Staphylococcus aureus is a major human pathogen whose characteristics support its success in various clinical settings including Cystic Fibrosis (CF). In CF, S. aureus is indeed the most commonly identified opportunistic pathogen in children and the overall population. S. aureus colonization/infection, either by methicillin-susceptible or methicillin-resistant strains, will become chronic in about one third of CF patients. The persistence of S. aureus in CF patients’ lungs, despite various eradication strategies, is favored by several traits in both host and pathogen. Among the latter, living in biofilm is a highly protective way to survive despite deleterious environmental conditions, and is a common characteristic shared by the main pathogens identified in CF. This is why CF has earned the status of a biofilm-associated disease for several years now. Biofilm formation by S. aureus, and the molecular mechanisms governing and regulating it, have been extensively studied but have received less attention in the specific context of CF lungs. Here, we review the current knowledge on S. aureus biofilm in this very context, i.e., the importance, study methods, molecular data published on mono- and multi-species biofilm and anti-biofilm strategies. This focus on studies including clinical isolates from CF patients shows that they are still under-represented in the literature compared with studies based on reference strains, and underlines the need for such studies. Indeed, CF clinical strains display specific characteristics that may not be extrapolated from results obtained on laboratory strains. Staphylococcus aureus is a major human pathogen whose characteristics support its success in diverse clinical settings like bacteremia, skin and soft tissue infections, necrotizing pneumonia, device-associated infections like catheter-related infections and ventilator-associated pneumonia, and Cystic Fibrosis (CF) [S. aureus against host defenses and eradication measures and, consequently, allows it to persist in the host [sis (CF) . Besidesthe host ,3. Biofithe host ,5. Biofithe host ,6. Biofithe host . Living Pseudomonas aeruginosa and S. aureus are the two major pathogens in CF. Although P. aeruginosa is dominant in the adult population, S. aureus is the most commonly identified opportunistic pathogen in children and the overall CF population [S. aureus is concerned, biofilm formation and the molecular mechanisms governing and regulating it have been extensively studied but have received less attention in the unique context of CF lungs.Cystic Fibrosis is an inherited disease affecting the CFTR (cystic fibrosis transmembrane conductance regulator) protein, marked by a vicious cycle of infection and inflammation which is highly deleterious for the patient’s lung function. pulation  with 60 pulation ,10,11. Apulation ,13,14,15pulation ,17. HoweStaphylococcus aureus”, “Biofilm” and “Cystic Fibrosis” in the PubMed database found 152 publications and a similar search for “Pseudomonas aeruginosa” found 1200 results and a combined search for “Staphylococcus aureus” and “Pseudomonas aeruginosa” gave 108 publications (8 October 2022)  . This hiound 30% .S. aureus on biofilm formation in CF. Among the S. aureus strains most often used in the literature we reviewed were: the community-acquired methicillin-resistant (MRSA) strain USA300 LAC [S. aureus reference strains were also noted in certain studies. These included strain ATCC 6538, a standard strain for biocide susceptibility testing [S. aureus strains [The main limitation was identified after reviewing the sections ‘Materials and Methods’ sections of the manuscripts selected, as many articles did not include any clinical strains from CF patients and only studied reference laboratory strains as material to support the observations reported . This waA300 LAC ,20 and iA300 LAC ,21 from A300 LAC ; strain A300 LAC ; strain A300 LAC ; and theA300 LAC ,25. Some testing ,27 and s testing , a strai strains .The use of these reference strains may not reflect the behavior of clinical CF strains as laboratory maintenance may affect their genomic integrity and alter phenotypic traits over the years ,30. CurrS. aureus colonize the toothbrushes of colonized patients and may represent a reservoir for patient reinfection [S. aureus on the sinus mucosa of CF patients supporting the fact that both upper and lower airways were the sites of S. aureus biofilm formation. In CF patients suffering from chronic rhinosinusitis, the sinuses may thus represent a reservoir for S. aureus, predisposing them to recurrent lung infection [A few of the studies we reviewed reported direct visualization of biofilm by scanning electron microscopy (SEM) and further viability evaluation by confocal laser scanning microscopy (CLSM) combined with Live/Dead stain. These approaches were applied to the toothbrushes of CF children and helped demonstrate that sessile nfection . Interesnfection . Similarnfection . Fluoresnfection .S. aureus biofilm whereas fewer, more recent, studies used ex vivo lung models. In both conditions, CF mucus-mimicking media, also called artificial sputum medium (ASM) or synthetic CF sputum medium (SCFM), were used in some studies to provide results more relevant to the CF lung than those obtained by using the media normally recommended, i.e., Trypticase Soja (TS) supplemented or not with glucose and NaCl, Brain Heart Infusion (BHI) or Luria Broth (LB) due to rheological properties closer to those of the CF mucus [S. aureus followed by P. aeruginosa in CF patients, and demonstrated that introducing the two species according to a timeline, and at various inoculation strengths, affects the formation of biofilm [However, the majority of currently available studies are based on in vitro approaches to studying CF mucus ,39,40,41CF mucus ,38. In pCF mucus . HoweverCF mucus ,41. SimiCF mucus . Finally biofilm .S. aureus from CF patients showing strains that did not produce EPS to EPS-overproducing strains, even in the same patient [S. aureus strains from CF patients  production using Congo red agar plates on a collection of  patient . Besides patient . They capatients . However® (BRT®)  and the BioFluxTM 200 to study biofilm formation in the presence or absence of antibiotics by MRSA from CF patients [® evaluates bacterial adhesion and early biofilm formation by immobilizing magnetic beads throughout the formation of biofilm. It is also dedicated to studying the capacity of antibiotics to inhibit biofilm formation through an approach called the Antibiofilmogram® [® which did not accept opaque media. They showed that adhesion was enhanced in ASM compared with BHI medium. They also brought new insights into CF MRSA’s ability to form biofilm, previoulsy little studied, showing that biofilm formation was strain-dependent, even for clonally related strains isolated from a same sputum sample, and differentially influenced by antibiotics. The importance of the patient’s colonization history was also highlighted as two MRSA strains, isolated three years apart in a chronically colonized patient, showed distinct patterns of biofilm formation (increased biofilm formation for the latest isolated strain) in the BioFlux™ 200 assay. More recently, Cheng et al., considering that in vivo biofilm may be formed in the absence of any surface for attachment, developed a surface-independent method to study biofilm formation based on a hanging-drop biofilm culture model with the aim of reducing the gap between in vitro predictions and in vivo responses [S. aureus biofilms: sdrC (Ser-Asp-Arg-rich fibrinogen-binding protein) encoding proteins for fibrinogen mediated cell adhesion, arcB (ornithine transcarbamylase) involved in extraction and catabolism of arginine, and ureC (urease accessory protein C) involved in metabolism of urea, showed that these three genes were differentially upregulated over time in hanging-drop S. aureus biofilms compared with surface-attached ones supporting that hanging-drop biofilm maturation occurred earlier than that of surface-attached biofilm. In addition, sdrC expression levels were unchanged in hanging-drop model but dramatically upregulated in surface-attached biofilm model suggesting that the biofilm formation in the hanging-drop formation may not depend on fibrinogen-mediated adhesion [Boudet et al. used both the Biofilm Ring Testpatients . BRT® evlmogram®  whereas lmogram® . Both aplmogram® ,48, partesponses . Using tS. aureus during co-infection with Respiratory Syncytial Virus (RSV) in live-cell imaging chambers. They observed that S. aureus biofilm growth was enhanced when cells were coinfected with RSV and that factors secreted during viral infection benefitted S. aureus biofilms. This observation obtained with RSV and S. aureus reference strains was also observed with human Rhinovirus 14 and confirmed with CF chronic rhinosinusitis clinical isolates [S. aureus biofilm formation after histological staining in an ex vivo model of CF infection comprising pig bronchiolar tissue  and the synthetic mucus SCFM in the presence or absence of antibiotics [S. aureus localized as aggregates in the mucus (unattached biofilm) rather than associated with tissue. Sweeney et al. also provided interesting comparative results for (i) a pair of S. aureus strains isolated during lung exacerbation and stable clinical status showing differences in both growth and the location of bacterial cells and (ii) three lungs showing that growth of the strains depended on the lung tissue inoculated and thus on the host [S. aureus isolates displayed an increased tolerance to antibiotics  with effects not correlated with the Minimum Inhibitory Concentration (MIC). Thus, the EVPL model was reported as a host-mimicking model suitable for accurate antimicrobial susceptibility testing of CF pathogens [Using both immortalized human CF airway epithelial cells and primary CF human bronchial epithelial cells (obtained from the explanted lungs of CF patients), Kiedrowski et al. quantified biofilm biomass produced by isolates . Two othibiotics ,51. Theythe host . Regardiathogens ,51.S. aureus strains according to the patient’s colonization history  or clinical status (stable or with pulmonary exacerbation). This should be borne in mind when analyzing the