Haemoproteus gallinulae are presented. Phylogenetic analysis of standard DNA sequences revealed a close relationship between Haemoproteus species and their classification into the subgenus Parahaemoproteus. However, three Plasmodium species were found to have a wide range of hosts. The phylogenetic tree of this parasite clearly showed individual clusters. For one of them, the species was not identified; however, it matched a previously reported species that was isolated from mosquitoes. Two of the Plasmodium species were identified as well-known, generalist parasites that cause severe diseases in hosts: P. collidatum and P. elongatum. This study revealed the role of White-breasted Waterhens as carriers of haemosporidian parasites. Our findings provide potential information for further research.Haemosporidian parasites infect terrestrial vertebrates as well as avian species worldwide. However, for resident birds of the Rallidae family, such as White-breasted Waterhens, few data are available regarding haemosporidian infection. Therefore, this study aimed to detect haemosporidian infection in White-breasted Waterhens, microscopic and genetic examinations of blood samples were performed. Four species of haemosporidians were identified; however, the morphological features of only Haemoproteus gallinulae, Plasmodium collidatum, Plasmodium elongatum, and an unidentified Plasmodium species. H. gallinulae was characterized with morphological features in White-breasted Waterhens for the first time; the morphological characteristics were consistent with previous descriptions. H. gallinulae was more closely related to Haemoproteus species of Passeriformes birds than to those of Gruiformes birds. The Plasmodium parasites infecting these White-breasted Waterhens previously caused severe avian malaria in other host species. The unidentified Plasmodium species had rarely been documented, although it was reported in the Culex vector and was possibly associated with specialist parasites either as host or habitat. Our findings reveal multiple haemosporidian species reflecting the role of this avian host as a carrier of haemosporidians. This study offers species records and molecular materials that may provide critical information for further targeted research into these haemosporidia.Haemosporidian parasites are vector-borne parasites infecting terrestrial vertebrates as well as avian species, such as the White-breasted Waterhen, a Gruiformes waterbird found in lowlands near wetlands and distributed throughout Thailand. However, information regarding haemosporidia infection in this species is lacking. To establish regional information, 17 blood samples were collected from White-breasted Waterhens. Four haemoparasite lineages were identified in six blood samples:  Haemoproteus, Plasmodium, Leucocytozoon, and Fallisia [Plasmodium homocircumflexum, which further caused anemia and cerebral paralysis and led to the death of experimentally infected canaries [P. elongatum (GRW6) and P. matutinum (LINN1) in New Zealand Kiwi (Apteryx spp.) have also been reported [Streptopelia turtur) [Alauda arvensis) [Haemosporidian parasites in all terrestrial vertebrate, likewise in avian species, are vector-borne blood parasites classified into four genera: Fallisia . These pFallisia  and of tFallisia . MoreoveFallisia ,5. Howevcanaries . Highly reported . Further turtur)  and Skylrvensis) . TherefoAmaurornis phoenicurus) is a waterbird of the family Rallidae in the order Gruiformes that is resident in south and southeast Asia. In Thailand, these birds are generally found near freshwater in agriculture fields [Plasmodium parasite in Malaysia [Haemoproteus gallinule, similar to other Rallidae such as Red-legged Crake  and Ruddy-breasted Crake (Porzana fusca) [Haemoproteus parasites have rarely been reported in wild birds of Thailand. Only eight morphospecies have been solely isolated from passerines [Haemoproteus parasites of Gruiformes were found to have only four morphospecies, and singular H. antigonis was available for molecular detection and identification (barcoding) [The White-breasted Waterhen  . Haemoprsserines . Furtherrcoding) . A surveAmaurornis phoenicurus) are a resident bird that frequently forages around lowland paddy fields where abundant with insects, small fish, aquatic invertebrates, and grains. This bird nestled in dry locations and colonized nearly freshwater. During the 2021 dry season, birds were decoy-trapped individually using bird sounds and mist nets in the Salaya suburb, Phutthamonthon district, Thailand . This location is represented and relates to the White-breasted Waterhen habitat. Seventeen birds were trapped and transported to The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals, Faculty of Veterinary Science, Mahidol University. Approximately 0.5 mL of blood was collected from the brachial vein and a thin blood smear was immediately prepared, allowed to air-dry, and fixed with absolute methanol. Duplicate or triplicate smears were prepared for each bird. The remainder of the blood sample was transferred into a 1.5-mL EDTA tube and stored at −20 °C until DNA extraction was performed. Then, all birds were released back into their original habitat at the sampling site. All procedures involving White-breasted Waterhens were reviewed and approved by the Faculty of Veterinary Science, Animal Care and Use Committee of Mahidol University (protocol no. MUVS-2020-11-55).White-breasted Waterhens  in accordance with the manufacturer’s recommended protocol with phosphate buffer (pH 7.2) at 5% concentration for 1 h. Microscopic examinations of at least 100 fields were performed under low magnification (×400) for 5–10 min and high magnification (×1000) using a Nikon/ECIPSE Ti2 Inverted Microscope . The intensity of infection was estimated from the number of infected erythrocytes per 10,000 cells . The morrasites” . To estirasites” .b (cytb) gene was amplified using nested polymerase chain reaction (PCR), as previously described [®  and visualized using a BioSens SC-Series 710 gel documentation system . The positive PCR products were subjected to nucleotide sequencing based on the Sanger sequencing method by a commercial company  using both strands of inner primers. The electropherograms of all obtained sequences were carefully checked for double bases, which indicate co-infection, using BioEdit version 7.0.5.3  [cytb sequences with no match found to those previously published were considered to be a new lineage of haemosporidia and named according to the MalAvi nomenclature [cytb sequences of all detected haemosporidian parasites were submitted and deposited to GenBank under accession numbers OQ565584–OQ565589.DNA was extracted from whole blood samples using the Genomic DNA Mini Kit (blood/cultured cells)  in accordance with the manufacturer’s recommended protocol. The partial cytochrome escribed , with thCA, USA) . SequencCA, USA) . SubsequCA, USA) . The obtnclature . The obtH. gallinulae and other Plasmodium parasites and the previously reported morphospecies of haemosporidian parasites in Asia and Oceania, which is the habitat of the avian host White-breasted Waterhen. The alignment was constructed with 27 partial cytb sequences (479 bp) of well-known morphospecies of Haemoproteus and Plasmodium species, 13 individual lineages, and Leucocytozoon majoris (as the outgroup) using MAFFT version 7.520 software [6) generations, with sampling every hundredth generation. The first 25% of the tree was discarded as burn-in. The remaining 22,500 trees were calculated to determine the majority rule consensus tree, which was then visualized using FigTree.v1.4.4 [A phylogenetic tree was constructed to determine the relationships between the newly obtained molecular characteristics of software . A Bayessoftware . The bessoftware . A Bayese.v1.4.4  with thee.v1.4.4 .t-test, after normal distribution and homogeneity of variance were confirmed using Shapiro–Wilk’s test and Levene’s test, respectively. A p-value of ≤0.05 was considered statistically significant.The heterophil-to-lymphocyte (H:L) ratio between uninfected and infected White-breasted Waterhens was compared using the Mann–Whitney U test. Comparisons of mean morphometric measurements between uninfected and infected erythrocytes as well as between macrogametocytes and microgametocytes were performed using Student’s independent p < 0.001) in infected White-breasted Waterhens than in uninfected ones  [cytb genes were amplified and the BLAST results were 100% identical to two known species: P. elongatum (lineage GRW06) and P. collidatum (lineage FANTAIL01) found in individual birds and an unknown Plasmodium species (GenBank no.: AB601445) found in two birds. Furthermore, the partial cytb sequences were deposited in GenBank under the accession numbers OQ565584–OQ565589 and one newly obtained sequence of H. gallinulae was assigned the lineage name AMPHO01 in the MalAvi database. Voucher specimen (accession no. G466274) of H. gallinulae from White-breasted Waterhens were deposited in the Queensland Museum, Queensland, Australia.The overall prevalence of haemosporidian infection was 35% (6/17). No birds in this study had signs of pathogenicity. The differential WBC count of haemosporidian infection is shown in ted ones . Two bironoides) . The intYoung gametocytes A–D: Thesp < 0.01). Erythrocyte nuclei were slightly atrophied (p < 0.05) in terms of the width and area but not in length (p > 0.05). Fully grown gametocytes distinctly laterally displaced the nuclei of the infected erythrocyte (Macrogametocytes E–L: Thesp > 0.05). The nuclei of infected erythrocytes were occasionally less displaced than those of macrogametocytes (p < 0.05).Microgametocytes M–P: Thescytb sequences belonging to genus Haemoproteus . Moreover, the other four sequences corresponded to three different lineages of Plasmodium parasites. Two sequences belonged to unidentified Plasmodium lineages (GenBank nos.: OQ565586 and OQ565587) and showed identity with the lineage CXNIG01. The other partial cytb sequence (GenBank no.: OQ565589) had 100% similarity to the P. collidatum lineage FANTAIL01, and the final sequence (GenBank no.: OQ565588) was closely related to P. elongatum. Only unique partial cytb sequences were included in the phylogeny  and Passeriformes. Furthermore, a closely related morphospecies of H. gallinulae was H. antigonis, belonging to the lineage GRUAME01, and described the only available molecular characteristics of Gruiformes birds. The genetic distance was 13.7% and likely higher than the lineage among the subgenus Parahaemoproteus. This indicated that the genetic distance of this cytb gene and the close relationship of Haemoproteus morphospecies found from Gruiformes birds were likely inconsistent. Regarding phylogeny, the Plasmodium lineage obtained in this study was clearly clustered individually (Plasmodium lineage (OQ565586) in this study was identical to Plasmodium lineage CXNIG01 and closely related to the P. elongatum GRW06 lineage, with a genetic distance of 4.3%, which was identical to the Plasmodium lineage (OQ565588) currently found. Another lineage of Plasmodium (OQ565589) was identical to P. collidatum FANTAIL01. The genetic divergence of lineage FANTAIL01 was