal death in low and middle-income countries, accounting for one third of all deaths in Ethiopia. A concerning issue is the increasing number of multidrug-resistant microorganisms facilitated by suboptimal antibiotic stewardship. The study aims to identify clusters of newborns switching antibiotic lines for sepsis in a neonatal intensive care unit (NICU) in Ethiopia, and to explore their potential association with sepsis outcomes.A retrospective cohort study was conducted including all newborns discharged with a diagnosis of probable neonatal sepsis from the St. Luke Catholic Hospital NICU between April and July 2021. The antibiotic management protocol included two lines according to WHO guidelines and a third line based on internal hospital guidelines. In the cluster analysis, the Gower distance was estimated based on the antibiotics employed in the different lines and the duration of each line. Mortality and respiratory distress (RD) were the response variables.In the study period, 456 newborns were admitted to the NICU and 196 (42.8%) had probable neonatal sepsis. Four antibiotic management clusters were identified. Cluster 1  had no antibiotic switches, using only the first line. Cluster 2  had one switch from the first to the second line. Cluster 4  had two switches: from first to second and then to third line. In cluster 3 , newborns were switched from ceftriaxone/cloxacillin as second line to off-protocol antibiotics. There were no differences in sex, age, weight on admission or crude mortality between clusters. Cluster 3 included a higher frequency of infants who did not breathe at birth  and that necessitated bag ventilation  compared to the other clusters.The first antibiotic line failed in one out of four newborns with probable sepsis while third-generation cephalosporins were insufficient in one in ten patients. Cluster analysis can provide valuable insights into antibiotic treatment patterns and their potential implications. This approach may support antibiotic stewardship and aid in contrasting antimicrobial resistance in limited resource settings.The online version contains supplementary material available at 10.1186/s13756-023-01325-w. Neonatal mortality is a concerning issue in low- and middle-income countries (LMIC) where most of the estimated 2.6 million yearly global deaths occur. Neonatal sepsis is the third leading cause of neonatal mortality being responsible for 13% of all neonatal deaths, and of 42% of deaths in the first week of life [Neonatal sepsis is defined by the systemic manifestation of infection, due to the presence in the bloodstream of a bacterial pathogen. Neonatal sepsis is classified as ‘confirmed’ if bacteremia is proved by a positive blood culture, ‘probable’ if signs and symptoms are supported by suggestive laboratory results, and ‘suspected’ if only clinical suspicion is present . DiffereMultidrug resistance for pathogens involved in sepsis is worsening, both globally and in LMICs, with greater attention advocated for antibiotic stewardship . AntimicCluster analysis is a machine learning technique that aims to group observations that are similar within the same cluster. This analysis includes a cluster construction phase and a subsequent validation phase. There may be an internal validation  and an external validation  . The devWe used a retrospective cohort study design to explore the use of cluster analysis in depicting antibiotic management of neonatal sepsis and its association with newborns’ outcomes in a limited resources NICU.The research was performed following the ethical standards of the 1964 Declaration of Helsinki and was approved by the Ethical Committee of St. Luke Catholic Hospital (SLCH) on the 14th of September (protocol number 1293/2021).woreda of Ameya, Wenchi, Waliso rural, Woliso town, Becho, and Goro representing the reference hospital for 743.797 individuals. The number of deliveries assisted at SLCH was 4455 in 2019 and 4015 in 2020. The SLCH NICU has 16 beds with an annual average bed-occupation rate of 112% in 2020 [SLCH is a referral hospital located in Wolisso Town, about one hundred km from the capital Addis Ababa. It is situated in the South-West Shoa Zone (SWSZ) of the Oromia region (Ethiopia), with an estimated population of 1,311,406 inhabitants, of which 15% are under five years of age . SLCH caAll newborns admitted in NICU between 1st April 2021, and 31st July 2021 and discharged with the diagnosis of probable neonatal sepsis were included. Exclusion criteria were missing information about antibiotic management or regarding the type of neonatal sepsis.Probable neonatal sepsis was defined as the presence of two or more of the