literature and trying to compare results from different studies.All these methodological development efforts underline the challenge of studying biofilm formation in the context of CF and it probably remains illusory to mimic all the environmental stresses and conditions found in CF airways . They also underline the different behavior of S. aureus strains through indirect measurement via CV staining.The studies reviewed in this part of the manuscript are summarized in S. aureus from CF and non-CF patients with divergent results. In the study by Molina et al., comparing 17 strains collected from CF respiratory samples and 20 strains from blood cultures of non-CF patients, nearly all strains formed biofilm and no significant difference in biofilm formation between strain categories was observed  [S. aureus (MSSA) compared with 57 non-CF S. aureus isolates collected from hospitalized patients with lower respiratory tract infection . Biofilm production was indeed detected in 96.8% of CF strains versus 47.4% of non-CF strains, suggesting that S. aureus may have a particular ability to form biofilm in the context of CF [First of all, we addressed the question of whether CF strains may have specific capacities for biofilm formation. We found two observational studies that compared the biofilm-forming ability of formers) . By contS. aureus strains including 16 MRSA and 70 MSSA. Biofilm production was detected in 74.4% of the strains distributed in 25.6% of weak, 41.9% moderate and 7% strong biofilm-producers [S. aureus isolates were minor biofilm-forming strains  , while 546 isolates (23.5%) were strong biofilm-producers [S. aureus strains examined by Pompilio et al. [® and ASM, 55.6% of 63 CF MRSA were shown to form early biofilm and were classified according to the biofilm formation index value generated by the device in 28.6% of intermediate and 27% of strong biofilm-producers [We then examined other studies reporting the overall proportion of CF strains displaying any ability to form biofilm. Taken together, three studies using the same classification criteria  examinedroducers ,55,56. Oroducers . Wienekeroducers . Biofilmroducers  and in 8o et al. . Finallyroducers .S. aureus strains collected early in the course of CF airway infection (early strains) and long-term adapted S. aureus strains (late strains). The seven studies retrieved showed different results, as four studies including few strains found a positive association between biofilm production and the persistence of S. aureus over time in CF patients [S. aureus strains, one reported no change [We finally examined studies evaluating biofilm formation over time during chronic colonization of CF airways by comparing biofilm formation of patients ,60,61,62o change  and two o change ,64. ThesS. aureus clinical isolates, one isolated at the beginning of infection and one at a chronic stage in another patient. Despite the limitation that the two strains were not isolated from the same patient, the late isolate produced almost five times more biofilm than the early isolate with the continuous-flow culture bioreactors [TM 200 system, whatever the experimental condition (BHI medium or ASM) and measured time point  [S. aureus strains (49 pairs of CF MRSA), Gilpin et al. did not observe any significant differences in biofilm production between early and late isolates collected at least two years apart, although all strains showed the capacity to form biofilm [S. aureus persistence was associated with variable evolution in biofilm formation. In a one-year observational prospective study, Wieneke et al. examined a total of 2319 CF S. aureus strains (methicillin-resistance not specified) collected from the sputum of 14 CF patients with long-term persistent infections (range: 10–21 years of persistence) [S. aureus (including 3.4% MRSA and 96.6% MSSA) collected from the sputum of CF patients with a mean persistence of 8.25 years (range: 5.1–13.6 years) [Ciornei et al. compared two reactors . Congrueor 36 h) . Similaror 36 h) . Of the or 36 h) . By stud biofilm . Finallyistence) . They ob6 years) . BiofilmP. aeruginosa is also a major factor of variability of S. aureus biofilm formation  [S. aureus survival to increase biofilm formation at the onset of chronic colonization to protect itself against phagocytosis and antibiotic treatment whereas, during long-term persistence, it might be more suitable to form less biofilm to be able to disseminate in the airways of CF patients [The variability observed in biofilm production both between and within studies might be explained by numerous confounding factors like the length time of observations and also the timeline of strain isolation regarding the patient’s colonization history . The influence of co-infecting pathogens such as  part 6) . Finallypatients .S. aureus isolates were associated with fewer pulmonary exacerbations in CF patients and, conversely, that exacerbations had a negative impact on biofilm formation [Altogether, biofilm formation was a common trait of both MRSA and MSSA strains in CF patients ranging from 55.6% to 100% of the strains studied according to the study with a proportion of strong biofilm-producing strains varying from 7 to 71.4%. As indicated, such variability may be associated with distinct methods of biofilm formation study or criteria applied for interpretation, to distinct studied strains and patient colonization history . The clinical impact of biofilm formation has yet to be investigated but Wieneke et al. showed that high biofilm-forming ormation .S. aureus collected from children with clinical symptoms of infection admitted to Children’s Medical Center Hospital in Tehran showing an association between biofilm formation and MRSA [ica) operon was shown to mediate biofilm formation in MSSA through the production of polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl-β-(1-6)-glucosamine (PNAG) whereas an ica-independent mechanism involving the fibronectin binding proteins, FnBPA and FnBPB, and the major autolysin Atl appears to play an important role in MRSA biofilm development [To date, we only found two studies that have compared MRSA and MSSA in their ability to form biofilm but only one included CF strains. In the latter study, Pompilio et al. showed a significantly higher median biofilm amount produced by MRSA compared with MSSA but this study was based on a few CF strains (three MRSA and three MSSA) . These rand MRSA . It thuselopment ,73.Regarding small colony variants (SCV), which are slow-growing auxotrophic subpopulations of bacteria that play an important role in persistence in the CF lung, Morelli et al. observed that 28 SCV strains (7 MRSA and 21 MSSA collected from CF patients) showed a significantly higher ability to form biofilm than 29 strains with normal phenotypes (100% versus 62%) but also comprised a higher proportion of strong biofilm-producers (21.4% versus 6.9%) . In thatS. aureus [S. aureus cultures, either MRSA or MSSA [S. aureus persistence (mean duration of 15.6 years) during which the number of SCVs increased longitudinally and the deep culturing conditions. Nevertheless, congruent results were observed in all three studies showing that mucoid S. aureus strains produced significantly higher amounts of biofilm compared to non-mucoid strains [S. aureus in patients’ airways.Finally, an unusual mucoid phenotype was described for . aureus  and furt or MSSA ,65,66. T or MSSA  compared strains ,65,66. C strains . MucoidyS. aureus biofilm formation, as well as the genes and proteins involved in biofilm production have been widely described in contexts other than CF [Sa G5-E repeat protein SasG, the Clf-Sdr family consisting of clumping factor A/B (ClfA/B) and the serine-aspartate repeat family (Sdr) proteins, ica operon (icaADBC) transcription resulting in the expression of PIA/PNAG. Similarly, different regulators were characterized such as the intracellular adhesin locus regulator (icaR), the teicoplanin-associated locus regulator (tcaR), the staphylococcal accessory regulator (sarA) and the accessory gene regulator (agr) [ than CF : FnBPA a than CF ,75, Sa Gor (agr) ,76,77,78or (agr) ).S. aureus strains from CF patients, i.e., host response, environmental factors , mutations and altered gene expression, and mucoidy, as well as the genes and molecular mechanisms that are critical for biofilm formation by S. aureus in the unique context of CF. These factors remain poorly explored in the literature and are promising targets for fighting persistent S. aureus infections especially in patients with CF.In the following subsections, we describe the different factors that were shown to influence biofilm formation of S. aureus strains (methicillin resistance not specified) trying to understand “frustrated phagocytosis”, i.e., the weak activity of phagocytic cells against microbial biofilm [S. aureus cell wall components, such as peptidoglycan and lipoteichoic acids, could influence the stimulation of leukocytes resulting in various amounts of cytokine production, including interleukin (IL)-10, IL-6 and tumor necrosis factor-α. However, although these immunomodulatory properties were observed for biofilm-forming CF S. aureus strains, they were not specific to these strains and also present in their planktonic counterparts, as already shown by Ciornei et al. [Sadowska et al. studied two CF  biofilm . They shi et al. .S. aureus in CF is affected by environmental factors including metal ions. Indeed, in an MRSA strain called CFSa36 (sequence-type not determined), Liu et al. identified a putative cobalt transporter ATP binding domain (CbiO) that was required for biofilm formation. They showed that the copper ions (Cu2+) entirely complemented the capacity of the cbiO knockout mutant to form biofilm in a dose-dependent manner without having any impact on bacterial growth. Conversely, iron ions (Fe3+) significantly decreased the ability of MRSA to form biofilms in a dose-dependent manner. Thus, they hypothesized that CbiO might mediate S. aureus biofilm formation by affecting the transport of copper ions [Biofilm formation by per ions ,81.S. aureus biofilm production. By studying 98 CF MRSA, Gilpin et al. showed that biofilm formation was significantly lower under anaerobic conditions than under aerobic conditions [ica gene transcription and PIA/PNAG expression by S. aureus and consequently, biofilm formation [S. aureus.Anaerobic conditions, typically observed in the CF lung due to mucus obstruction, can also influence nditions . It shouormation ,83. BecaS. aureus strains from CF children (specimen collection site and methicillin resistance not specified), Ulluwishewa et al. demonstrated that physiologically relevant concentrations of bile and, more precisely, sodium cholate and sodium deoxycholate bile acids enhanced biofilm formation [S. aureus develops an adaptive response by increasing biofilm production. Although bile-stimulated biofilm formation was also observed in two community-acquired MRSA strains from non-CF patients, suggesting that it is not a CF-specific phenomenon, it may be more prevalent in CF patients as up to 40% of CF children and 80% of CF adults can suffer from gastro-oesophageal reflux disease resulting in the presence of bile acids into the lungs [Bile acids are another environmental factors affecting biofilm formation. On two ormation . Regulathe lungs ,84.S. aureus strains (one pair of MSSA and two pairs of MRSA) collected from chronically infected CF patients. Mutations and altered gene expression occurred over time, some of which seems to give CF S. aureus strains greater ability to produce biofilm, i.e., mutations in the fakA (fatty acid kinase A) gene and mutations in the saeR (Staphylococcus exoprotein expression protein R) gene causing an up-regulation of SdrD adhesin involved in biofilm production, down-regulation of the agr regulon resulting in an overexpression of adhesins [fakA gene currently little described in S. aureus, these genes have already been reported as being involved in S. aureus biofilm production [dacA and gpdP genes correlated with increased biofilm formation over time. The dacA gene encodes a deadenylate cyclase required for the synthesis of cyclic-di-AMP (c-di-AMP), a multifunctional secondary metabolite involved in biofilm formation whereas gpdP encodes a phosphodiesterase responsible for c-di-AMP hydrolysis [Sa biofilm formation by Corrigan et al., and are not specific to CF strains [saeR gene [S. aureus in CF patients: increased expression of specific metabolic genes including gapR , zwf (involved in the pentose phosphate pathway) and oxidative tricarboxylic acid (TCA) cycle genes like sdh and mqo1 as well as decreased expression of gltA, all of which lead to biofilm production [fumC gene expression that functions primarily as a malate dehydratase increasing fumarate concentrations not significantly assimilated by MRSA strains. Thus, in CF patients, MRSA strains undergo metabolic reprogramming with minimal consumption of TCA cycle substrates (which limit pro-oxidant generation) and conversely increased assimilation of pyruvate and glucose polymers which promote the production of extracellular polysaccharide-composing biofilm, such as PIA/PNAG . By proS. aureus strains isolated from CF patients were hyper-producers of PIA-associated biofilms but also described the underlying molecular mechanism for mucoidy and its impact on biofilm formation [icaR and icaA. Deletion affected binding of the repressor of biofilm “rob” [S. aureus strains, Schwartbeck et al. identified non-mucoid and non-biofilm-forming late isolates without PIA/PNAG hyperexpression, and carrying the 5 bp-deletion. They demonstrated that various compensatory mutations in icaA, icaD and icaC caused the abrogation of biofilm formation and a non-mucoid phenotype. Thanks to whole genome sequencing, they confirmed that mucoid isolates evolved from the non-mucoid S. aureus clonal strain and that the isolates with both 5 bp-deletion and compensatory mutations evolved from the mucoid isolates. For some, but not all mucoid isolates, mucoidy might thus confer a short-term advantage but, due to mucoidy-associated fitness loss, not a long-term advantage for persistence, resulting in the emergence of compensatory mutations. This corroborated certain observations of a decrease in biofilm formation over time and the hypothesis drawn by Hirschhausen et al. that, after an initial increase in biofilm formation to enhance protection during establishment in the CF lungs, decreasing biofilm formation could be more appropriate for dissemination and persistence [Schwartbeck et al. not only showed that mucoid ormation . The 115lm “rob” , resultisistence .Pulmonary infections in CF patients are considered as polymicrobial ,90,91 anS. aureus-P. aeruginosa as this is the co-infection most frequently observed in CF patients. Although co-infections with Haemophilus influenzae are observed before adulthood (less than 10% of patients colonized by Sa), the majority of co-infections are indeed those observed with P. aeruginosa. A study on 134 patients from 2004 to 2017 managed to identify S. aureus-P. aeruginosa co-infections in 30–50% of patients [S. aureus being mostly studied in association with P. aeruginosa. Nevertheless, among all these studies, we noted that (i) most studies on the P. aeruginosa-S. aureus combination focused on P. aeruginosa with few or no results on S. aureus [P. aeruginosa co-studied strains were mostly reference strains, i.e., PA14, originating from a wound on a burns patient [S. aureus and P. aeruginosa clinical strains, with the aim of reporting results as close as possible to the specific clinical context of CF. A single exception was made for the publication by Barraza & Whiteley that studied laboratory strains of P. aeruginosa (PA14) and S. aureus (LAC) but used synthetic CF sputum medium (SCFM2), to be as close as possible to CF lung conditions [S. aureus-P. aeruginosa studies, we noted a major lack of studies on clinical strains from CF patients as the majority of studies—even the most recently published ones—only included reference strains. The most relevant study among all those we selected is probably the one by Fugère et al. which included S. aureus and P. aeruginosa clinical strains co-isolated from the same patient. It showed that some results were specifically obtained when studying these pairs of strains, hence the need for such studies which are extremely rare in the literature [Diverse associations of pathogens may be observed in CF patients , the mospatients . More gepatients . A simil. aureus  and (ii) patient ,103, PAO patient ,39 and s patient . As in oterature .S. aureus co-cultured with P. aeruginosa with (i) fewer S. aureus aggregates, S. aureus planktonic cells being more numerous, and (ii) S. aureus aggregates of smaller size [First, regarding the overall architecture of biofilm, results obtained for laboratory strains in SCFM2 highlighted a modification in the structural frame of biofilm formed by ler size .S. aureus produces various exoproducts that can be sensed by P. aeruginosa [S. aureus virulence, and the Staphylococcal protein A of S. aureus (SpA), a secreted virulence factor that affects host immune response and inhibits phagocytosis (P. aeruginosa isolates. StP was shown to inhibit biofilm formation by P. aeruginosa by reducing the availability of zinc [S. aureus (chronic colonization) and P. aeruginosa (sporadic colonization) from the same CF patient via the CV-based method and showed a decrease in P. aeruginosa biofilm formation in S. aureus-P. aeruginosa co-culture compared with P. aeruginosa monoculture [S. aureus-P. aeruginosa interplay has only been reported for strains originating from CF patients. On the other hand, SpA interacts with two important determinants of P. aeruginosa biofilm formation: i) exopolysaccharide, encoded by the polysaccharide synthesis locus (psl), which is the predominant polysaccharide of the extracellular matrix of P. aeruginosa biofilm and a crucial component for its formation and ii) PilA, a component of the type IV pili of P. aeruginosa [P. aeruginosa biofilms and the interaction between SpA and Psl was associated with an increase of P. aeruginosa tolerance towards tobramycin, an antibiotic commonly used against this pathogen in aerosol therapy [On a molecular level, ruginosa ,105. Thoocytosis . Both ex of zinc . Interesoculture . So far,ruginosa . SpA was therapy .S. aureus side, interacting with P. aeruginosa led to several modifications in characteristics, presented hereafter, all favoring the persistence of clinical CF S. aureus isolates  quorum sensing system of P. aeruginosa and increased biofilm formation by S. aureus strains [P. aeruginosa isogenic mutants deficient in PQS and HQNO production which significantly stimulated less biofilm formation by S. aureus [P. aeruginosa supernatants to S. aureus biofilms grown either on epithelial cells or on plastic also significantly decreased the susceptibility of clinical CF S. aureus strains to vancomycin [P. aeruginosa siderophores, pyoverdine and pyochelin, which also induce a decrease in S. aureus growth correlated with the decrease in susceptibility to certain antibiotics observed [S. aureus and P. aeruginosa strains co-isolated from the same CF patient were of great interest, showing that results vary depending on the origin of the strains, with less HQNO production by P. aeruginosa and a less stimulated S. aureus biofilm formation for co-isolated strains. These results highlighted the within-host co-evolution of S. aureus and P. aeruginosa adapting to the specific abiotic conditions of CF lungs and also to other pathogens present in the niche [Several studies have observed a correlation between HQNO (2-heptyl-4-hydroxyquinoline N-oxide) molecules of  strains ,109. The. aureus . Adding ncomycin . HQNO moncomycin , just asobserved . The obshe niche .S. aureus, a form adapted to intracellular survival [S. aureus were significantly more frequently co-colonized with P. aeruginosa than patients colonized by S. aureus of normal phenotype (75% vs. 37.9%) [S. aureus regulator Sigma factor B (SigB), a crucial factor for adaptation in chronic infections, which was previously linked to an increased expression of both the FnBPA-encoding gene and the biofilm-associated sarA gene [P. aeruginosa selected for mucoid phenotype of S. aureus that was associated with high biofilm formation and this represented another adaptive characteristic of S. aureus for protectin and survival against attacks by small molecules produced by P. aeruginosa such as HQNO [HQNO molecules also induce SCVs of survival . Interes. 