related to P. ashfordi GRW02 with a genetic distance of 7.9%.The phylogenetic analysis revealed that the lineage of oproteus , box Aa.vidually , box B. H. gallinulae and describe its morphological characteristics in White-breasted Waterhens. Of the 15 Rallidae species found in Thailand, the White-breasted Waterhen is the only one surveyed for haemosporidian parasites [Porzana bicolor), Grey-headed Swamphen , Red-legged Crake , and White-browed Crake (Porzana cinerea) or winter migratory species such as Baillon’s Crake (Porzana pusilla), Band-bellied Crake (Porzana paykullii), Common Moorhen , Eastern Water Rail , Eurasian Coot (Fulica atra), Ruddy-breasted Crake (Porzana fusca), Slaty-breasted Rail , Slaty-legged Crake , Spotted Crake (Porzana porzana), and Watercock  [This is the first report on haemosporidian species from White-breasted Waterhens in Thailand; furthermore, we herein report the molecular characteristics of arasites . Moreovearasites . Moreovecinerea) . These mcinerea) . The H:Lcinerea) ,33. SignHaemoproteus parasite for which the characteristic morphology was consistently identified to be Haemoproteus gallinulae. This Haemoproteus species was considered the first record in Thailand. Moreover, there were no data for GenBank accession and lineage code available for H. gallinulae [Rallina eurizonodes) and American Coot (Fulica americana) [Gallinula chloropus) [H. gallinulae was hypothesized to depend on the other genera of hosts in the Rallidae family. In this study, the morphological features of the parasite were characterized in another host species, Amaurornis phoenicurus  broadly halteridial form in fully grown gametocytes   or type loropus)  exhibiteenicurus . The effetocytes E–P; (2) es birds . Howeveracharovi . TherefoH. gallinulae was distinctly classified into the subgenus Parahaemoproteus. Although the morphological characteristics between H. gallinulae and H. antigonis were indistinguishable, the details of pigment granules differed [H. asymmetricus (9.6%) and H. sanguinis (9.8%), indicating that close genetic relationships exist between distinguishable morphospecies found in different hosts at the order level [cytb sequence as well between H. janniae and H. iwa, but the morphospecies of these two Haemoproteus species are distinguishable [cytb sequence was not sufficient for distinguishing between some species of Haemoproteus parasites. Therefore, Haemoproteus species identification was performed based on morphological and molecular features.The phylogeny of the haemosporidian parasite in White-breasted Waterhens revealed that differed ,36. Howeer level ,38. Simiuishable ,40. ExamPlasmodium parasite in this study should be discussed. First, the P. collidatum (FANTAIL01) lineage was considered a generalist lineage that infects a wide range of hosts and had previously infected 16 bird species, including Rufous Fantail (Rhipidura rufifrons) [Plasmodium lineage had never been reported in White-breasted Waterhens. Subsequently, this Waterhen was added to one of several host species of the FANTAIL01 lineage in south and southeast Asia and the Oceania region [Spinus spinus) [Plasmodium parasite caused significantly virulent avian malaria.Furthermore, the three obtained lineages of fifrons) ,42. Howea region ,43. MorePlasmodium lineage was the well-known P. elongatum GRW06 lineage, which is also presently found to infect White-breasted Waterhens. Although there are previous reports of infection in other species of the Rallidae family, such as Corncrake (Crex crex) and Purple Gallinule (Porphyrio martinica), this Plasmodium lineage was distributed worldwide in resident and migratory birds such as Great Reed-warbler , Madagascar Swamp Warbler , Eurasian Marsh-Harrier (Circus aeruginosus), and Carrion Crow (Corvus corone) [Spheniscus magellanicus) [P. elongatum GRW06 was a cause of severe avian malaria [Plasmodium lineage had not been identified at the species level belonging to the CXNIG01 lineage that was isolated from Culex nigropunctatus mosquitoes in Japan [Plasmodium (GenBank no.: AB601445) was previously reported to infect White-breasted Waterhens. This lineage of Plasmodium parasite was rarely recorded in islands of Japan. Therefore, to the best of our knowledge, this is the first report of this Plasmodium lineage in additional geographic regions in Salaya suburb, Thailand.Another  corone) ,45,46,47lanicus) . Further malaria . Anotherin Japan . MoreoveH. gallinulae AMPHO01 lineage was characterized morphologically from different host species in Rallidae. Phylogenetic analysis revealed close lineages of Haemoproteus species classified into the subgenus Parahaemoproteus. Although morphospecies between H. gallinulae and H. antigonis (Haemoproteus of Gruiformes host) were difficult to distinguish, the genetic distance of partial cytb sequences was probably distinctly different. Moreover, this study found three different lineages of Plasmodium parasite. Although one of them was not identified as a species found in Culex mosquitoes, it was found in the avian host White-breasted Waterhen. Furthermore, two lineages, namely, P. collidatum and P. elongatum, were identified for the first time in this avian host. These findings reveal the role of White-breasted Waterhens in possibly harboring multiple species or serving as a reservoir host of the haemosporidian parasite. Knowledge of the relationship between the host and haemosporidian parasites can provide a principle for disease management.This is the first study of haemosporidian infection in White-breasted Waterhen resident birds. The findings present a new record of haemosporidian parasites in Thailand, and the


Portable computerized devices represent a potential source of healthcare infections. The objective was to assess the knowledge, attitudes, and practices (KAP) of healthcare workers (HCWs) toward infection control practices used with Close Loop Medication Administration (CLMA) devices. Additionally, to quantify the impact of education and training on the bacterial burden on CLMA devices.The study design consisted of two steps: a cross-sectional study was conducted among HCWs working in a tertiary care center in Riyadh, Saudi Arabia. A 32-item questionnaire was used to assess KAP information. The second step was environmental samples collected from the surfaces of CLMA devices before and after implementing a multifaceted intervention.P<0.001). It was also better among those with a longer duration of work experience (P<0.001) and those who received related training (P<0.001). Approximately 75% of HCWs expressed their need for more information about CLMA. Post-interventional samples had much lower bacterial burden, with the positive rate reduced from 51.4% before intervention to 16.8% after intervention (P<0.001).A total of 325 HCWs were included in the study. The mean age was 32.6±7.4 years. The majority were females (92%) and nurses (91.3%). The overall KAP score was 74.8%, 74.2% adequate knowledge, 79.3% positive attitude, and 71.3% appropriate practices. KAP score was better (≥ median KAP score) among HCWs working in laboratory and organ transplant units (Awareness and behavior of HCWs about appropriate infection control practices related to portable devices is still inadequate. A multifaceted intervention including education and training significantly reduces the bioburden on portable devices. Healthcare-associated infection (HAI) remains a major healthcare challenge, threatening patients' safety in healthcare settings globally ,2. HAI aContaminated surfaces play an important role in the transmission of HAI and antimicrobial resistance . They arThe use of portable computerized devices  are becoming very prevalent in today's healthcare services . They arThe study design consisted of two steps: the first was a cross-sectional study conducted in April 2019 among HCWs to assess KAP toward IPC practices related to the use of CLMA devices. The second step was pre- and post-interventional assessment of a multifaceted intervention, including education and training, on bacterial contamination of these devices, using environmental samples from the surfaces of CLMA devices taken on May 2019 and again in March 2020.The present study has been carried out in different units/departments of King Abdulaziz Medical City, Riyadh (KAMC-R). It is an approximately 1730-bed tertiary care facility composed of two hospitals. KAMC-R provides healthcare services for almost 1.15 million eligible Saudi National Guard soldiers, employees and their families. Approximately 13% of the hospital beds in both hospitals were serving ICU patients, through 15 different ICUs. The facility is accredited by Joint Commission International (JCI). The following units/departments were included in this study: emergency department, intensive care unit, oncology/hematology, organ transplant, general pediatric wards, and laboratory.Previous studies that focused on the awareness of IPC practices related to fomites including medical equipment showed variable knowledge level ,15,19. THCWs closely working with CLMA devices in the target units/departments including nurses and technicians were included. Physician and administrative staff were excluded. There were no exclusions based on gender or the duration of professional experience. The participants were recruited conveniently through emails, phone messages, and personal visits to the target units/departments.An anonymous self-administered questionnaire was electronically sent to targeted HCWs. In addition to demographic and professional data, the questionnaire covered the three KAP domains of CLMA-related information. These included the pattern of use, IPC practices, and disinfection practices. They were arranged in 10 knowledge questions, 10 attitude questions, and 12 practice questions. Knowledge questions had the following responses “true”, “false”, and “don't know”. Attitude questions had the following responses; “strongly agree”, “agree”, “uncertain”, “disagree”, and “strongly disagree”. Practice questions were multiple-choice questions with variable responses. One-point was given to correct knowledge and practice questions. One-to five-points were given to increasing agreement of positive attitude. Face and content validity of the questionnaire was evaluated by infection control, infectious disease, and epidemiology professionals. The questionnaire was piloted on 15 HCWs, with very positive feedback. The questionnaire had good reliability with Cronbach's Alpha 0.76. A copy of the questionnaire is attached as supplementary material.A pre and post-interventional study was carried out between May 2019 and March 2020. Environmental samples were collected from the surfaces of CLMA devices in May 2019 and again in March 2020. The samples were tested for bacterial contamination using standard microbiological methods. A multifaceted intervention was implemented between May 2019 and February 2020. The intervention included education and training of HCWs in target units/departments, re-defining cleaning and disinfection responsibilities of HCWs, emphasizing the auditing role of clinical resource nurse, adding a checklist and schedules for cleaning and disinfection of CLMA device, and updating of the IPC manual.P-values