following clinical signs and symptoms: hypo-hyperthermia (BT < 35.5 °C or > 37.5 °C), heart rate > 180 or < 100 bpm, respiratory rate > 60 breaths/min with grunting or desaturations, lethargy or altered mental status, glucose intolerance (plasma glucose > 10mmol/l), feed intolerance; plus at least one of the following laboratory results at birth: leukocytosis , leukopenia , thrombocytopenia  [Neonatal sepsis was defined as early-onset (EOS) if the sepsis symptoms started within 72 h of birth and late-onset (LOS) if they began later than72 hours from birth .Respiratory distress was defined as the presence of two or more of the following signs: abnormal respiratory rate , grunting, nasal flaring, intercostal recessions, xiphoid recessions, with or without cyanosis . RD was For each subject, the following data were collected: sex, age and weight at admission, date of hospital admission and of hospital discharge, number of antenatal care (ANC) visits, mode of delivery, delivery place, presence of maternal chorioamnionitis, occurrence of premature rupture of membranes (PROM), maternal pre-eclampsia/eclampsia, Apgar score at 1st, 5th, 10th minute, respiratory status at birth, use of oxygen and/or positive pressure ventilation with AMBU bag at birth. We also collected data on type of neonatal sepsis, type of every antibiotic used during hospitalization, start and end date of every antibiotic type, presence of RD, number, type and length of every respiratory support device used during hospitalization and the outcome at NICU discharge. Due to the study setting , microbiological tests (pathogen typing and AMR profiling) were not available and were therefore not included in the analysis.The antibiotic management protocol at the SLCH NICU had three different antibiotic lines. The first two lines were based on WHO and Ethiopian guidelines : the firThe primary endpoints were the number of newborns assigned to each cluster of antibiotic line switch and the development of respiratory distress or death in NICU. The secondary endpoints were the proportion of patients diagnosed with early and late-onset neonatal sepsis subtypes.For descriptive purposes, frequency rates and percentages were used for categorical variables and medians with interquartile range (IQR) for continuous variables. Proportions for categorical variables were compared by the χ2 and Fisher’s exact test. Continuous variables were compared via Mann-Whitney-U non-parametric test.The optimal number of antibiotic management clusters to be imputed in the algorithm was evaluated through silhouette coefficient fitted on Gower distance computed as the average of partial dissimilarities across individuals included in the study. Variables included to be used in the Gower distance estimation were the type of antibiotics used as the first, second, or third line, respectively, and the length of therapy  of each antibiotic line. The Gower distance was selected because data had both continuous and categorical variables, and it allows for mixed variables to be used simultaneously. The individuals were assigned to the different clusters through the partitioning around medoids (PAM) technique with the k-medoids algorithm fitted on the previous computed optimal number of clusters and Gower distance .The PAM algorithm is based on the search for medoids (‘k’ representative objects) among the observations of the database. It has two phases. Build phase: (1) select k objects that will become the medoids; (2) calculate the dissimilarity matrix; (3) assign each object to the nearest medoid. Swap phase: (4) for each cluster, assess whether one of the objects decreases the average dissimilarity coefficient; if so, this is selected as the new medoid for this cluster; (5) if at least one medoid has swapped, go to step 3, otherwise end the algorithm. The goal of the algorithm is to minimize the average dissimilarity of objects with respect to the closest selected object.The differences between the clusters in terms of descriptive socio-demographic and clinical characteristics were assessed via Fisher’s exact test and the non-parametric Mann-Whitney-U test for categorical and continuous variables, respectively.The median survival time was examined by Kaplan–Meier estimates. To assess the effect of the antibiotic management clusters and the clinical variables on mortality a Cox Proportional-Hazards model was used with type of neonatal sepsis, age, weight, antibiotic management cluster, and presence of RD as potential determinants. Results were presented as hazard ratio (HR) with 0.95 confidential interval (CI). Since time at RD onset was not available, to explore its association