37.9%) . Both tharA gene ,111. Add as HQNO .P. aeruginosa, S. aureus may also undergo a switch to a Viable But-Non-Cultivable (VBNC) state that favors its survival and persistence in the co-colonized host. Molecular investigations revealed that several S. aureus genes involved in virulence  were overexpressed during this switch [S. aureus pathogenicity and be involved in the clinical outcome of the co-infection [Finally, in the dual-species biofilm with s switch , suggestnfection .S. aureus was studied in mixed biofilm assays with Streptococcus anginosus in the presence or absence of P. aeruginosa in a mucin-containing medium [S. aureus secreted compound(s) protected sessile S. anginosus from antibiotic killing whereas the antibiotic killing of P. aeruginosa was not influenced by the presence of S. aureus or S. anginosus and more S. aureus cells were killed by antibiotic treatment when grown together with S. anginosus and P. aeruginosa [Introducing a third partner add complexity to the interactions. We found only one study on three species—the study by Tavernier et al.—in which g medium . The comruginosa .S. aureus and P. aeruginosa correlates with a decline in lung function and a higher number of exacerbations and intravenous antibiotic treatments compared to infection with S. aureus or P. aeruginosa alone [P. aeruginosa and MRSA is associated with the most severe clinical pictures compared to co-infections with P. aeruginosa and MSSA [P. aeruginosa and MRSA, an increase in markers of lower airway inflammation was observed [S. aureus and P. aeruginosa affecting biofilm formation during Cystic Fibrosis but also that CF clinical isolates may display distinctively different traits from reference laboratory strains, thereby supporting the need for additional studies including clinically documented S. aureus strains from CF patients. These observations also warrant the need for studies of multispecies biofilms formed by S. aureus and other clinically relevant species in CF, either bacterial species such as members of the Burkholderia cepacia complex or emerging pathogens Stenotrophomonas maltophilia and Achromobacter spp., or fungal species. Indeed, interactions highlighted between S. aureus and these species in planktonic cultures [Despite growing interest in characterizing species interactions, their potential implications in the progression of polymicrobial pathologies are still poorly understood ,114. Howsa alone . Particuand MSSA . Furtherobserved . These sobserved , particucultures ,118,119 cultures  should bS. aureus and CF, several compounds including natural ones and antibiotics were shown to display anti-biofilm activity, whether limiting the formation of biofilm or affecting preformed biofilm.Limiting biofilm formation has thus become an important tactic considered in the development of new antimicrobial strategies. Regarding Antimicrobial peptides (AMPs) are natural products of the immune system of particular interest with their broad-spectrum antimicrobial activity due to their disruptive mode of action against bacterial membranes occurring after electrostatic interaction between AMPs and bacterial cells . Three cLimnonectes fujianensis, was shown to be particularly effective against two sessile MRSA CF strains through membrane permeabilization [Galleria mellonella larvae infected by MRSA whereas no death of larvae were observed after injection of two doses of Japonicin-2LF. This AMP appeared to be a potential candidate for further evaluations as cytotoxicity, haemolytic activity and lactate deshydrogenase release were observed with high concentrations of 64 μM of Japonicin-2LF alone [More recently, Japonicin-2LF, an AMP secreted by the skin of the amphibian lization . InteresLF alone .S. aureus, unlike the observations made for P. aeruginosa and B. cepacia, proteins modified by the addition of negatively charged residues in the α1−α4 region showed increased anti-biofilm activity against S. aureus. Despite the origin of the clinical S. aureus strains used in this study is unclear, the study showed the importance of this region of the protein in anti-biofilm activity against S. aureus. A further study addressed the antibiofilm activities of SPLUNC1-modified proteins and SPLUNC1-derived peptides against six MRSA strains isolated from CF patients [Human SPLUNC1  is a protein of the innate immune system secreted in the human respiratory tract. It acts as a surfactant and regulates the epithelial sodium channel (ENaC) whose deregulation in CF worsens the mucus dehydration and ion imbalance due to a defect in CFTR . Althougpatients . A focusv/v concentration and it was found that several EOs had the ability to inhibit biofilm formation by one or several S. aureus CF strains. EOs from Piper nigrum (black pepper) (EO45) and Mentha suaveolens (sweet mint) (EO58) were selected for an in-depth study based on their constant activity on the studied S. aureus strains and biofilm reduction of up to 40% or more of its initial value, despite their different compositions. We may note that neither EO45 nor EO58 had any antibacterial activity on the CF clinical isolates showing an antibiofilm effect unrelated to the inhibition of bacterial growth. SEM analyses made it possible to visualize the highly disruptive action of both EOs on S. aureus biofilm showing a deconstructed surface and EPS disintegration. However, the dose-dependent effect of EOs was also highlighted as lower concentrations (0.05% v/v) mostly ended up abolishing the inhibition effect on biofilm formation and even enhanced biofilm growth in certain cases. Finally, chemical analysis led to the identification of the EO constituents related to the most effective biofilm growth inhibition as being eugenol, β-caryophyllene and, partially, β-pinene. Because of their various distinct components, essential oils probably have a multi-target action as each of the EO compounds may exhibit a different mechanism of action against biofilm formation by S. aureus strains from CF patients [S. aureus P8-AE1 biofilms) associated with G. mellonella larvae protection from S. aureus-induced killing [Essential oils are complex mixtures derived from plants and may display certain antimicrobial properties. Papa et al. studied the anti-biofilm effect of 61 essential oils (EOs) on three CF clinical isolates 4S, 5S and 19S (including two MRSA and one MSSA) compared with two reference laboratory strains using microtiter plate biofilm assay and CV staining . All EOspatients . In a gepatients . Other p killing . EncapsuPulmonaria officinalis was investigated for anti-adhesion and anti-biofilm properties on 20 clinical isolates from the sputum of children (1.5–19 years) with CF and chronic respiratory tract infection. The assay used inert polystyrene surfaces conditioned or not with mucin and elastin to mimic respiratory tract mucosa [S. aureus viability were suggestive of variable reduction in surface adhesin expression. However, subsequent observation of biofilm reduction did not reach significance and biofilm may even be increased under certain conditions; the extract was not effective in eradicating preformed biofilm either [S. aureus in several studies [S. aureus enhancing adhesion and inducing the production of biofilm components [S. aureus strategy as they might reduce not only epithelial toxicity but also biofilm formation on host tissues.Among the numerous complementary, alternative medicine practices, CF patients may use herbal therapy. In this context, the phenolic-rich fraction of an extract of the aerial parts of t mucosa . Adhesiom either . Howeverm either  making Sm either . Alpha-t studies ,131,132. studies . Others S. aureus strains by Pompilio et al., 2013. Usnic acid and atranorin, which displayed antimicrobial activity on six S. aureus strains from CF patients , were evaluated for their activity against adhesiveness, biofilm formation and preformed biofilm using CV staining. Both secondary metabolites at 1/2x MIC affected adhesion of all strains and biofilm formation in all strains except one (unaffected by usnic acid only). Atranorin also decreased bacterial adhesion at lower concentrations (1/4x and 1/8x MIC) (75% reduction of adhesiveness compared with control for most conditions). At these subinhibitory concentrations, usnic acid and atranorin displayed variable results on biofilm formation depending on the S. aureus strain and metabolite concentration, with the highest activity for atranorin on MRSA and usnic acid on MSSA. Both metabolites were also significantly active on biofilm preformed by the two strongest biofilm-producing strains in this study (Sa3 and Sa15 MRSA) whatever the concentration tested  [Secondary metabolites from lichens, more precisely, the fungal component of lichens, were first challenged for antibiofilm activity on CF 10x MIC) . The eff10x MIC) . Regardi10x MIC) . However10x MIC) .P. aeruginosa. For S. aureus, an antibiofilm effect was observed with tobramycin, the comparator used to evaluate AMP’s anti-biofilm activity against S. aureus CF strains [S. aureus strains tested), up to the lowest sub-inhibitory concentration evaluated (1/8x MIC). The Antibiofilmogram® approach by BRT® in filtered ASM was also used to study the effect of five antibiotics  on biofilm formation by 17 strongly adherent/strong biofilm producers MRSA strains from CF patients [TM 200 system and ASM confirmed the activity of these three antimicrobial agents in limiting biofilm formation and showed that ceftaroline and ceftobiprole had a significantly greater effect than linezolid [S. aureus (two MRSA and four MSSA) isolated from CF patients [10 decreases in 24-h-old and 12-day-old biofilms, respectively) than with clarithromycin ; iii) there was no influence from combining tobramycin with clarithromycin [Despite antimicrobial resistance may be drastically increased in biofilm ,135,136, strains . In thispatients . Trimethinezolid . Finallypatients . The maihromycin .S. aureus therapeutic options have been yet evaluated for their anti-biofilm activity, such investigations have to be pursued to completely characterize the anti-biofilm potential and mechanisms of the different treatments against S. aureus since results