were two-tailed. A P-value <0.05 was considered significant. SPSS  was used for all statistical analyses.Categorical variables were presented as frequencies and percentages while continuous variables were presented as means and standard deviations (SD). The overall KAP score was divided into two categories based on the median score: ≥ median and < median. Demographic and professional characteristics were compared between KAP groups. Chi-square or Fisher exact tests were used to compare categorical data while t-test was used to compare continuous data. The correlation between practices with both knowledge and attitude were done using Spearman coefficient. All As shown in P<0.001) and in non-Saudi compared with Saudi nationals (P<0.001). It increased with increasing age groups (P<0.001), increasing duration of work experience (P=0.004), and higher nursing titles (P=0.003). Awareness was highest among those working in the laboratory (87.5%), organ transplant units (72.0%), and intensive care unit (52%) and lowest in those working in general pediatric wards (32.8%) and emergency department (41.2%).A total 325 HCWs were included in the study. As shown in P<0.001), especially training covering cleaning and disinfection (P=0.002) and training provided by the company (P=0.027) but not colleagues (0.004). Additionally, awareness was better among HCWs who did not ask for more information about CLMA (P=0.023).As shown in P=0.011). Additionally, there was moderate positive correlation between attitude score and practice score (r=0.275 and P<0.001).As shown in Pseudomonas aeruginosa (16.4%), Coagulase-negative staphylococci (10.9%), Klebsiella pneumoniae (9.4%), Escherichia coli (7.0%), Diphtheroids (6.3%), Acinetobacter spp. (5.5%), Enterobacter spp. (3.9%), Staphylococcus aureus (2.3%), and Streptococcus spp. , and no Gram-negative pathogens were isolated , Out of 111 CLMA devices tested in the pre-interventional assessment, 57 (51.4%) were culture positive . Out of p. 1.6%, . Out of p. 1.6%, . They inp. 1.6%, . Post-inThis study assessed the KAP of HCWs (mainly nursing staff) towards IPC practices related to the use of CLMA devices. The overall average KAP score was 75%, which is probably inadequate. Approximately 96% of the current participants were aware of the role CLMA devices can play in transmitting infection while 82% were reporting frequent cleaning. However, there were doubts about the reported cleaning habits. For example, 76% of HCWs were not aware of the appropriate disinfectant, 42% were unaware that the cleaning is the responsibility of all device users, and 28% were not aware that cleaning should be done before and after use of the device. Such doubts were confirmed by the relatively high (51%) pre-intervention bacterial contamination of these devices.Comparing the current finding to existing literature is challenging due to lack of similar studies focusing on CLMA devices. Nevertheless, the current finding was consistent with previous studies that assessed awareness and behavior of HCWs, regarding cleaning and disinfection practices of hospital fomites. These studies focused on personal touch-screen devices such as mobile phones and tablets  and portAs expected, awareness and appropriate practices in our study was better in more experienced HCWs. For example, higher KAP scores were associated with longer duration of work experience, older age, and higher nursing titles. Previous studies showed that more experienced healthcare workers, specially nurses, are more likely to have better knowledge of and adherence with IPC practices . The lonClostridium difficile rooms from 20% to 100% [As expected, receiving CLMA-related training in this study was associated with better awareness and cleaning practices. Similarly, previous studies were consistent on the positive impact of education and training on cleaning practices ,24,25. FThe multifaceted intervention in this study was very successful in reducing the bio-burden of CLMA devices from 51% to 17%. The pre-intervention contamination is not surprising as previous studies reported bacterial growth in more than 85% of portable patient equipment, such as stethoscopes, otoscopes, and diagnostic ultrasound . HoweverBacterial examination before and after cleaning has been used by multiple studies in assessing the efficacy of cleaning methods . For exaOur study has some limitations. As the study used a cross-sectional design to assess CLMA-related awareness and practices, bias cannot be excluded, and causality cannot be proved. Additionally, the studied HCWs were recruited from a single institution which may limit the generalizability of the findings. Despite those limitations, this study investigated the reported cleaning practices using environmental samples. The combined study design allowed assessment of the impact of a multifaceted intervention. Finally, the intervention did not focus only on education and training but also monitoring, auditing, engagement, process adjustment, and manual update.In conclusion, awareness and behavior of IPC practices related to the use of CLMA devices were assessed in a group of HCWs. The overall KAP score was 75%, including 74% adequate knowledge, 79% positive attitude, and 71% appropriate practices. Better awareness and practices were associated with older age, longer duration of work experience, higher nursing titles, and receiving related training. A multifaceted intervention including education, training, auditing, engagement, process adjustment, and manual update significantly improved the CLMA-related cleaning practices. This was confirmed by reduction of positive bacteria cultures from 51% in pre-intervention samples to 17% post-intervention samples, with removal of pathogenic bacteria.Kassem Abou Yassine, Aiman El-Saed, Majid M. Alshamrani conceived the study idea. Kassem Abou Yassine; Aiman El-Saed, Majid M. Alshamrani, Sarr Ramou, Bassam H. Al Alwan, Kholoud Ameer, Mustafa Hawthan,and Mohammed Al Zunitan were involved in the methodology, data collection, Data curation, and analysis plan. Aiman El-Saed and Fatmah Othman analyzed the data. Majid M. Alshamrani,Kassem Abou Yassine; Aiman El-Saed, Sarr Ramou, Bassam H. Al Alwan, Kholoud Ameer, Mustafa Hawthan, Fatmah Othman and Mohammed Al Zunitan were involved in visualization, investigation, and validation of the study. All authors contributed to the first draft of the manuscript and critically revised the manuscript. All authors contributed to the drafting and editing of the final manuscript and have provided their permission to publish the manuscript. All authors agree to take responsibility for the work.The study obtained all required approvals from the IRB committee of King Abdullah International Medical Research Center (KAIMRC), Riyadh, Saudi Arabia. protocol number NRC23R/248/03.The authors received no financial support related to this research.All authors have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Solar energy, a prominent renewable resource, relies on photovoltaic systems (PVS) to capture energy efficiently. The challenge lies in maximizing power generation, which fluctuates due to changing environmental conditions like irradiance and temperature. Maximum Power Point Tracking (MPPT) techniques have been developed to optimize PVS output. Among these, the incremental conductance (INC) method is widely recognized. However, adapting INC to varying environmental conditions remains a challenge. This study introduces an innovative approach to adaptive MPPT for grid-connected PVS, enhancing classical INC by integrating a PID controller updated through a fuzzy self-tuning controller (INC-FST). INC-FST dynamically regulates the boost converter signal, connecting the PVS’s DC output to the grid-connected inverter. A comprehensive evaluation, comparing the proposed adaptive MPPT technique (INC-FST) with conventional MPPT methods such as INC, Perturb & Observe (P&O), and INC Fuzzy Logic (INC-FL), was conducted. Metrics assessed include current, voltage, efficiency, power, and DC bus voltage under different climate scenarios. The proposed MPPT-INC-FST algorithm demonstrated superior efficiency, achieving 99.80%, 99.76%, and 99.73% for three distinct climate scenarios. Furthermore, the comparative analysis highlighted its precision in terms of control indices, minimizing overshoot, reducing rise time, and maximizing PVS power output. Given the current economic crises, which have increased in severity with Covid 19, it has become imperative to integrate renewable energy sources (RESs) into the power grid and maximize the utilization of the available RESs to alleviate these crises . A futurThe main elements that limit the quantity of electrical energy that could be captured include atmospheric parameters, dirt, temperature (T), rainfall, cloud cover, and geography. Because of the radiation’s varying angle of azimuth per hour, the level of irradiance (I) usually varies during the daytime. Any PVS produces different amounts of electricity based on the load of the panel, which reduces the quantity of electricity generated even at the same I and T. The maximum power point (MPP) at a particular load occurs and changes throughout the course of a day with Ts and I variations, rendering it difficult to locate and compute the MPP . The priMPPT systems require a method of control to be able to boost their efficacy. Because of its ease of use and versatility in execution, the PID controller is frequently used in MPPT devices. With its straightforward framework, PID, nevertheless, exhibits a low efficiency for MPPT uses . The mosA number of the latest and most significant modern MPPT methods that potentially address some of the problems brought on by conventional MPPT controllers include soft calculation (SC), artificial intelligence (AI), and bio-inspired technology (BT) , 15. SopIn these studies, various advanced algorithms and techniques are presented to optimize power generation in PVSs. They include the use of FL based on particle swarm optimization in , adaptivThere is a wealth of research available in the literature aimed at addressing the drawbacks of existing techniques and trying to enhance them. PVSs are suggested for a unique FL-based mix MPPT strategy. To calculate the MPP, an offline current from a short circuit is employed, and the FL is then applied to get the precise magnitude of the highest possible power. The findings show that, in a range of climatic circumstances, the suggested method surpassed the combination technique (open-circuit voltage and P&O methodology) . A revolThe typical MPPT techniques, fractional open-circuit voltage (FOCV), and fractional short-circuit current (FSCC) were proposed in many MPPT applications, however, they did not demonstrate high efficiency and accuracy in reaching the MPP . Ref. 3 used a PThe PID controller, which is widely used in numerous MPPT approaches as well as other RES applications, is the most simple . CertainAccording to a cursory review of the scientific literature, CTs are simple to set up and yield beneficial outcomes for MPPT, but they have the problem of exhibiting unfavorable fluctuations close to MPP. However, they are difficult, costly, and time-consuming to use, SC techniques are