with type of neonatal sepsis, age, weight, and antibiotic management clusters a logistic regression model was applied. Results were presented as odds ratio (OR) with 0.95CI. Additionally, the main clinical features were tested between clusters through Fisher’s exact test and Mann-Whitney-U non-parametric test. Dunn’s test with Bonferroni adjustment was used for nonparametric pairwise multiple comparisons. Post-hoc multiple comparison between clusters in the logistic regressions was carried out through Tukeys’ test.Finally, the same set of analyses  was conducted to assess differences in the distribution of the main clinical characteristics between patients with EOS and LOS.A p-value < 0.05 was considered significant. All analyses were performed using the R software (version 4.1.1) using package “cluster”, “ggplot2” and “Rtsne” to perform the cluster analysis and data visualization .In the study period 456 newborns were admitted to the NICU of SLCH and 196 (42.8%) were discharged with a diagnosis of probable neonatal sepsis. One newborn was excluded due to missing data  and 15 (7.7%) neonatal sepsis cases, respectively. Four antibiotic management clusters were identified  and was higher in the cluster 3 group when compared with clusters 1 and 2 (Table Since less than 50% of the cohort died (death rate = 13.3%), the Kaplan-Meier estimate median survival time was not computed Fig. . Death HThe main clinical features are listed in Table In this proof-of-concept study we explored the use of cluster analysis as a tool to discern the antibiotic lines used for neonatal sepsis in a resource-limited setting. Clustering allowed to identify a group of patients needing advanced or off-protocol antibiotic lines, and to assess frequency and types of switching between antibiotic lines in complex critically ill newborns. While three of the four clusters reflected the three lines of antibiotic therapy set out in the protocol , cluster 3 was characterized by the use of ceftriaxone/cloxacillin as a second-line antibiotics and the use of off-protocol antibiotics for the third line. Interestingly, cluster 3 had a higher odd of RD compared to both cluster 1 and 2. The severity of the disease impacts patient management as it trickles urgent use of broader spectrum antibiotics. As ceftriaxone broad spectrum focuses on gram-negative bacteria, clinicians decided to extend the spectrum by adding cloxacillin for gram-positive bacteria. When considering the survival probability, no differences were found based on antibiotic cluster assignment. The small sample size and the low number of patients in clusters 2, 3 and 4 may have led to this result. At the same time, it could also be related to the fact that prompt switch to a different line of antibiotics or the use of broader-spectrum antibiotics may have offset more severe infections or to the fact that some newborns may have died before switching antibiotic lines.Cluster analysis also identified the frequency of antibiotic lines used, estimating the need to switch between lines due to ineffectiveness related to sepsis severity or antibiotic-resistant pathogens. In one out of four newborns of these cases , the first line antibiotic therapy with ampicillin plus gentamicin was not sufficient, in line with an increase in resistance to WHO-suggested first line therapy. The 5 common groups of Gram-negative bacteria , the main cause of neonatal sepsis in LMIC, are resistant to ampicillin and gentamicin in 40–100% of cases, depending to the bacteria involved –23.The second line including a third-generation cephalosporin (i.e. ceftriaxone) required additional antibiotics in 12.3% of cases, leading to the emergence of clusters 3 and 4 in the present study. A review and meta-analysis in sub-Saharan Africa showed Gram-negative resistance to ceftriaxone ranging between 33% and 49%  while stCompared with high-income countries, the incidence of neonatal sepsis has been reported to be 40 times higher in LMICs . In the In our study, EOS had a prevalence three times higher than LOS. A study in Nepal showed a similar prevalence of EOS (71.2%) when considering clinically suspected neonatal sepsis. Higher EOS prevalence (between 84.1% and 90.2%) was reported in two studies conducted in different areas of Ethiopia , 27. LOSMortality rate was higher for infants with EOS (16.4%) compared to those with LOS (4.1%). A similar case fatality ratio was reported in literature for EOS (18%) and it was higher when compared to both community and hospital acquired LOS . When evThe main limitation of the study is the retrospective design and the small sample size of patients with neonatal sepsis and the small number