might be helpful to guide the choice of the most effective therapy against CF airway infection.As not all anti-Bdellovibrio bacteriovorus species is a member of the Oligoflexia class found in the human gut microbiota and known to prey on Gram-negative bacteria. Iebba et al. showed that the B. bacteriovorus strain HD100 was also able to prey on a S. aureus isolate from a chronically mono-colonized CF patient. However, its mode of attack was however specific compared with Gram-negative prey as direct contact was observed during the whole predation process and three non-released bacteriolytic enzymes that remain to be characterized appeared to have a role in this predator-prey interaction [S. aureus biofilms preformed during 24 h either statically (microtiter plate CV-straining) or under flow  were exposed to B. bacteriovorus in predatory assays. Epibiotic predation led to decreases in the amounts of preformed biofilm observed, both in static and dynamic settings, with a significant reduction in biofilm of 74% after 24 h of contact with the predator in the static assay and a 33% reduction after 14 h of contact, increasing to 46% after 20 h in the dynamic assay. The destruction of S. aureus cells seemed to occur by breaking down the bacterial wall and releasing the intracytoplasmic content of S. aureus. B. bacteriovorus was also shown to be a predator for CF P. aeruginosa, with an even higher reduction observed for preformed P. aeruginosa biofilm than S. aureus. Considering its presence in healthy human gut microbiota and its inability to infect mammalian cells, its ability to reduce established biofilms appeared of interest for in vivo applications during CF [Among the bacterial genera described as having predatorial activity, the eraction . S. aureuring CF .S. aureus strains despite the growing interest in bacteriophage-mediated control of biofilm, based on the ability of these bacteriophages to penetrate existing biofilm and eliminate its structure [S. aureus colonization [Surprisingly, our search criteria did not find any publications on bacteriophage anti-biofilm activity on CF tructure . This totructure , was sucnization .S. aureus strains only  studied in ASM supplemented with 0.25% glucose and in two oxygen conditions for biofilm eradication assays with the aim of mimicking CF conditions more accurately. Biofilms grown in ASM for 48 h were challenged with NO-releasing alginates for 24 h under aerobic and anaerobic conditions and the results were compared to those obtained with tobramycin and vancomycin. The anti-biofilm effect of NO-releasing alginates was clearly demonstrated, particularly for Alg5-PAPA-DPTA/NO, with a 5-log reduction in biofilm viability observed after 24 h and alginates performed more efficiently than tobramycin and vancomycin in both oxygen conditions. As NO disrupts vital bacterial cell functions and structures through protein, DNA, and metabolic enzyme alterations, NO-releasing alginates display a broad-spectrum activity, including P. aeruginosa and B. cepacia in addition to S. aureus, making them promising candidates for future therapeutic options [Ahonen et al. also studied the development of nitric oxide (NO)-releasing alginate oligosaccharides, considering NO’s anti-bacterial activity both on planktonic bacteria and biofilms and the need to decrease its toxicity through finely-controlled release . Alginat options .S. aureus strain (Sa2) from a CF patient, showing the effectiveness of AgNPs against biofilm viability, with a dose-dependent effect and a maximum biofilm-killing rate of 98.2 ± 0.5% at 2x MIC [P. aeruginosa and shown to be non-toxic in in vivo studies on G. mellonella larvae. They require further investigation to elucidate their mode of action on S. aureus biofilms and their therapeutic potential for CF patients.Pompilio et al. evaluated electrochemically synthesized silver nanoparticles (AgNP) formulation on a strong biofilm-forming t 2x MIC . These nS. aureus strains from CF patients [S. aureus strains  to form biofilm on titane (Ti-6Al-4V) and zirconium oxide biomaterials commonly used in dental implants [S. aureus biofilm reduction, affecting both adhesion  and S. aureus survival (live/dead fluorescence microscopy) as well as detaching biofilm-forming S. aureus from the biomaterial. Indeed, biofilm structure damage was visible after 10 min and attributed to an increase in the repulsive electrostatic forces between S. aureus and the biomaterial [Physical stimulations, either electrical or magnetic, have also been applied to biofilms formed by patients ,145. In S. aureus adhesiveness and the subsequent formation of biofilm was also the hypothesis drawn from the observation that applying a magnetic field of extremely low frequency to three S. aureus strains from CF patients (strong biofilm formers and multidrug resistant) significantly decreased biofilm formation and viability [2+, Cu2+, Fe2+/Fe3+, K+, Mg2+, Na+, Zn2+) showed that stimulating nearly all ions caused a significant reduction in S. aureus biofilm biomass formation (at least 25% compared with control) and in viability of the biofilm formed, suggesting that there were modifications in the attraction between the S. aureus surface, normally negatively-charged, and the positively-charged polystyrene surface used in this study. The high potential of such an intervention has been suggested to prevent biofilm formation or to eradicate biofilm on medical devices like nebulizers used by CF patients.Altering the electrostatic interactions that might reduce iability . Indeed,S. aureus clinical isolates from CF patients is given in A summary of the strategies limiting biofilm formation by S. aureus strains isolated from patients with CF, were reported in the text. However, observational studies are the main studies performed in the CF context and affected targets by the anti-biofilm strategies reviewed in our manuscript remain largely unidentified in this context but also more generally in the literature.In our review of the literature, we found no strategies with a specific action on the dispersal stage of biofilm but it is obvious that each affected stage of biofilm formation  will impact the subsequent ones . When avS. aureus is a major opportunistic pathogen in CF patients and biofilm production is a determining factor in the onset of persistent S. aureus respiratory infections in these patients. During CF, a broad panel of biotic and abiotic factors influences the different stages of biofilm development by S. aureus due to the specific, stressful conditions found in CF lungs. In this unique environment, S. aureus strains display specific features driven by bacterial adaptation and within-host evolution during persistence. Biofilm formation is one of these important traits representing a major obstacle, protecting S. aureus from host defenses and eradication attempts. Greater knowledge of the specific characteristics of biofilm formation by well documented S. aureus strains originating from CF patients is required as modulating or inhibiting biofilm formation is an important strategy for infection control and a target for the development of new therapeutic agents [S. aureus in CF patients, none of them has currently been currently evaluated during clinical trials. However, yet available results pave the way to further research that should address the more promising strategies for managing S. aureus infections in these patients but also their advantages and limitations. Indeed, biofilm eradicating strategies that kill bacteria independently of their physiological stage and are thus also active on persister cells within biofilm are of great interest [c agents . Althouginterest . The potinterest . Finallyinterest ,148.


Staphylococcus lugdunensis endophthalmitis is an uncommon intraocular infection with potentially visually devastating consequences. S. lugdunensis endophthalmitis have been reported following cataract surgery, trauma, intravitreal injections of anti-vascular endothelial growth factor agents and dexamethasone implant. We report four cases of postoperative S. lugdunensis endophthalmitis after cataract extraction (three patients) and combined pars plana vitrectomy and cataract extraction (one patient). The onset of presentation of endophthalmitis was acute (within 2 weeks) in two patients, subacute (2 to 6 weeks) in one patient, and chronic (more than 6 weeks) in one patient. All patients had presenting visual acuity (VA) of hand motions or worse and were treated with pars plana vitrectomy with intravitreal antibiotics. The final VA was 20/50 in two patients, 4/200 in one patient with pre-existing myopic maculopathy, and no light perception in one patient with retinal detachment. In antibiotic susceptibility testing, S. lugdunensis isolates were resistant to penicillin , but all were susceptible to vancomycin, oxacillin, teicoplanin, tigecycline, and sulfamethoxazole-trimethoprim. S. lugdunensis may be associated with acute or chronic endophthalmitis. Favorable visual outcomes can be achieved with prompt diagnosis and management. Staphylococcus species. The incidence of postoperative endophthalmitis ranged from 0.02 to 0.2% after cataract extraction and from 0.016 to 0.083% after intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents or corticosteroids [Staphylococcus lugdunensis [S. lugdunensis accounted for a total of 5.9% of coagulase-negative Staphylococcus specimens cultured from both intraocular and extraocular sites, whereas S. epidermidis accounted for 81.9% [S. lugdunensis is a Gram-positive, coagulase-negative bacterium, and can cause infective endocarditis, bone and joint infections, and skin and soft tissue infections [S. lugdunensis endophthalmitis have been reported following cataract surgery, trauma, and intravitreal injections of anti-VEGF agents and dexamethasone implant [S. lugdunensis at a tertiary referral center in Northern Taiwan. We also conducted a literature review and compared the clinical characteristics with our study.Bacterial endophthalmitis is the most feared complication of intraocular surgery that can result in severe visual loss. Acute postoperative endophthalmitis is mainly caused by patients’ own flora on the ocular surface, with the majority being associated with dunensis . The EVSor 