the most effective at monitoring MPPT. Consequently, this inspired the author to create an adaptive regulator that may mitigate the shortcomings of CT and SC methods. It is therefore extremely difficult to significantly enhance the MPPT technique’s capabilities in terms of level of complexity, monitoring rapidity, precision, oscillations around MPP, and monitoring effectiveness in changing circumstances.The paper introduces an innovative INC-FST to improve MPPT efficiency, which will reduce system management complexities and effectively handle instabilities and disturbances in the investigated PVS. This work improves the INC-MPPT algorithm with a fuzzy self-tuning-based PID controller (INC-FST) for regulating the MPPT control of the studied system. This self-tuning algorithm will drive the DC-DC PC D to be updated to track the MPP with changes in I and T. The suggested method is simple, has higher dynamic responsiveness, barely oscillates near MPP, tracks quickly, and performs better in variable weather circumstances. The MATLAB/Simulink is used to investigate the proposed technique. Three tests are applied (variable I with constant T (C1), variable T with constant I (C2), and variable I with variable T (C3)) to assess the proposed effectiveness. The suggested technique efficacy under C1, C2, and C3 are 99.80%, 99.76%, and 99.73%, respectively. The PVS output, including power, voltage, current, and DC bus voltage, shows that the proposed technique performs admirably under a variety of conditions, including I and T fluctuations. The key finding of the suggested work is summarised as follows:For PVSs to enable effective MPPT, an innovative INC-FST is suggested.The suggested controller has low instabilities around MPP, excellent efficacy, faster rapid responsiveness, and quick converging time.The INC-FST is resistant to I and T change since it is adaptable.The enhanced algorithm is contrasted with three control approaches . The improved INC algorithm outperformed previous algorithms in some control indices, such as overshoot, rise time, and settling time, and demonstrated a quicker dynamic reaction for MPPT.The rest of this paper is organized as follows: Section 2 gives a synopsis of the system under study and the PVS modeling. Section 3 demonstrates P&O and INC MPPT algorithms. Section 4 explains a modified INC algorithm with an INC-FST controller. The simulation’s work findings are reported in Section 5. Finally, conclusions and discussions are presented in section 6.The grid-connected PVS consists of PV solar cells, PCs, filters, and grid interfaces control system as shown in The PV cell is the most main component of a PVS. A PV module is a group of connected cells to form a PV panel; many panels make up a PV array. Power generated by the PV generator is affected by T and I. The practical electrical circuit of the solar cell and the inverted diode is depicted in The output current model by the solar cell can be expressed in Calculations for the diode’s altered current based on the Shockley diode equation can be expressed as :ID=IOeThe PV cell current can be determined as:sh is the cell’s shunt resistance in Ohms, Io is the saturation current diode in ampere, q is the electron charge in coulomb and K is the Boltzmann gas constant. Both single-diode and double-diode types of PV modules are widely used today. Double-diode models can be used to correctly represent the solar panel. Because of its simplicity and accuracy, the single-diode model is utilized in this paper. The following equation can be used to express the current (Imp) at the point of maximum power.In which m is the diode quality factor, V is the PV cell output voltage, Tc is the cell’s absolute temperature in Kelvin, Rs is the cell’s series resistance in Ohms, and RImp is the maximum panel current and Vmp is the maximum panel voltage.However, the maximum power s (Pmax) is given by:VO, VPV), respectively, are related as:The output of the PVS is connected to the grid through a boost DC\DC-PC and DC\AC-PC as given in By adjusting the D, the output voltage of the boost PC is regulated to track the voltage at which the MPP is achieved.The primary role of MPPT is to maximize the amount of energy from the PVS by adjusting the system operating voltage at the most efficient value corresponding to the MPP at different Ts and Is . As the The P&O algorithm is the most popular MPPT algorithm. This method has many benefits, including low cost, rapid deployment, fewer parameters, and the flexibility to make changes , 34. TheIncremental variations in PV array voltage and current are sensed by the controller in INC algorithms to estimate the impact of a voltage variation. To keep up with rapidly changing conditions, this technique requires more controller computing than the P&O algorithm , 37. LikNormally, a source’s output voltage is positive. Consequently, this algorithm’s primary goal is to locate the voltage operating point where conductance equals incremental conductance. These ideas are expressed in Some modifications in the INC have been presented to update the controller parameters in light of the changes in the environmental conditions to track the maximum power rapidly.FL control is a method for developing nonlinear controllers based on heuristic data derived from expert expertise, as illustrated in E(K), which determines where the MPP is in the PV module. The CE(K) input determines whether or not the operating point is moving in the MPP direction. The increase in D is the output variable, which can take positive or negative values depending on where the operational point is located. To drive the load, this output is delivered to the DC-DC PC. An accumulator was used to calculate the D using the value of D provided by the controller.The slope of the P-V curve is the input The main drawback of this FL controller is that the PID controller parameters are not updated with variations in the Ts and Is which will lead to a non-guarantee of extracting the maximum power from the PVS at these environmental variations.The INC and INC-FL are not adaptive control methods, as inferred from the foregoing discussion. In other words, under specific environmental conditions, their controller parameters are adjusted. This signifies that the best operation is only provided under these conditions, and if they are changed, the grantee will not be able to reach the best operations. This calls for adaptive control techniques. These methods provide for optimal performance by allowing the controller parameters and, in turn, the control signal, to be adjusted. These adaptive techniques surpluses the non-adaptive ones in many applications , 44. UsikP1, kI1, kD1) are the outputs. The FL controller is added to the traditional PID controller to adjust the parameters  of the PID controller online according to the error along with the change in the error. As indicated in FL self-tuning algorithm for MPPT is developed to guarantee obtaining the greatest amount of possible power from the PVS by updating the controller parameters with any changes in the environmental conditions. The error and change of error are employed as inputs to the FL self-tuning while the PID controller gains  and temperature (T) changes, such as shading, dust, and component degradation.Comparison with emerging techniques: Compare the INC-FST method with emerging MPPT techniques, including artificial intelligence-based algorithms and other state-of-the-art controllers, to assess its competitiveness and advantages.Grid Interaction: Examine the algorithm’s behavior when integrated with grid systems, focusing on its impact on power quality, grid stability, and interactions with grid-tied inverters.Cost-Benefit Analysis: Conduct a cost-benefit analysis to evaluate the economic feasibility of implementing the INC-FST algorithm in real-world PV systems, considering factors like initial investment and long-term savings. 10 Oct 2023
 PONE-D-23-29962

 A New Adaptive MPPT Technique Using an Improved INC Algorithm Supported by Fuzzy Self-Tuning Controller for a Grid-linked Photovoltaic System

 PLOS ONEDear Dr. MBADJOUN WAPET,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Nov 24 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at
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Please ensure that you refer to Figure 10 in your text as, if accepted, production will need this reference to link the reader to the figure.Additional Editor Comments:Dear Author,The manuscript needs to be revised as per the reviewers' suggestions.I suggest adding few more recent references, related to modelling of PV cell, 10.1007/s13198-022-01658-6Related to comparison of various technoqies, 10.1109/INCET57972.2023.10170183, 10.3390/en15228776There are lot many recent literature which were not referred, 10.1007/s11356-023-27261-1, 10.3390/en15176172[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. 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However, authors should include following papers in literature review section as:1. DOI:10.1109/JSYST.2018.2817584https://doi.org/10.1049/iet-epa.2017.0804.2. 3. doi: 10.1109/JSYST.2019.2949083.4. doi: 10.1109/JSYST.2019.2948899.https://doi.org/10.1049/rpg2.125055. Reviewer #2: 1- Abstract should be improved to reflect novelty and some specific results should be given.2- Please mention the novelty of this study and suggest further studies.3- Introduction must be expanded and significantly improved. Novelty is not well reported in the manuscript.4- Please compare your results and research with the results of other authors and underline what is the scientific novelty in this work.