of newborns assigned to clusters 2, 3 and 4. Cluster analysis, as an unsupervised learning technique, works best with large numbers. In addition, the small sample size did not allow the cluster to be used as a time-varying covariate making survival analysis to be interpreted with caution in the first 6 days since infants assigned to cluster 1 might have died before they had a chance to switch antibiotics and thus be assigned to another cluster. Nevertheless, to the best of our knowledge, this is the first study that seeks to apply an indirect technique to study antibiotic stewardship in settings where disease severity, patient complexity and lack of microbiological laboratory capacity leads to piling up of antibiotic prescriptions. Second, the lack of blood culture and pathogen isolation has prevented the diagnosis of confirmed neonatal sepsis and characterization of the antibiotic resistance profile. In addition, because of the lack of more specific blood tests  and blood cultures, some of the infants diagnosed with neonatal sepsis, based on symptoms and white blood cells, may actually have had perinatal asphyxia. Moreover, respiratory distress was collected as binary data; this prevented the application of a competing risk analysis. Finally, the study was conducted in a referral hospital and therefore involved a population of newborns that may have had more severe characteristics than those in peripheral health centers.The present proof of concept study used cluster analysis to depict the challenges of antibiotic management in a LMIC NICU. First-line antibiotics recommended by the WHO were not sufficient in one quarter of probable sepsis cases. Second-line treatment with third-generation cephalosporins was also insufficient in one in ten patients. The use of a machine learning technique allowed disentangling a patient group that received a different treatment than protocolized and more frequently evolved in RD. The use of different statistical methodologies should be encouraged to collect data where laboratory tests for sepsis typing and bacteria identification are not available so as to expand data pools from low resourced settings.Below is the link to the electronic supplementary material.Supplementary Material 1


Streptococcus salivarius (25%), Streptococcus vestibularis (19%), Streptococcus oralis (13%), and Staphylococcus aureus (6%) was identified while in the case of AT, Streptococcus salivarius (11%), Streptococcus parasanguinis (11%), Staphylococcus epidermidis (12%), Enterococcus faecalis (9%), Staphylococcus hominis (8%), and Candida albicans (6%) were identified. Notable to specified that the Candida albicans was noticed only in AT samples, indicating a negative impact on the antibiotic therapy.In the present research, the MALDI-TOF MS technique was applied as a tool to rapidly identify the salivary microbiome. In this fact, it has been monitored the changes occurred in molecular profiles under different antibiotic therapy. Significant changes in the composition of the salivary microbiota were noticed not only in relation to the non antibiotic (non-AT) and antibiotic treatment (AT) groups, but also to the used media, the antibiotic therapy and co-existed microbiota. Each antibiotic generates specific changes in molecular profiles. The highest number of bacterial species was isolated in the universal culture medium (72%) followed by the selective medium (48% and 38%). In the case of non-AT patients, the prevalence of The accuracy of the MALDI-TOF MS technique was performed by the 16S rRNA gene sequencing analysis—as a reference method. Conclusively, such an approach highlighted in the present study can help in developing the methods enabling a faster diagnosis of disease changes at the cellular level before clinical changes occur. Once the MALDI tool allows for the distinguishing of the microbiota of non-AT and AT, it may enable to monitor the diseases treatment and develop a treatment regimen for individual patients in relation to each antibiotic.The salivary microbiota of antibiotic-treated patients was more bacteria varietyMALDI-TOF MS is a promising tool for recording of reproducible molecular profilesOur data can allow to monitor the treatment of bacterial diseases for patients Since the time when the first antibiotics were introduced to treat bacterial infections, drug resistance to pathogens has become a serious health problem. The reasons include various factors such as an irresponsible dosage of antibiotics, naturally occurring mutations, and the transmission of drug-resistant strains. Microorganisms can have both positive and negative impacts. Many of them can make food go bad