81.9% . S. lugdfections . Several implant ,11,12,13S. lugdunensis endophthalmitis from January 2009 to April 2019 at a tertiary referral center in Northern Taiwan. The institutional review board of Chang Gung Memorial Hospital in Taoyuan, Taiwan, approved this retrospective study protocol  and waived the need for written informed consent by these patients. All clinical procedures were conducted according to the principles of the Declaration of Helsinki. A review of all patients with a diagnosis of acute-onset endophthalmitis at the Chang Gung Memorial Hospital Microbiology Department was performed. Patients were included in this study if vitreous and/or aqueous samples taken at the time of diagnosis grew isolates of S. lugdunensis. The availability of medical records used electronic systems. Data collected and reviewed included demographic information, medical history, presenting signs and symptoms, duration of symptoms before diagnosis of endophthalmitis, intervals between event, and diagnosis of endophthalmitis, culture sites, antibiotic sensitivities and resistance patterns, management administered, and final visual acuity (VA). The doses of intravitreal agents were as follows: vancomycin, 1 mg/0.1 mL; ceftazidime, 2.25 mg/0.1 mL; and dexamethasone, 0.4 mg/0.1 mL. Poor visual outcomes were defined as VA worse than 20/200.This study is a retrospective, consecutive case series of patients diagnosed with All microbiological investigations were performed at the Microbiology Department of Chang Gung Memorial Hospital, Taoyuan, Taiwan. Bacterial culture isolates were identified using conventional microbiological methods and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Conventional microbiological methods included Gram staining and biochemical tests. In MALDI-TOF-MS, automated measurement of the spectrum and comparative analysis against reference bacteria spectra were performed using an Ultraflextreme mass spectrometer and in MALDI Biotyper 3.0 software . The reliability of identification in the MALDI Biotyper system was expressed in terms of points. A log(score) of ≥2.0 indicated identification to the species level. The antibiotic susceptibility testing results were based on the choice of antibiotics tested in our hospital during the study period. The isolates were tested for susceptibility to various antibiotics using the Kirby–Bauer disc diffusion method on Mueller–Hinton blood agar. Clinical and Laboratory Standards Institute  standards were used for interpretation and quality control for each corresponding year.S. lugdunensis endophthalmitis during the study period. The mean age of patients was 71.0 ± 3.5 years (range: 67 to 75 years). All patients were female. A total of four patients were diagnosed with S. lugdunensis isolates. Most S. lugdunensis isolates were resistant to penicillin (3/4), but all were susceptible to vancomycin, which is currently being used empirically to treat Gram-positive bacterial endophthalmitis. The isolates were also susceptible to oxacillin, teicoplanin, tigecycline, and sulfamethoxazole-trimethoprim. One isolate was resistant to clindamycin and erythromycin.S. lugdunensis is a constituent of the human normal skin flora and an infrequent human pathogen. Early studies established S. lugdunensis as a skin, nasal cavity, conjunctival commensal [S. aureus, but this has been rectified by recent routine use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in diagnostic laboratories [S. lugdunensis represents an ambiguous staphylococcal species, and, since its first description in 1988 [S. lugdunensis may be related to its tendency to form a biofilm, which has a role in bacterial colonization and interferes with the phagocytosis-associated activities of neutrophils and the production of enzymes  [S. lugdunensis represents the most aggressive species of CNS [S. lugdunensis endophthalmitis often had a worse final visual outcome and a higher frequency of post-vitrectomy retinal detachment [ommensal ,14,15,16 in 1988 . The aggS. lugdunensis endophthalmitis occurred within 3 weeks. However, in our series, two patients occurred within 2 weeks of surgery, and another two patients developed endophthalmitis on postoperative day 41 and 83. This could represent delayed onset endophthalmitis or secondary infection from unknown reasons. During vitrectomy, diffuse retinal vasculitis and hemorrhages were identified in all patients. One patient (patient 3) had condensed posterior pole preretinal exudates and retinal detachment, and became phthisic in last visit. We previously reported that acute endophthalmitis with severe vitritis are typically associated with condensed posterior pole preretinal exudates, which might be associated with poor visual outcomes [Previous studies showed that cases of outcomes .S. lugdunensis endophthalmitis following cataract surgery, Chiquet et al. [S. lugdunensis endophthalmitis caused by surgery  or trauma (two patients), Garoon et al. [S. lugdunensis endophthalmitis after intravitreal injection of anti-VEGF or dexamethasone implant (Ozurdex), a favorable visual outcome (≥20/200) was achieved in four patients receiving intravitreal antibiotics or pars plana vitrectomy. In our study, three eyes were successfully managed with pars plana vitrectomy, and two eyes achieved favorable visual outcomes. Given the mixed results, it is possible that pars plana vitrectomy may have impacted the final visual outcome in patients with worse presenting VA, and intravitreal injections of antibiotics alone could have resulted in favorable outcomes in patients with better presenting VA.The comparisons of our study with the literature are listed in t et al.  reportedt et al. . In anotn et al.  reportedn et al. ,9,10,12 S. lugdunensis isolates were susceptible to vancomycin, but most isolates (75%) were resistant to penicillin. All isolates were also susceptible to oxacillin, teicoplanin, tigecycline, and sulfamethoxazole-trimethoprim. In previous studies in patients with S. lugdunensis endophthalmitis, all isolates identified were susceptible to vancomycin [S. lugdunensis demonstrated resistance to oxacillin and one isolate demonstrated resistance to ciprofloxacin [S. lugdunensis was not performed in our hospital; therefore, there was no susceptibility testing data available for levofloxacin.In our study, all ncomycin ,9,13. GaS. lugdunensis isolates were not routinely tested for fluoroquinolones at our hospital. Third, follow-up periods were not standardized. Despite these limitations, our findings contribute to the literature in providing detailed analysis and review of S. lugdunensis endophthalmitis.The study has some limitations. First, this was a single-center retrospective small case series. Second, S. lugdunensis may be associated with acute or chronic endophthalmitis. Prompt diagnosis and management can result in favorable visual outcomes. Even though vancomycin-resistant Staphylococcus species are emerging in certain regions, vancomycin provides consistent coverage against S. lugdunensis, which has no known resistance at present, and remain a first line agent in treating endophthalmitis caused by CNS [In conclusion, d by CNS ,19.


Genetic mutations in SARS-CoV-2 have resulted in variants with more transmissibility and partial resistance to COVID-19 vaccines, as seen in the recently classified variant of concern (VOC) “Omicron”. The rapid spread has raised concerns about Omicron being airborne, which leads to a high risk of contamination in public premises, particularly among the frontline healthcare workers. Mandatory usage of protective face masks and respirators is highly recommended in order to break the chain of transmission. Furthermore, health authorities need to reassess the modes of transmission of VOCs and provide updated guidelines to the general public for its prevention. Coronavirus disease 2019 (COVID-19) is an illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). SARS-CoV-2 has evolved over time during viral replication due to genetic mutations that have given rise to multiple “variants” all around the world. The center for disease control and prevention (CDC) has defined a variant as “a viral genome (genetic code) that may contain one or more mutations” .Recently classified VOC, Omicron, consists of 50 mutations in its viral genome and its spike (S) protein has 30 mutations that brought about transmissibility greater than other VOCs, along with partial resistance to COVID-19 vaccines .ConsequePrevention from inhalation of potentially infectious contaminants has become possible by respirators which are manufactured as personal protective equipment (PPE) . CDC hasIn conclusion, SARS-COV-2 cases are potentially rising in multiple countries, resulting due to evasion from immunity from vaccination and higher transmissibility. To cut down the spread of SARS-CoV-2, appropriate PPE is essential requirement for public and healthcare professionals. Health authorities must consistently analyze and update guidelines for the use and selection of PPEs according to the latest findings regarding VOCs. This can help in increasing compliance as well as a reduction in SARS-CoV-2 transmission. Along with the vaccination campaigns, there is a need of raising awareness about appropriate use of PPEs among general public and medical community.Rate of prevalence and incidence of Covid-19 cases all over the world is given below Fig. 1)..Fig. 1).To overcome the deadly wave of omicron variant infections, public awareness should be spread regarding its rapid transmissibility. Use of personal protective equipments, circulation of fresh air in indoor settings, and immunization against the pernicious virus should be promoted. Not required.None.Z.Y and M.J.T conceived the idea, M.A.K, Z.Y, M.Z, and M.S.A retrieved the data, did write up of letter and finally Z.Y, M.J.T, and M.S.A reviewed and provided inputs. All authors approved the final version of manuscript.None.Not required.1.Name of the registry: Not required.2.Unique Identifying number or registration ID: N/A3.Hyperlink to your specific registration (must be publicly accessible and will be checked):Muhammad Sohaib Asghar.Externally peer reviewed, not commissioned.No financial support was acquired for this article.The authors declare no conflict of interest.