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  13 Oct 2023***Technical response to the reviewers*** October 14th, 2023Journal: PLOS ONEManuscript No.: PONE-D-23-29962Title: “A New Adaptive MPPT Technique Using an Improved INC Algorithm Supported by Fuzzy Self-Tuning Controller for a Grid-linked Photovoltaic System” Nagwa F. Ibrahim1, Mohamed Metwally Mahmoud2, Hashim Alnami3, Daniel Eutyche Mbadjoun Wapet4, *, Sid Ahmed El Mehdi Ardjoun5, Mohamed I. Mosaad6.7, , Ammar M. Hassan8, and H. Abdelfattah11Electrical Department, Faculty of Technology and Education, Suez University, Suez 43533, Egypt2Department of Electrical Engineering, Faculty of Energy Engineering, Aswan University, Aswan 81528, Egypt3Electrical Engineering Department, Jazan University, Jazan, 45142, KSA4*National Advanced School of Engineering, Universit´e de Yaound´e I, Yaound´e, Cameroon5IRECOM Laboratory, Faculty of Electrical Engineering, Djillali Liabes University, Sidi Bel-Abbes, Algeria6Electrical & Electronics Engineering Technology Department, Yanbu Industrial College (YIC), Royal Commission Yanbu Colleges & Institutes, Yanbu 46452, Saudi Arabia7Electrical Engineering Department, Faculty of Engineering, Damietta University, Damietta 34511, Egypt8Arab Academy for Science, Technology and Maritime Transport, South Valley Branch, Aswan 81516, Egypt.Nagwa.ibrahim@ind.suezuni.edu.eg, Metwally_M@aswu.edu.eg, halnami@jazanu.edu.sa, eutychedan@gmail.com, m_i_mosaad@hotmail.com, elmehdi.ardjoun@univ-sba.dz, ammar@aast.edu, and hany.abdelfattah@ind.suezuni.edu.eg*Corresponding author: Daniel Eutyche Mbadjoun WapetDear Editors and ReviewersThe authors are thankful to the learned Editor and Reviewers for their thoughtful and detailed comments to improve the quality of the manuscript. The authors have given reviewer comments a lot of interest in the revision process in an attempt to address all of the reviewers’ concerns and corrections as you will already find them incorporated in the revised manuscript. Moreover, a reply to each of the reviewers’ comments is provided below.Kindly find the response to the reviewer’s comments in the following paragraphs. We hope this revised version of the manuscript meets the editor and reviewers’ expectations, and the standards of publication in the PLOS ONE Journal. The changes carried out by the authors are incorporated in the revised manuscript and highlighted in YELLOW.Editor's Comments:Comments to the Authors:Comment-1: Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. The paper should be revised thoroughly incorporating the comments offered by reviewers.Response-1: Our sincere thanks and appreciation to the editor for considering our manuscript for publication in PLOS ONE Journal, and the recommending submission of the revised manuscript with major revisions. To improve the quality of the manuscript, the reviewer's queries are addressed and their suggestions are incorporated into the revised manuscript. The changes carried out by the authors are incorporated in the revised manuscript and highlighted in YELLOW to be easily viewed by the editors and reviewers. A cover letter is provided and prepared to explain, point by point, the details of the revisions to the manuscript. Kindly, check the revised version.Comment-2: Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. We look forward to receiving your revised manuscript.Response-2: Our sincere thanks and appreciation to the editor for his comment. The required items are attached during submission process. A cover letter is provided and prepared to explain, point by point, the details of the revisions to the manuscript. The changes carried out by the authors are incorporated in the revised manuscript and highlighted in YELLOW to be easily viewed by the editors and reviewers. An unmarked version of the revised paper without tracked changes is also provided.Comment-3: Please ensure that your manuscript meets PLOS ONE's style requirements.Response-3: The authors are extremely thankful to the editor for this thoughtful point. The revised manuscript meets PLOS ONE's style.Comment-4: The manuscript needs to be revised as per the reviewers' suggestions.I suggest adding few more recent references, related to modelling of PV cell, 10.1007/s13198-022-01658-6Related to comparison of various techniques, 10.1109/INCET57972.2023.10170183, 10.3390/en15228776There are lot many recent literatures which were not referred, 10.1007/s11356-023-27261-1, 10.3390/en15176172Response-3: As recommended by the esteemed reviewer, all the suggested recent references are considered. Actually, these references strength the current work. Kindly, check the revised paper. Comment-4: Please ensure that you refer to Figure 10 in your text as, if accepted, production will need this reference to link the reader to the figure.Response-4: The authors are thankful to the esteemed editor upon his valuable comment. All the esteemed editor suggestions are done in the updated paper. Reviewers Comments:Reviewer#1 Comments to the Authors:Comment-1: The manuscript has some merits, but it lacks a performance comparison with state-of-the-art solutions. Experimental results supporting this comparison are welcome.Response-1: First, the authors would like to thank the respected reviewer for his insightful comment and useful feedback that will enhance the presentation and quality of the paper. From our side, we are very keen to answer and take into account the reviewer comment. The proposed technique is investigated and compared with three methods, and a comparison with previously published works is presented in Table 5 in the updated paper. The authors would like to emphasize that the application significance of the proposed approach was tested and confirmed using data gathered from a test distribution network. The authors are now working on testing the proposed technique in a Typhoon HIL real-time simulator. The Typhoon HIL real-time simulator has ultra-low-latency, high-high-fidelity, and only single-vendor solutions for real-time application. Typhoon HIL also features very high-resolution (12bit DAC) output pins, allowing the user to obtain highly precise real-time data. Furthermore, the Typhoon HIL operates in a unified environment, ensuring that there are no compatibility concerns at any moment.In our laboratory, the test bed was in the process of being prepared. Unfortunately, the COVID 19 epidemic and accompanying limitations have prevented this from happening until today. As a result, the authors are constantly working in this direction for the experimental setup, which may be included in future work of the proposed approach. The honorable reviewer reviewer's understanding and cooperation in this regard will be greatly appreciated.Comment-2: More experiments should be done to demonstrate the superiority of the proposed method over existing methods.Response-2: The authors are thankful to the honorable reviewer for the words of encouragement and trust in our work. The proposed technique is investigated and compared with three methods, and a comparison with previously published works is presented in Table 5 in the updated paper. Kindly check the revised manuscript.Table 5. Comparative performance of the suggested technique with recently published works.Ref. Year Publisher Method Complexity Efficacy Oscillations Sensed variables[40]2021 Elsevier Quantized sliding mode M 98.90% Very L Voltage (V) and current (It)[41]2023 Elsevier Grey wolf-based PID M 99.50% Very L V&It[42]2020 MDPI Genetic-INC M 99.09% Very L V&It[43]2020 Wiely&Hindawie A self-constructing Lyapunov NN M 98.14% L V&It[44]2021 Elsevier Hybrid whale algorithm-based ANFIS H 99.35% L I, T, and V[45]2022 Elsevier Reduced oscillations-based P&O M 99.49% Very L V&ItCurrent study INC-FST L 99.80% Very L I, and Thttps://doi.org/10.1049/iet-epa.2017.0804., 3. doi: 10.1109/JSYST.2019.2949083. 4. doi: 10.1109/JSYST.2019.2948899., 5. https://doi.org/10.1049/rpg2.12505Comment-3: Paper is well written. However, authors should include following papers in literature review section as: 1. DOI:10.1109/JSYST.2018.2817584, 2. Response-3: The authors thank the reviewer for his recommendation, which would enhance the quality of the paper. The article's references are updated and added the references suggested by reviewer. Kindly refer to the highlighted lines in the reference section.[16] N. Priyadarshi, S. Padmanaban, P. Kiran Maroti, and A. Sharma, “An Extensive Practical Investigation of FPSO-Based MPPT for Grid Integrated PV System under Variable Operating Conditions with Anti-Islanding Protection,” IEEE Syst. J., vol. 13, no. 2, pp. 1861–1871, 2019, doi: 10.1109/JSYST.2018.2817584.[17] N. Priyadarshi, S. Padmanaban, M. S. Bhaskar, F. Blaabjerg, and A. Sharma, “Fuzzy SVPWM-based inverter control realisation of grid integrated photovoltaicwind system with fuzzy particle swarm optimisation maximum power point tracking algorithm for a grid-connected PV/wind power generation system: Hardware implementation,” IET Electr. Power Appl., vol. 12, no. 7, pp. 962–971, 2018, doi: 10.1049/iet-epa.2017.0804.[18] N. Priyadarshi, S. Padmanaban, J. B. Holm-Nielsen, F. Blaabjerg, and M. S. Bhaskar, “An Experimental Estimation of Hybrid ANFIS-PSO-Based MPPT for PV Grid Integration under Fluctuating Sun Irradiance,” IEEE Syst. J., vol. 14, no. 1, pp. 1218–1229, 2020, doi: 10.1109/JSYST.2019.2949083.[19] N. Priyadarshi et al., “A Hybrid Photovoltaic-Fuel Cell-Based Single-Stage Grid Integration with Lyapunov Control Scheme,” IEEE Syst. J., vol. 14, no. 3, pp. 3334–3342, 2020, doi: 10.1109/JSYST.2019.2948899.[20] N. Priyadarshi, P. Sanjeevikumar, M. S. Bhaskar, F. Azam, I. B. M. Taha, and M. G. Hussien, “An adaptive TS-fuzzy model based RBF neural network learning for grid integrated photovoltaic applications,” IET Renew. Power Gener., vol. 16, no. 14, pp. 3149–3160, 2022, doi: 10.1049/rpg2.12505.Reviewer#2 Comments to the Authors:Comment-1: Abstract should be improved to reflect novelty and some specific results should be given.Response-1: At the beginning, the authors are thankful to the honorable reviewer for the words of encouragement and trust in our work. The abstract is improved and have numerical data in the updated version. Kindly check the revised manuscript.Solar energy, a prominent renewable resource, relies on photovoltaic systems (PVS) to capture energy efficiently. The challenge lies in maximizing power generation, which fluctuates due to changing environmental conditions like irradiance and temperature. Maximum Power Point Tracking (MPPT) techniques have been developed to optimize PVS output. Among these, the incremental conductance (INC) method is widely recognized. However, adapting INC to varying environmental conditions remains a challenge. This study introduces an innovative approach to adaptive MPPT for grid-connected PVS, enhancing classical INC by integrating a PID controller updated through a fuzzy self-tuning controller (INC-FST). INC-FST dynamically regulates the boost converter signal, connecting the PVS's DC output to the grid-connected inverter. A comprehensive evaluation, comparing the proposed adaptive MPPT technique (INC-FST) with conventional MPPT methods such as INC, Perturb & Observe (P&O), and INC Fuzzy Logic (INC-FL), was conducted. Metrics assessed include current, voltage, efficiency, power, and DC bus voltage under different climate scenarios. The proposed MPPT-INC-FST algorithm demonstrated superior efficiency, achieving 99.80%, 99.76%, and 99.73% for three distinct climate scenarios. Furthermore, the comparative analysis highlighted its precision in terms of control indices, minimizing overshoot, reducing rise time, and maximizing PVS power output.Comment-2: Please mention the novelty of this study and suggest further studies.Response-2: The authors express their gratitude to the reviewer for providing a valuable comment that will enhance the quality of the paper. In addition, future research direction section is added. Kindly check the revised manuscript.The paper introduces an innovative INC-FST to improve MPPT efficiency, which will reduce system management complexities and effectively handle instabilities and disturbances in the investigated PVS. This work improves the INC-MPPT algorithm with a fuzzy self-tuning-based PID controller (INC-FST) for regulating the MPPT control of the studied system. This self-tuning algorithm will drive the DC-DC PC D to be updated to track the MPP with changes in I and T. The suggested method is simple, has higher dynamic responsiveness, barely oscillates near MPP, tracks quickly, and performs better in variable weather circumstances. The MATLAB/Simulink is used to investigate the proposed technique. Three tests are applied (variable I with constant T (C1), variable T with constant I (C2), and variable I with variable T (C3)) to assess the proposed effectiveness. The suggested technique efficacy under C1, C2, and C3 are 99.80%, 99.76%, and 99.73%, respectively. The PVS output, including power, voltage, current, and DC bus voltage, shows that the proposed technique performs admirably under a variety of conditions, including I and T fluctuations. The key finding of the suggested work is summarised as follows:• For PVSs to enable effective MPPT, an innovative INC-FST is suggested.