and cause serious diseases. For this reason, it is extremely important to search for quick and reliable methods to identify the basic infectious agents such as bacteria, which is particularly important in the medical diagnostics  once we were restricted in the sample number collection. Generally, all the patients were instructed to avoid eating, drinking, and brushing their teeth for 2 h before the saliva collection.Brain Heart Infusion (BHI)  and two selective mediums such as the Vancomycine Resistant Enterococci Agar Base (VRE) , and the Azide Blood Agar BASE (AZB)  were chosen. The saliva was diluted with sterile peptone water  in a 1:9 (v/v) ratio. The cultivation was performed by the serial dilution method based on the procedure of Abouassi and co-workers  were then used for the identification. Owing to problems with the identification by the MALDI-TOF MS on AZB (considered as a selective medium), the respective medium was changed to the Tryptic Soy Agar . TSA is considered a universal medium and applied as a routine diagnostic medium. The colonies isolated on the AZB medium were transferred to the TSA medium, incubated for 24 h at 37 °C in aerobic conditions, then identified using the MALDI tool.The standard extraction protocol was adopted from our previous study, Pauter et al. with some changes  each of the identified microbial genus. Moreover, Bacillus subtilis was also chosen, based on the unreliable low score (1.6) generated by the MALDI-TOF MS.To correlate the data obtained from the identification by the MALDI-TOF MS, the 16 rRNA sequencing method was performed. There was an attempt to select one species . The polymerase chain reaction amplification was performed using universal primers as forward: 27F(5-AGAGTTTGATCMTGGCTCAG-3) and reverse primers: 1492R(5-GGTTACCTTGTTACGACTT-3). After that, the PCR amplification products were purified and the sequencing of the amplified fragments was performed by using the Sanger dideoxy method by Genomed . Then, from the received sequences, the contigs were submitted using the BioEdit Sequence Alignment Editor ver. 7.2.5 software (12.11.2013) Hall . FinallyThis study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Bioethical Commission of Collegium Medicum in Bydgoszcz of Nicolaus Copernicus University in Torun, Poland, according to the agreement number 477/2021 – 14.09.2021. A written informed consent was obtained from all the participants.In the present research, the emphasis is put on the microbiota differences in patients with non- and under-antibiotic therapy. Moreover, to date, no work has been focused on the investigation of protein profile changes of isolate species identified in the patient’s microbiota not undergoing and undergoing the antibiotic therapy.5 and 107 CFU/mL. However, the number of CFU in the non-AT patients was noticed more than 107 CFU/mL (64% of patients). Only in the case of non-AT2, non-AT6, non-AT7, non-AT8, and non-AT10, (36%) the bacterial count was around 106 CFU/mL. The slight differences in the abundance can be associated with various lifestyles and distinct genotypes of the hosts  in Table B. subtilis (AT12) was found to be below 1.7 (log (score) = 1.6). Moreover, it is necessary to underline that S. aureus and S. pneumoniae were noticed only in non-AT group, whereas E. faecalis, E. faecium, S. epidermidis, B. subtilis, S.cohnii and yeasts C. albicans, C. glabrata, and C. dubliniensis were found only in AT group. Based on the shown date, it can be noticed that the identification for S. salivarius cultivated on various medium was similar , . In contrast, this signal disappeared in the case of the VRE medium, and the new signal was registered at 2984 m/z. Moreover, the signal m/z= 7888 was observed in the protein profile of S. salivarius identified on the BHI and VRE growing media in the non- and treated group. It is notable that some signals are present only in one mass spectra of non-AT4 in the case of each medium: 9030 and 10268 m/z in BHI; 9443 m/z in VRE and 10840 m/z in TSA. Based on the mass spectra of S. salivarius in the patient group treated with antibiotics (AT13 and AT1), the common signals  can be noticed. Furthermore, the m/z = 10068 was noticed in the universal media (BHI and TSA). However, the differences in the protein profile of the studied bacteria strain were also recorded. The signals at 9090 m/z were observed in BHI and at 9444 m/z in the VRE growing medium.The only one - btilis AT2 was fou), Table . The comS. salivarius can be correlated with the salt mixture present in this selective culture medium. Karamonová and co-workers , BA), and selective cultivation medium Enterobacter sakazakii Isolation