Ixodes arboricola, I. frontalis, I. ricinus, Haemaphysalis concinna and Hyalomma marginatum) were also selected for statistical comparisons. Differences were demonstrated between these tick species regarding their association with bird species that typically feed from the ground and those that rarely occur at the soil level. The ecology of these five bird-infesting tick species is also illustrated here according to avian orders, taking into account the ecology (habitat type) and activity (circadian rhythm and feeding level) of most bird species that represent a certain order.Hard ticks (Acari: Ixodidae) are considered the most important transmitters of pathogens in the temperate zone that covers most of Europe. In the era of climate change tick-borne diseases are predicted to undergo geographical range expansion toward the north through regions that are connected to southern areas of the continent by bird migration. This alone would justify the importance of synthesized knowledge on the association of tick species with avian hosts, yet birds also represent the most taxonomically and ecologically diverse part of urban vertebrate fauna. Birds frequently occur in gardens and near animal keeping facilities, thus playing a significant role in the dispersal of ticks and tick-borne pathogens in synanthropic environments. The primary aim of this review is to provide a comprehensive reference source (baseline data) for future studies, particularly in the context of discovering new tick-host associations after comparison with already published data. The records on the ixodid tick infestations of birds were assessed from nearly 200 papers published since 1952. In this period, 37 hard tick species were reported from 16 orders of avian hosts in Europe. Here we compile a list of these tick species, followed by the English and Latin name of all reported infested bird species, as well as the tick developmental stage and country of origin whenever this information was available. These data allowed a first-hand analysis of general trends regarding how and at which developmental stage of ticks tend to infest avian hosts. Five tick species that were frequently reported from birds and show a broad geographical distribution in the Western Palearctic ( Ixodes ricinus) to even 5 weeks or longer . While If thermophilic ticks carried by birds from south to north detach after engorgement, and find themselves in a suitable (warm enough) environment, they can molt to the next developmental stage. These ticks may then survive through mild winters, potentially establishing new, northern populations . This maIxodes species occur in the Western Palearctic, most of which associate with sea birds in Western Europe . These ticks occur in and near avian nests, or are frequently endophilic and live in tree holes or burrows inhabited by nesting birds, particularly in the subgenus Pholeoixodes   lividus) . Since tontalis: ] and thuThis review focuses on hard ticks reported from birds in Northern, Western, Central, and Southern Europe, in the hope that the checklist will provide useful baseline information for future scientific research in the topics of ticks and tick-borne pathogens. Nearly 700 scientific papers published since 1952 were checked for this review. Soft ticks (Acari: Argasidae) were not included in this review, because in the majority of cases these are reported from the environment and thus data on their association with avian host species are not representative.Ixodes” OR “Hyalomma” OR “Haemaphysalis” AND “birds” AND “Europe,” followed by a search with names of bird-specialist tick species . The following databases were used: Web of Science, Zoological Record, and Google Scholar. These records were imported into an Excel file, followed by screening the publications and references cited within. After exclusion of duplicates, we extracted each individual bird host-tick record from these references, noting the location (country), host and parasite species and developmental stage of ticks.The primary corpus of publications used in this review was collated with database search using the following keywords: “ticks” OR “Ixodidae” OR “Ixodes redikorzevi is considered as a synonym of I. acuminatus. Data were only included in the checklist if the tick collected from a bird was reported as identified to the species level. E.g., in Norway a rare finding was mentioned, the occurrence of a Dermacentor sp. on a willow warbler (Phylloscopus trochilus)  , but thihttps://bou.org.uk/british-list/bird-names/). When higher avian taxa are mentioned, these follow the phylogenetic order based on Kuhl et al. FringillaPhasianus colchicus Turdus merula [L: ]- Common starling – aris Parus major Aegithalos caudatus Corvus moPoecile montana Corvus cornix [NA: ]- Rock dove –via [NA: a]Corvus monedula [NA: (- Western jackdaw – ula [NA: ]a).Distribution of reported cases: United Kingdom , 30, Far59Ixodes canisuga is primarily a parasite of mammals Athene noRiparia riparia Turdus meButeo buteo ErithacusTurdus pilaris Carduelis cabaret [F: Asio otusotus [L: ]Aegithalos caudatus AlectorisTurdus iliacus [L: Sylvia mehala [L: ]Amblyramphus holosericeus Gallus gallus domesticus [M: Falco tinulus [L: ]Carduelis cannabina [L: Carduelismmea [L: ]Phoenicurus phoenicurus [L: Oriolus oUpupa epops [L: HaematopuAcrocephalus scirpaceus [L: HippolaisLarus canus [L: Corvus fregus [L: ]Emberiza rustica [NA: Sylvia me Locustella luscinioides [L: Corvus modula [L: ]Motacilla flava [L: MotacillaLagopus lagopus [L: Phalacrocarbo [L: ]Falco rusticolus - Common murre – alge - Razorbill – orda - Red-faced cormorant –ile [NA: b]Uria lomvia Oenanthe oenanthe [NA: (- Wheatear – the [NA: ]a), Norway   175a, , 179, NoIxodes ventalloi, commonly known as the rabbit tick as it mostly feeds on the European rabbit (Oryctolagus cuniculus), is a rare finding on birds Turdus merula [N: ]Phasianus colchicus Luscinia Phasianus colchicus [NA: Emberiza Sturnus vulgaris [NA: Carduelisoris [L: ]Erithacus rubecula Lanius collurio [NA:  Acrocephenus [L: ]Turdus philomelos Turdus phAnthus trivialis [NA:  is mainly a parasite of hedgehogs and small mammals ]- Alpine swift – elba [N: a]Grus grus [M: (- Common Crane – grus [M: ]Bubo bubo [F: (- Eurasian eagle-owl – bubo [F: ]Merops apiaster [M: (- European Bee-eater – ster [M: ]a), Bosnia and Herzegovina Corvus frCorvus monedula [L: Sylvia counis [L: ]Prunella modularis [L: HaematopuAcrocephalus scirpaceus [N: Corvus frAlauda arvensis [NA: Sturnus uMuscicapa striata [L: Meleagrisvo – [N: ]Corvus monedula [N: Corvus modula [N: ]Motacilla flava [NA: ]]Turdus mStruthio camelus [A: Tyto alba Hirundo rustica [N: ]- Bluethroat – cica - Common kestrel – ulus Alcedo atthis - Common nightingale – chos Phoenicurus phoenicurus - Common redstart – urus - Eurasian blackcap – illa - Eurasian hoopoe – ops [NA: d]Garrulus glandarius Otus scops [L: Otus scopSaxicola torquatus [L: Oenanthe Acrocephalus arundinaceus [L: (193a)]- Great reed warbler – ceus - Marsh warbler – tris Turdus viscivorus [N: ]- Ortolan bunting – lana [N: b]Milvus milvus [L: Sylvia me Locustella luscinioides [N: MuscicapaStrix aluco [N: ]- Tree pipit – alis Muscicapa striata [L: ]- Western yellow wagtail – lava L: , 191f]fMotacillOenanthe oenanthe [L: PhylloscoPhylloscopus sibilatrix [L: , Cyprus MeleagrisDistribution of reported cases: Portugal .Rhipicephalus pusillus is found in the Palaearctic region, mainly in Mediterranean climatic areas where birds are considered to be exceptional hosts .- House sparrow – icus [F:  RhipicepAccipiter gentilis [A:  or those that prefer mammalian hosts, such as rodents (I. acuminatus), carnivores, or insectivores . Some of the tick species have a clear host preference for sea birds and predominate in Western-Northern Europe . A significant number of ixodid species are rarely found in association with birds, as exemplified by D. reticulatus, D. marginatus, H. erinacei, H. parva, H. sulcata, R. annulatus, R. turanicus, R. sanguineus, and H. aegyptium. The latter is a tortoise-associated tick species, whereas R. turanicus and R. sanguineus are mostly reported from birds of prey, most likely transferring from rodent and other prey items to these birds. In addition, exotic (non-indigenous) tick species transported by birds from Africa to Europe include H. truncatum, A. lepidum, A. marmoreum, A. nuttalli and A. variegatum.In this review data on the ixodid tick infestation of birds were collected from nearly 200 papers published since 1952. In this period, 37 hard tick species (17 from Prostriata and 20 from Metastriata) were reported from 16 orders of avian hosts in Europe. These include endophilic tick species that are ornithophilic  (The primary aim of this checklist is to provide a comprehensive reference source (baseline data) for future studies, particularly in the context of discovering new tick-host associations after comparison with already published data. Nevertheless, these data also allow a first-hand analysis of general trends regarding how and which developmental stage of ticks tend to infest avian hosts in general. Therefore, five tick species that were frequently reported from birds and show a broad geographical distribution on this continent were selected for statistical comparison (ginatum) .I. ricinus, H. concinna, and H. marginatum . This is not the case for ornithophilic tick species  from which adult ticks were collected significantly more frequently (n = 301 vs. 115 adults) (P < 0.0001). Interestingly, males of generalist tick species occurred significantly more frequently on birds  than males of ornithophilic tick species  (P = 0.009).Considering developmental stages, larvae and nymphs predominate on birds in the case of I. arboricola was significantly (P < 0.0001) more frequently collected from bird species that typically feed above the ground (n = 134) than from those feeding at the ground level (n = 60). This was not the case for I. ricinus , I. frontalis (121 vs. 216) and H. concinna (36 vs. 63) . This is in line with its active host seeking (hunting) strategy  . This gend level , 127. Thlibrated 2 vs. 84.I. arboricola significantly more frequently occur on bird species feeding above the ground (P < 0.0001-0.045), than corresponding stages of I. frontalis, I. ricinus and H. concinna. Females of I. ricinus are more likely to infest ground-feeding than arboreal bird species (18 vs. 4) compared to nymphs of this species (507 vs. 336) (P = 0.046). Interestingly, females of H. marginatum significantly more frequently associate with bird species feeding above the ground level than with those collecting food on the soil surface (4 vs. 1) compared to those of I. ricinus (4 vs. 18) (P = 0.017) and I. frontalis (27 vs. 61) (P = 0.041).In addition, considering data from references that included the number of tick developmental stages collected from various bird species, comparison of tick infestations according to five habitat categories of avian hosts  did not I. arboricola was only reported from species of avian orders whose members typically use forested habitats and (with the exception of Columbiformes) typically feed above the ground  feed from the ground level, in both open and forested habitats  funding scheme. SH and AS were supported by OTKA K-132794 of the National Research, Development and Innovation Office. The Project is supported by the European Union and co-financed by the European Social Fund .