• The suggested controller has low instabilities around MPP, excellent efficacy, faster rapid responsiveness, and quick converging time.• The INC-FST is resistant to I and T change since it is adaptable.• The enhanced algorithm is contrasted with three control approaches . The improved INC algorithm outperformed previous algorithms in some control indices, such as overshoot, rise time, and settling time, and demonstrated a quicker dynamic reaction for MPPT.In the future, this research can be extended and refined in the following ways:1. Real-world implementation: Implement the proposed innovative INC-FST in a physical PV system to validate its performance under practical conditions.2. Hardware validation: Test the algorithm on actual hardware components and assess its effectiveness in improving the efficiency of MPPT under real-world weather fluctuations.3. Robustness and adaptability: Investigate the robustness of the INC-FST algorithm to various environmental factors beyond current (I) and temperature (T) changes, such as shading, dust, and component degradation.4. Comparison with emerging techniques: Compare the INC-FST method with emerging MPPT techniques, including artificial intelligence-based algorithms and other state-of-the-art controllers, to assess its competitiveness and advantages.5. Grid Interaction: Examine the algorithm's behavior when integrated with grid systems, focusing on its impact on power quality, grid stability, and interactions with grid-tied inverters.6. Cost-Benefit Analysis: Conduct a cost-benefit analysis to evaluate the economic feasibility of implementing the INC-FST algorithm in real-world PV systems, considering factors like initial investment and long-term savings.Comment-3: Introduction must be expanded and significantly improved. Novelty is not well reported in the manuscript.Response-3: The authors thank the reviewer for pointing this point out. kindly check the updated manuscript. The introduction section is expanded and the novelty is presented in the contributions part in the introduction. Kindly check the revised manuscript.Comment-4: Please compare your results and research with the results of other authors and underline what is the scientific novelty in this work.Response-4: The authors are thankful to the honorable reviewer for the words of encouragement and trust in our work. The proposed technique is investigated and compared with three methods, and a comparison with previously published works is presented in Table 5 in the updated paper. Kindly check the revised manuscript.Table 5. Comparative performance of the suggested technique with recently published works.Ref. Year Publisher Method Complexity Efficacy Oscillations Sensed variables[40]2021 Elsevier Quantized sliding mode M 98.90% Very L Voltage (V) and current (It)[41]2023 Elsevier Grey wolf-based PID M 99.50% Very L V&It[42]2020 MDPI Genetic-INC M 99.09% Very L V&It[43]2020 Wiely&Hindawie A self-constructing Lyapunov NN M 98.14% L V&It[44]2021 Elsevier Hybrid whale algorithm-based ANFIS H 99.35% L I, T, and V[45]2022 Elsevier Reduced oscillations-based P&O M 99.49% Very L V&ItCurrent study INC-FST L 99.80% Very L I, and TThe authors once again thank the learned Editors and Reviewers for their valuable comments for improving the quality of the manuscript.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 17 Oct 2023A New Adaptive MPPT Technique Using an Improved INC Algorithm Supported by Fuzzy Self-Tuning Controller for a Grid-linked Photovoltaic SystemPONE-D-23-29962R1Dear Dr. MBADJOUN WAPET,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Praveen Kumar BalachandranAcademic EditorPLOS ONEAdditional Editor Comments :All the suggested comments were incorporated and addressed properly.I recommend, the manuscript may be accepted in the present form.Reviewers' comments: 26 Oct 2023PONE-D-23-29962R1 A New Adaptive MPPT Technique Using an Improved INC Algorithm Supported by Fuzzy Self-Tuning Controller for a Grid-linked Photovoltaic System Dear Dr. Mbadjoun Wapet:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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Arenaviridae is a family for ambisense RNA viruses with genomes of about 10.5 kb that infect mammals, snakes, and fish. The arenavirid genome consists of two or three single-stranded RNA segments and encodes a nucleoprotein (NP), a glycoprotein (GP) and a large (L) protein containing RNA-directed RNA polymerase (RdRP) domains; some arenavirids encode a zinc-binding protein (Z). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Arenaviridae, which is available at www.ictv.global/report/arenaviridae. Arenavirids produce virions that are spherical or pleomorphic in shape and 40–200 nm in diameter, with dense lipid envelopes . The virArenavirid genomes consist of two or three single-stranded, typically ambisense, RNA segments . Some of these RNAs encode two proteins in non-overlapping open reading frames of opposite polarities that are separated by non-coding intergenic regions (IGRs) . The S RArenavirions attach to cell-surface receptors or attachment factors and enter via the endosomal route. Some viruses engage intracellular receptors in endosomes. pH-dependent fusion with late endosomes releases the virion RNP complex into the cytoplasm. The virus RNP directs both RNA genome replication and gene transcription. During replication, L protein reads through the IGR transcription–termination signal and generates uncapped antigenomic and genomic RNAs. Transcription of mRNAs encoding GPC and Z occurs only after the first round of virus replication, during which S and L antigenomes are produced. Arenavirid mRNAs lack 3′-terminal poly(A) tracts but have several non-templated 5′ bases, consistent with the use of a cap-snatching mechanism to initiate transcription. Virion budding occurs from the cellular plasma membrane, thereby providing the virion envelope .ictv.global/taxonomy. The family Arenaviridae is included in the negarnaviricot order Bunyavirales. Arenavirids are most closely related to mypovirids, nairovirids, phenuivirids and wupedevirids. Arenavirids differ from most other bunyavirals by having segmented genomes with ambisense organization. The family includes several genera and >59 species. Some arenavirids can cause severe diseases in humans (e.g. Lassa fever) [Current taxonomy: a fever) . Other aa fever) .Arenaviridae: www.ictv.global/report/arenaviridae.Full ICTV Report on the family


Pseudomonas syringae pv. tomato DC3000 regulates its response to environmental stimuli, including interactions with hosts and neighboring bacteria. Despite the importance of transcriptional regulation during these agriculturally significant interactions, a comprehensive understanding of the TRN of P. syringae is yet to be achieved. Here, we collected and decomposed a compendium of public RNA-seq data from P. syringae to obtain 45 independently modulated gene sets (iModulons) that quantitatively describe the TRN and its activity state across diverse conditions. Through iModulon analysis, we (i) untangle the complex interspecies interactions between P. syringae and other terrestrial bacteria in cocultures, (ii) expand the current understanding of the Arabidopsis thaliana-P. syringae interaction, and (iii) elucidate the AlgU-dependent regulation of flagellar gene expression. The modularized TRN yields a unique understanding of interaction-specific transcriptional regulation in P. syringae.The transcriptional regulatory network (TRN) of the phytopathogen Pseudomonas syringae pv. tomato DC3000 is a model plant pathogen that infects tomatoes and Arabidopsis thaliana. The current understanding of global transcriptional regulation in the pathogen is limited. Here, we applied iModulon analysis to a compendium of RNA-seq data to unravel its transcriptional regulatory network. We characterize each co-regulated gene set, revealing the activity of major regulators across diverse conditions. We provide new insights on the transcriptional dynamics in interactions with the plant immune system and with other bacterial species, such as AlgU-dependent regulation of flagellar genes during plant infection and downregulation of siderophore production in the presence of a siderophore cheater. This study demonstrates the novel application of iModulons in studying temporal dynamics during host-pathogen and microbe-microbe interactions, and reveals specific insights of interest. Pseudomonas syringae pv. tomato DC3000 is a gram-negative phytopathogen that infects tomato plants, causing major crop losses worldwide  of P. syringae is required. Understanding the TRN is imperative to improving our knowledge of the fundamentals of its pathogenesis and survival strategies at a systems level.orldwide . P. syriIndependent component analysis (ICA) was developed to identify statistically independent components from complex data sets . SpecifiE. coli  to understand competition and survival strategies of the pathogen in the presence of neighbors it often coexists with in natural habitats  database of NCBI using our previously established pipeline   . These dAfter characterization, the 45 iModulons were classified into four broad categories: functional, genomic, uncharacterized, and noise. The iModulons classified as “Functional” were further divided into six subcategories: “Exometabolites,” “Metabolism,” “Stress response,” “Motility,” “Translation,” and “Metal uptake” . Due to P. syringae pv. syringae B728 and P. aeruginosa PAO1, are members of the AlgU iModulon  were upregulated. Although AlgU is known to regulate genes in these functional categories, the differential regulation of these genes may be due to indirect transcription factor interactions  18). We. We18). iModulon . FurtheriModulon .Unlike the traditional differential expression of genes (DEG) analysis which only compares two conditions and therefore captured signals from several related transcriptional mechanisms, our approach used all available data to separate each signal into its own iModulon. This global use of available data allows us to provide and strengthen evidence of gene-regulator interactions and more easily quantify the global transcriptional state of the cell. The key iModulons that are used to interpret the transcriptomic changes are summarized in the Data S1.P. syringae, B. thailandensis, and C. violaceum  and Major Facilitator Superfamily efflux pumps that confer antibiotic resistance.Over a period of 45 h , iModulon activity levels were notably altered in cocultures containing response . B. thaieighbors , 11. As B. thailandensis as well as C. violaceum (Supplementary Note S1). We believe that while the upregulation of the Resistance-1 iModulon is necessary in the presence of more harmful compounds such as antibiotics produced by B. thailandensis, the Resistance-2 iModulon