Agar (ESIA). It was observed that the intensity of the recorded mass spectra was lower in the case of the ESIA medium than in the universal media . It was suggested that the unsatisfied intensity of the protein profile of Cronobacter sakazakii CB03 can be caused by the deficient composition of the selective medium rather than the universal media and the presence of specific (selective) substances  as inhibitors of growing microbial competitors. Finally, the TSA medium was chosen to further analysis by the MALDI-TOF MS  and the antibiotic treatment (AT7) patients were also compared , the non-common signals m/z 4923     LeBel .Micrococcus, Enterococcus, Serratia, Escherichia, Rothia, Candida, Staphylococcus, Bacillus, Streptococcus, Lactobacillus, and Neisseria) were noticed. The relationship between the identified microorganisms and the reference strains was found. The showing longest distance level correlation between the bacteria strain was observed in the cluster belonging to the Staphylococcus genus. Moreover, the cluster describing the Enterococcus genus indicates the shortest distance among the identified species. According to the dendrogram, a close relationship between the Serratia and Escherichia genus can be noticed. Moreover, the Bacillus genus was also included in the phyloproteomic tree , and the phyloproteomic tree was created. Figure ree Fig. , and (maBacillus subtilis was obtained while the standard method for the bacteria identification (16S rRNA gene sequencing) was performed. Furthermore, to confirm the accuracy of the MALDI results for all the identified 11 genus of bacteria, the one species from each cluster was selected . In addition, the Streptococcus pneumoniae as a serious pathogen and Staphylococcus cohnii showing the high level of the antibiotic resistance were also chosen. However, the genus with the low abundance of percentage in the identification including Candida, Rothia, Escherichia, Serratia, and Micrococcus was considered in the PCR analysis.Considering the manufacturer’s guidelines, the low score value of 1.6 Table , only foBacillus subtilis). However, the low identification level was verified in the PCR analysis. On the basis of the data from the PCR assay, the value of identification was over 99.5% for all the studied bacteria species. Moreover, the following accession numbers were given to the bacteria: B. subtilis (MZ336018); N. perflava (MZ191898); Lactiplantibacillus plantarum (the previous form Lactobacillus plantarum) (A taxonomic note on the genus Lactobacillus) (MZ411566); S. salivarius (MZ191906); S. aureus (MZ191908); S. epidermidis (MZ411533); E. faecalis (MZ191905); S. pseudopneumoniae (MZ191882), and S. cohnii (MZ191897).Then, the results received by the MALDI-TOF MS (score > 2.00) were correlated with the 16S rRNA gene sequencing method  and Streptococcus vestibularis (19%) dominated in the non-antibiotic treatment patients. Another predominant bacteria species in patients with the normal salivary microbiome were Streptococcus oralis and Staphylococcus aureus.Regarding the investigated radar chat, the differences between the salivary microbiota of non-AT and AT patients were found. It can be observed that the S. salivarius to R. mucilaginosa) were identified. Compositionally, the most abundant microorganism present in the AT patients were Streptococcus salivarius(11%) Streptococcus parasanguinis (11%), Staphylococcus epidermidis (12%), Enterococcus faecalis (9%), Staphylococcus hominis (8%), and Candida albicans (6%).The blue area of the web chart Fig.  down shoThe orange color indicates that the saliva of patients under the antibiotic therapy was more bacterially rich than the non-AT group. It was also observed that pathogenic microorganisms dominated in the group. It can be assumed that the type of the antibiotic treatment influenced the salivary bacteria composition in the AT patients. In comparison with patients with normal  salivary microbiota, more diversity of microorganism and more abundance of pathogenic bacteria can be noticed in the AT group, which can be associated with stress conditions under the antibiotic treatment.The correlation between the identified pathogen and the antibiotic was shown in the cluster analysis Figs.  and 5. FS. vestibularis is shown  and antibiotic treatment (ciprofloxacin). A similar effect was observed for S. oralis , azithromycin (AT1), and clindamycin (AT5). Regarding the other two strains found in both patients group—S. salivarius and S. parasanguinis—the comparative analysis revealed the highest proteomic variations; however, grouping according to drug-treated and untreated patients was not as evident as in the former cases. Nonetheless, S. parasanguinis strains isolated from the AT patients were placed into different clusters according to the type of antibiotic used, where strains from piperacillin-treated patients (AT10 and 12) were much more similar to each other than those from ciprofloxacin, amoxicillin, and clindamycin with levofloxacin  or Bacillus subtilis (AT12) demonstrated a more unique proteins pattern than those comprised of streptococci only (AT2 or non-AT6). A similar phenomenon was noted for S. salivarius, where the strains showing extremely different protein profiles came from samples significantly different in the species composition—non-AT7  vs. non-AT8 —or the antibiotic used—AT13 (clindamycin with levofloxacin) vs AT4 (clindamycin with metronidazole). All in all, comparing species derived from the saliva of treated and untreated patients, the impact on the proteomic diversification increases as follows: the type of the antibiotic used>coexistent microbiota>culture medium type.The analysis of the intraspecific proteomic variation within distinct microbial species derived from the saliva of patients treated and untreated with antibiotics revealed the impact of the treatment undertaken, associated microbiota, and applied culture conditions on the generated protein profiles Figs.  and 5. Rown Fig. , such dilis Fig. , where icin Fig. . AdditioC. albicans  most likely resulted from the type of antibiotic treatment undertaken—ciprofloxacin with levofloxacin vs. clindamycin in the case of former and piperacillin vs. clindamycin or cefotaxime in the latter one. Moreover, among S. epidermidis strains the most distinct proteins profile was demonstrated by the isolate AT12—the only one that derived from the sample in which the presence of Candida spp. was not observed.Similarly, a comparison of the proteomic diversity among microbial species that occurred only in the AT patients was performed Fig. . The effans Fig.  along wiins Fig. . InteresS. aureus strains based on their protein patterns. As the authors pointed out, it may result from the induction of some new metabolic pathways in bacteria leading to the appearance of more discriminant signals.Intraspecific differences in microbial protein profiles depending on culture media compositions were recognized as significant since 50% of the peaks of all bacteria are non-ribosomal proteins, which are more or less metabolic status dependent  leading to misidentification. Knowing that in the case of closely related species, the genomic and proteomic differences are very small, even slight variation in the culturing conditions may matter.According to previous reports  and Streptococcus vestibularis (19%), Streptococcus oralis (13%) and Streptococcus parasanguinis (6%)) and in the AT , Streptococcus parasanguinis (11%), Staphylococcus epidermidis (12%), Enterococcus faecalis (9%), Staphylococcus hominis (8%), and Candida albicans (6%)) groups. The salivary microbiota of antibiotic-treated patients was characterized by a more bacteria variety; the appearance of the Candida albicans species was noticed only in the AT patient indicating a negative impact of the antibiotic administration on the patient microbiota. Moreover, the proteomic analysis showed the influence of the antibiotic therapy, composition, and abundance of saliva microbiota and used growth medium on the recorded protein profiles. Remarkably, the MALDI-TOF MS analysis represents a promising method for a large-scale, less labor intensive, rapid, and cost-effective record of reproducible molecular profiles of microorganisms, particularly the salivary bacteria. It is notable that the proposed approach enables the early administration of the specie-specific antimicrobial therapy in the patients. Therefore, our data can allow a medical diagnosis to be confirmed and they may also enable us to monitor the treatment of diseases and develop drugs for individual patients. Moreover, the presented study can be the base to develop methods enabling a faster diagnosis and the detection of disease changes at the cellular level before clinical changes occur. To summarize, the obtained results suggest that tracking proteomic intra-specific variation within microorganisms may be a promising tool for the future use to examine the effectiveness of the applied antibiotic curation and to check whether one deals with mono- or polymicrobial cultures including the presence of the other pathogenic species.Our results indicated significant differences in the saliva microbiota between non-antibiotic treatment and antibiotic treatment patients. We noticed the dominated and characteristic microorganisms in the non-AT (