is initially upregulated to confer antibiotic resistance up to a certain extent. The Resistance-2 iModulon contains genes involved in the oxidative stress response, copper efflux, and a gene encoding an RND efflux pump neighboring the copper efflux genes.The activity of a second resistance-associated iModulon, Resistance-2, was found to have a strong positive correlation with that of the Resistance-1 iModulon in the monoculture and coculture samples . NotablyB. thailandensis indicates that B. thailandensis creates oxidative stress  through the Fenton reaction.Cocultures containing tivities . Throughandensis . These puisition . Based ouisition , P. syriynthesis . It is nP. syringae common to the monoculture and coculture conditions, albeit to varying degrees  by pattern recognition receptors (PRRs) , 31. UpoP. syringae. In agreement with previous studies, we found that exposure to PTI also upregulated motility, as seen through the upregulation of the Chemotaxis, Flagella-1, and Flagella-2 iModulons . It is not clear as to whether this upregulation of motility is due to misregulation in transcription caused by PTI or evolved mechanisms attempting to move to a more favorable environment.Compared to naive host infection, exposure to PTI through pretreatment with flg22 caused a downregulation of pathogenesis-associated iModulons (T3SS and Coronatine) and translation-associated iModulons (Translation-1 and Translation-2) . In contModulons . It is A. thaliana suppresses the T3SS of P. syringae is through the production of the T3SS-suppressing electrophilic compound sulforaphane . As these genes allow the bacteria to resist the effects of sulforaphane, we analyzed the activities of the T3SS and Resistance-1 iModulons over a period of 5 h post inoculation (hpi) during naive host infection and pre-induced PTI  and motility  in P. syringae -mediated signaling pathways contribute to the PTI response in plants, and its accumulation has been found to enhance the sensitivity of plants to PAMPs , 37. Hensyringae  12, 37), 37P. sysyringae . Previouapoplast . As SA iP. syringae elicited a strong response in the bacteria  for P. syringae pv tomato DC3000 as of August 20, 2020. A total of 202 samples were collected from previous studies (https://github.com/ncbi/sra-tools/wiki/HowTo:-fasterq-dump), after which Trim Galore  was used for read trimming. The trimmed reads were checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and then aligned to the reference genome using Bowtie (2 transcripts per million (TPM)  .R2 <0.95 for non-coculture samples, R2 <0.90 for coculture samples). Additionally, samples with no replicates were discarded. Finally, log-TPM data within projects were normalized to reference conditions specific to each project.Hierarchical clustering was performed to visualize global correlations between each sample with all other samples, after which samples with atypical expression profiles were discarded. Further, sample metadata were manually curated, and biological replicates with low correlation were dropped (−7 (ICA was performed to decompose the matrix of gene expression profiles (X) into a matrix of independent components  (M) and their activities in all conditions (A) . Specifi−7 . The num−7 . To ensu−7 . The thrhttps://github.com/SBRG/pymodulon/blob/master/docs/tutorials/creating_the_gene_table.ipynb. Gene annotations were primarily obtained from AE016853.1, with additional information being collected from KEGG (and Cluster of Orthologous Groups (COG) using EggNOG mapper  as well as KEGG and GO enrichment (FDR < 10−2). iModulons were subsequently named based on their enriched regulators or functional characteristics.For each iModulon, Fisher’s exact test with Benjamini-Hochberg false discovery rate (FDR) correction was used to calculate regulator enrichment against known regulons (FDR = 10P-values were determined by calculating the absolute value of the difference between mean iModulon activities and comparing with the log-normal distribution of the iModulon. This was followed by Benjamini-Hochberg correction. A threshold of 5 was considered to be significant.Differences in iModulon activation between two or more conditions were calculated by using a log-normal distribution. To determine the statistical significance of these differences, iModulon activity levels were clustered hierarchically using the average linkage method. Additionally, the minimum activity level for https://pymodulon.readthedocs.io/en/latest/tutorials/creating_an_imodulondb_dashboard.html.We generated the iModulonDB dashboards using the PyModulon package , 17. The


Streptococcus suis are an extensive economic problem as well as an animal welfare concern for the global swine industry. Previous studies have evaluated the genomic diversity and population structure of S. suis isolates, however, the majority of these studies utilized isolates obtained from countries other than the U.S. This study applied whole genome sequencing and cgMLST-based typing to evaluate the population structure and genetic relatedness among S. suis isolates obtained within the U.S. The established high-resolution phylogenomic framework revealed extensive genomic variation and diversity among the sampled S. suis isolates, with isolates from the U.S. and from countries outside the U.S. found interspersed in the phylogeny. S. suis isolates obtained within the U.S. did not cluster by state or geographic location, however, isolates with similar serotypes, both obtained from within and outside the U.S., generally clustered together. Average nucleotide identity (ANI) values determined for the S. suis genomes were extensively broad, approaching the recommended species demarcation value, and correlated with the phylogenetic group distribution of the cgMLST-based tree. Numerous antimicrobial resistance (AMR) elements were identified among both U.S. and non-U.S. isolates with ble, tetO, and ermB genes identified as the most prevalent. The epf, mrp, and sly genes, historically used as markers for virulence potential, were also observed in the genomes of isolates that grouped together forming a subclade of clonal complex 1 (CC1) isolates. Collectively, the data in this report provides critical information needed to address potential biosurveillance needs and insights into the genetic diversity and population structure of S. suis isolates obtained within the U.S.Diseases caused by the zoonotic pathogen  Streptococcus suis is one of the leading causes of bacterial infection in swine and contributes to significant economic losses to the swine industry worldwide , extracellular protein factor (epf), and suilysin (sly)  ,b, 2017.in (sly) . Additioin (sly) ,b, 2017. S. suis . Genotypountries .S. suis isolates. Isolates are serotyped based on structural differences in the CPS and to date there are 27 recognized serotypes based on the antigenicity of CPSs and 28 novel CPS loci  are the most common methods used to differentiate CPS loci . Among tCPS loci . Additioconcerns . A standng genes , with isng genes . For exang genes .S. suis isolates (S. suis isolates derived from countries other than U.S. (S. suis isolates obtained within the U.S. using a core genome (cg) MLST-based methodology. Additionally, S. suis isolates obtained from various countries outside the U.S. were included in our analyses to provide a broader perspective and examine how the population structure of U.S. S. suis isolates compares with isolates obtained from various countries outside the U.S.Phylogenetic studies employ whole-genome sequencing (WGS) and data analysis to better understand the epidemiology, origin, and evolution of bacteria . WGS anaisolates . The U.Shan U.S. . The goaStreptococcus suis isolates obtained from swine samples collected from pigs throughout the U.S. and submitted to the University of Minnesota Veterinary Diagnostic Laboratory between 2015 and 2017 were selected for the project with an effort to include isolates from as many states and an effort to include as many total isolates as possible without duplication (n = 103)  (n = 103) . This se(n = 45) . All isoS. suis isolates obtained from across the United States were generated as previously described (n = 94), Illumina HiSeq data was obtained from libraries created using the NEB Ultra II DNA Library Prep Kit  sequenced on a HiSeq 3000 instrument generating 2 × 150 bp paired-end reads. Sequencing reads were assessed for quality using FastQC.1 Reads were randomly subsampled using seqtk2 to target a genome coverage of 150x considering an average expected genome length of 2.1 Mbases. Sequence data were assembled using MIRA v. 4.9.63 (Draft and complete genomes for escribed . Briefly. 4.9.63 . The aven = 9), whole genome sequencing was performed using the Pacific Biosciences (PacBio) platform. Library preparation for PacBio sequencing was performed following the PacBio 10-kb insert library preparation protocol. The 10 kb library for each strain was sequenced using the PacBio RSII platform with two SMRT® cells for each isolate. Closed whole-genome assemblies for isolates ISU1606, ISU2660, ISU2414, ISU2515, ISU2614, ISU2714, ISU2812, and SRD478 were generated using the hybrid assembler Unicycler v. 0.4.4  strain ISU1606 (GenBank accession CP030017) as seed. Core genome and accessory MLST targets were identified according to the inclusion/exclusion criteria of the SeqSphere + Target Definer. The allele information from core, accessory, and combined gene sets were used to establish phylogenetic hypotheses using the neighbor-joining method  for gene-by-gene alignment, allele calling, and comparison . A core g method . The resg method .S. suis strain ISU1606  and the analyzed genomes were calculated using FastANI, based on MinHash mapping (Average nucleotide identity (ANI) between the designated reference genome  mapping  in Galax mapping .5 was used to identify antimicrobial resistance genes using the AMR gene databases from the Comprehensive Antibiotic Resistance Database (CARD) (PRJNA313047), which was downloaded in April 2019. A minimum percent identity threshold of 80% was used to identify AMR genes in the assembled genomes. Statistical analysis was completed using GraphPad Prism 9.0 . Comparisons of AMR resistance gene frequency between U.S. and non-U.S. isolates were completed using Mann-Whitney tests. A P-value less than 0.05 was considered significant.Abricate e (CARD) , ResFinde (CARD) ] and themrp and sly were identified by BLASTN searches and the percent identity for each gene was determined for each isolate relative to the S. suis strain ISU1606  orthologs. In silico PCR using primers from epf gene and distinguishment between epf or epf*, the long form of extracellular protein factor, based on product size. Percent identity was determined by alignment with either the epf gene from strain D282 (accession X71881) or the epf* gene from strain 1890 (accession X71881) (Genes  X71881) .S. suis from the U.S. and various countries outside the U.S. were comprehensively analyzed in this study. International isolates were included if complete genome assemblies were publicly available, allowing for the in silico screening for AMR- and virulence-factors, as well as sequence type (ST), CPS serotype, and clonal complex (CC) determination. Strain ISU1606 was used as the reference or seed strain because it shares numerous traits and characteristics with S. suis strain P1/7 (S. suis isolates obtained from the U.S. (n = 103) and from various countries outside the U.S. (n = 45), a neighbor-joining tree was constructed from cgMLST targets using the S. suis strain ISU1606 as the seed  to 94.42% (29995) with an average of 97.12%  and CC16 (ST16) isolates and included isolates 861160 and GD-0001 , isolate GD-0088 , as well as isolates 40439 and 40458 . The larger subclade consists of 20 isolates, all CC1  except for isolate LSM178, an ST1005 isolate not assigned to a CC and therefore listed as a singlet. Four of the twenty isolates are U.S. isolates , while the majority (16/20) of isolates within this subclade are non-U.S. isolates. Additionally, 17 isolates of this subclade were serotype 2 and three isolates were serotype 14 . Isolates within this subclade were mainly ST1 (9/20) or ST7 (6/20).P = 0.038) lower than number of AMR elements harbored by an individual U.S. isolate (ble (100% in both U.S. and non-U.S.) followed by tetO (87% U.S. and 49% non-U.S.), and ermB . Both the tetO and ermB loci are absent in 27 genomes, eleven of which were serotype 2 isolates grouped within the larger subclade CC1 isolates in Genomes were screened for chromosomal mutations and genes conferring antimicrobial resistance. For the U.S. isolates, the number of AMR elements harbored by an individual isolate ranged from one to twelve . The ave isolate . While nepf, mrp, and sly have historically been used to predict the virulence potential of S. suis  with a 99.9% identity to the reference ortholog. None of the four isolates encode the long form of extracellular protein factor (EF*), which can be expressed by some serotype 2 isolates and produce a high molecular weight variant (>110 kDa)  of the 37 isolates was predicted to encode a frameshift resulting in a pseudogene. Extensive nucleotide diversity with the mrp gene has been previously reported   . This gea) (epf* . The mrpf 80.21% . Among nidentity . The mrpreported . The slyidentity . Twenty-.4–100%) .epf, mrp, and sly genes]. These nineteen isolates form a larger subclade mainly comprised of serotype 2 isolates, as previously mentioned  to 94.42% (29995), and approach the recommended species demarcation value .Average nucleotide identity has increasingly been utilized as a robust method for assessing genomic similarity between two genomes, with organisms belonging to the same species typically having ANI values of ≥95%. The ANI values determined for the on value . The broS. suis isolates obtained within the U.S. did not cluster by state or geographic location, in general all isolates with similar serotypes, regardless of their origins, cluster together in the cgMLST phylogeny. An exception to this general trend were the serotype 2 isolates, which clustered into two distinct groups. One group mostly consisted of ST28 and ST961 and contained three serotype 1/2 isolates and one isolate with an undetermined serotype. The second group, consisting of ST1 and ST7, had three serotype 14 isolates clustered within the group. The clustering of these isolates harboring the 1/2, 2, and 14 serotypes is reasonable and consistent with the high degree of similarly among these serotypes. Specifically, the CPS loci of serotypes 1 and 14 and serotypes 2 and 1/2 are almost identical and most current PCR methods are unable to differentiate these specific serotypes  can be found in the article/Ethical approval was not required for the study involving animals in accordance with the local legislation and institutional requirements because all isolates were either obtained from samples collected as part of previous studies or were obtained from samples submitted as part of field case investigations and did not require Institutional Animal Care and Use Committee (IACUC) approval.All authors conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents, materials, and analysis tools, wrote the manuscript, approval of the final version to be published, and agreed to be accountable for all aspects of the work.


Francisella tularensis, the causative agent for tularaemia, is a Tier 1 select agent, and a pan-species pathogen of global significance due to its zoonotic potential. Consistent genome characterization of the pathogen is essential to identify novel genes, virulence factors, antimicrobial resistance genes, for studying phylogenetics and other features of interest. This study was conducted to understand the genetic variations among genomes of F. tularensis isolated from two felines and one human source. Pan-genome analysis revealed that 97.7 % of genes were part of the core genome. All three F. tularensis isolates were assigned to sequence type A based on single nucleotide polymorphisms (SNPs) in sdhA. Most of the virulence genes were part of the core genome. An antibiotic resistance gene coding for class A beta-lactamase was detected in all three isolates. Phylogenetic analysis showed that these isolates clustered with other isolates reported from Central and South-Central USA. Assessment of large sets of the F. tularensis genome sequences is essential in understanding pathogen dynamics, geographical distribution and potential zoonotic implications. Francisella tularensis is the causative organism of tularaemia, a deadly disease in over 100 species. Genome analysis and frequent genome surveillance would be helpful to assess the genomic alterations and their effect on pathogenicity, virulence and trends in antimicrobial resistance. This study aims to assess three genomes of F. tularensis, giving insight into the dynamics of this pathogen. Whole genomes of bacteria isolated from one human (archived), and two felines were sequenced and analyzed in this study.Francisella tularensis is a Gram-negative coccobacillary intracellular bacterium belonging to the class Gammaproteobacteria. F. tularensis is the causative agent of tularaemia, a vector-borne zoonotic disease commonly called ‘rabbit fever’. Tularaemia has been reported in all states of the USA except Hawaii  and a human isolate [OADDL_FT3-Bates (FT3)] were sequenced and assembled. In brief, all three isolates (OADDL-FT) were assembled into two contigs with a genome size ranging between ~1.89 and 1.96 Mb with an average GC content of 32.23 %. The ANI between these microbes was 99.97%, indicating that these genomes are highly similar. However, the genome coverage of these microbes for ANI was <80 %, which indicated the diversity in OAADL-FT isolates. The genome sequences have been deposited in the NCBI GenBank database and their assembly accession numbers along with genome statistics are listed in F. tularensis genomes from GenBank  were identified between the OADDL-FT genomes . FifteenF. tularensis subsp. tularensis SCHU S4 as a representative genome for comparison. These virulence factors are listed in Table S2. Various virulence factors belonging to different classes, namely Adherence and Invasion factors , Intracellular survival factors , Iron uptake factors , Magnesium uptake factors (FmvB), Nutritional virulence factors , Secretion systems (T6SS), Serum resistance and immune evasion factors (capsular factors and LPS) and Toxins were annotated. All OADDL-FT genomes shared all but one virulence factor with F. tularensis subsp. tularensis SCHU S4. The three OADDL-FT genomes had an annotated toxin virulence factor, phytotoxin phaseolotoxin (cysC1), which was absent in SCHU S4. Phaseolotoxin is a plant toxin commonly produced by Pseudomonas spp. and is known to induce chlorosis in plants [as iglC, iglD, pdpA, iglI, vgrG, carA, carB, pyrB, acpA, acpB, acpC and hapA and bacterial factors for cytosolic replication [dipA, iglD, purMCD, ggt, ripA, iglA, iglB and pdpB were also identified (Table S2).VFDB was used to predict and identify ORFs for possible virulence factors in the draft genomes, using n plants . Variousn plants  such as lication  such as https://isfinder.biotoul.fr/), insertion sequences were annotated. The insertion sequences that had blast alignments to segments of the draft genomes are listed in Using databases from ISfinder (sdhA) was identified and assessed for typing. Our analysis confirmed the presence of A at nucleotide position 465 of the sdhA gene in all three genomes, which confirmed that OADDL-FT isolates are Type A F. tularensis, which was further validated by CanSNPer. The CanSNPer pipeline aims to identify single nucleotide polymorphisms (SNPs) of diagnostic significance. Based on CanSNPer typing, the genomes FT1, FT2 and FT3 were assigned as types A.1.14, A1.11, and A.1.13 respectively. Two antibiotic resistance genes, blaFTU-1 and blaA, were detected in all OADDL-FT genomes. blaFTU-1 codes for a class A beta lactamase and confers resistance to beta-lactams in F. tularensis [gyrA gene for chromosomal mutations that confer resistance to fluoroquinolone [gyrA were observed in OADDL-FT genomes [F. tularensis gyrA gene (NC_007880), we observed multiple mutations in the OADDL-FT gyrA gene that lead to amino acid substitutions at positions 395 (Glu-395-Lys), 486 , 501 (Cys-501-Arg) and 670 (Thr-670-Lys).The coding sequence for the gene succinate dehydrogenase  shared a close common ancestor with high similarity to FT2, one of the feline isolates.The maximum likelihood phylogenetic tree with a GTR model of the OADDL-FT genomes and 243 other tral USA . The lacericanum , a knownFrancisella species contain gene clusters that are homologous to a set of gene clusters of Neisseria and Pseudomonas [cysC1 – Phytotoxin phaseolotoxin. Based on previous studies, phaseolotoxin is a toxic agent for plants [Pseudomonas spp., and there are no known reports of phaseolotoxin pathogenicity in animals. Further experimentation will be needed to confirm the presence and function of the gene in OADDL-FT genomes. The three draft genomes shared many virulence factors with F. tularensis subsp. tularensis SCHU S4, indicating similar pathogenicity between these isolates. The annotation and identification of bacterial factors needed for phagosomal escape and cytosolic replication further emphasize the pathogenic potential of these isolates sequenced in this study.It has been established that many omonas . These aomonas . One of r plants  secretedet al. [transformation, conjugation and transduction.’ In general, MGEs include insertion sequences, phage sequences, conjugative transposons, integrons and unit transposons [Frost et al.  discussensposons . Insertinsposons . These insposons . MGEs ainsposons . ISFtu1,blaFTU-1 and blaA) genes in all the OADDL-FT isolates. These genes are intrinsic in the genus Francisella and blaFTU-1 confers narrow-spectrum resistance to β-lactams in most Francisella strains [Francisella [gyrA genes of all OADDL-FT genomes, are associated with fluoroquinolones resistance.Genome analysis for antibiotic resistance genes revealed the presence of two β-lactamase ( strains . Amino aisella . FurtherFrancisella spp. to better understand bacterial evolution and dynamics.The annotation of genetic elements such as insertion sequences, AMR genes and virulence factors underscore the need for regular sequencing of Click here for additional data file.