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a serious pest of crops in different regions of the world. Our recent studies on the joint application of Akanthomyces attenuatus (a pathogenic insect fungus) and matrine  against B. tabaci have shown promising results. Using RNA sequencing (RNA-Seq), we identified differentially expressed genes involved in whitefly responses to single or mixed applications of A. attenuatus and matrine.B. tabaci treated with individual and combined treatments of A. attenuatus and matrine to determine variations in gene expression among whiteflies in response to different treatments.In this study, we compared the transcriptome profiles of A. attenuatus (BtA), matrine (BtM), and A. attenuatus + matrine (BtAM) treatment, respectively. A total of 65 common differentially expressed genes (DEGs) were identified between whiteflies treated with A. attenuatus (BtA) and matrine (BtM). A comparison of DEGs across the three treatments  revealed two common DEGs. The results also revealed that AMPK signaling, apoptosis, and drug metabolism pathways are likely involved in whitefly defense responses against A. attenuatus and matrine infection. Furthermore, a notable suppression of general metabolism and immune response genes was observed in whiteflies treated with A. attenuatus + matrine (BtAM) compared to whiteflies treated with individual A. attenuatus (BtA) or matrine (BtM) treatments.Transcriptomic data analysis showed differential expression of 71, 1194, and 51 genes in response to B. tabaci subjected to different treatments . To the best of our knowledge, this is the first report on the molecular interactions between whitefly and individual or combined treatments of A. attenuatus and matrine. These results will further improve our knowledge of the infection mechanism and complex biochemical processes involved in the synergistic action of A. attenuatus and matrine against B. tabaci.Dynamic changes in the number of differentially expressed genes were observed in The online version contains supplementary material available at 10.1186/s12864-022-09048-9. Bemisia tabaci, is an invasive pest and disease vector with a rapidly increasing global population of over 50 cryptic species known to date [B. tabaci Middle East-Asia Minor 1 (MEAM1) (formerly known as 'Biotype B') replaced the indigenous B. tabaci populations in China [Bemisia tabaci MEAM1 can cause direct (via feeding on phloem sap) or indirect damage (by secreting honeydew) to its host plants [B. tabaci MEAM1 adults have also been found to transmit over 150 plant viruses to commercial crops [B. tabaci management has resulted in development of insecticide resistance [Akanthomyces attenuatus Zare & Gams is a well-known pathogen of insect pests, including whiteflies, aphids, and thrips [A. attenuatus and other entomopathogenic fungi has received little attention owing to their slow action [A. attenuatus and Beauveria brongniartii) against different insect pests have shown promising results [The whitefly,  to date . Over thin China . Bemisiat plants , 3. B. tal crops . The usesistance . Recentlsistance . Over thsistance . Entomopd thrips –12. Howew action . Recentlw action –17. Our  results .A. attenuatus and matrine. The recently available whitefly transcriptome sequences [B. tabaci to single or mixed applications of A. attenuatus and matrine. Using RNA-Seq, we identified differentially expressed genes in B. tabaci nymphs treated with single or mixed applications of A. attenuatus and matrine and analyzed how the whitefly orchestrates its defense responses to these applications. Altogether, our work provides the first report to reveal the molecular mechanisms of B. tabaci responses to single or mixed applications of A. attenuatus and matrine and new insights into the whitefly infection process during the synergistic action of the interaction between A. attenuatus and matrine.Innate immunity is the first line of protection against numerous pathogenic illnesses. Conserved signal cascades within the innate immune system against insecticides and natural pathogens have been well documented in whiteflies –21. Howeequences , 23, in equences , 25, havnd instar B. tabaci nymphs treated with A. attenuatus and matrine alone or in combination was significantly higher than that of the control. After 3 d of application, B. tabaci mortality in response to the combined treatment with A. attenuatus and matrine increased substantially compared to the individual treatments. After 5 d of treatment, the mortalities of 2nd instar B. tabaci nymphs treated with A.attenuatus (1×106 conidia/mL), matrine (1.0 mg/L), and A.attenuatus + matrine (1×106 conidia/mL + 1.0 mg/L) were 65.67%, 43.67%, and 100%, respectively , the second largest gene length was 201–400 bp , and the smallest gene length was 1,601–1,800 bp  . A total of 16,016 (0.73%) unigenes were annotated in this study through GO database matching , followed by KEGG , COG , NR , Swiss-Prot , and Pfam  databases Fig. .Fig. 2FuA. attenuatus compared to whiteflies from the control treatment (BtA-Vs-Bt) . The number of genes with Log2FC values <-1 was 2 (2.81%) . There were 146 genes (12.22%) with Log2FC values >2, and 258 genes (21.60%) with Log2FC values <-1 were observed  , whereas 16 genes (31.37%) with Log2Ratio values <-1 were observed .Our results showed differential expression of 71 (69 upregulated and two downregulated) DEGs in whiteflies treated with Bt) Fig. A, among Bt) Fig. C, among 2FC values of most genes in the treatment groups were positive, which indicated that these genes were upregulated compared with those in the control groups . A group of upregulated DEGs in whiteflies treated with A. attenuatus was fatty acyl CoA reductase I consisting of three genes   and CTLMA2 (Bta05757)  were similar to those differentially expressed in whiteflies treated with A. attenuatus  were observed in whiteflies treated with matrine compared with those from the control treatment. Among the 936 upregulated DEGs, five major categories were identified, namely 245 orphan genes, 21 cathepsins, 19 cuticle proteins, 17 cytochrome P450, and five fatty acyl-CoA reductase 1 genes. Two categories, orphan genes and cuticle proteins, were also differentially regulated in whiteflies treated with matrine compared to whiteflies from the control treatment, 60 were classified as orphan genes. Another category of downregulated DEGs in whiteflies treated with matrine consisted of three cuticle proteins . One group of downregulated DEGs in whiteflies treated with matrine was cytochrome P450, which consisted of three genes . The two downregulated DEGs involved in the response of B. tabaci to matrine were von Willebrand factor C domain-containing protein 2-like (Bta01761) and CTLMA2 (Bta05757)  was similar to those differentially expressed in whiteflies treated with matrine. Furthermore, two upregulated DEGs (Bta09545: Protein LEA-1and Bta05911: Cathepsin F-like protease) were also differentially regulated in whiteflies treated with individual treatments of A. attenuatus and matrine compared to whiteflies from the control treatment  and CTLMA2 (Bta05757)  18 orphan genes; 2) four cuticle proteins ; 3) three cathepsins ; 4) three fatty acyl-CoA reductase 1 ; and 5) two glucose dehydrogenase genes   and 2) cathepsin F-like protease (Bta05911)) were differentially expressed between all three treatments  between both variables.The correlation analysis between DEGs and mortality of whiteflies treated with individual and combined treatments of A. attenuatus; matrine; A. attenuatus + matrine). In whiteflies treated with A. attenuatus, the most enriched pathways were “Longevity regulating pathway - worm,” “Renin secretion,” and “Antigen processing and presentation” of Organismal Systems; “Peroxisome,” “Lysosome,” and “Apoptosis” of Cellular Processes, and “Glycosphingolipid biosynthesis - ganglio series, Glycosphingolipid biosynthesis - globo and isoglobo series of Metabolism  metabolism, 2) organismal systems, and 3) cellular processes  data, the transcript levels of eight randomly selected genes (six upregulated and two downregulated) were quantified by qRT-PCR, using RNA extracted from three independent biological replicates. In all three biological replicates, ten genes showed concordant changes between the DGE and qRT-PCR data Figure , suggestAkanthomyces attenuatus Zare & Gams is a well-known pathogen of insect pests, including whiteflies, aphids, and thrips. Recently, many studies have investigated the joint application of pathogenic insect fungi and natural enemies, synthetic oils, bacterial metabolite avermectins, and other biopesticides. Our recent studies on the combined application of A. attenuatus and matrine  against B. tabaci have shown promising results. To date, no study has been conducted to elucidate the immune response of B. tabaci to the combined application of A. attenuatus and matrine at the molecular level. In the present study, we obtained a transcriptome dataset containing 35,895 unigenes of B. tabaci using RNA-Seq. The analysis of DEGs involved in the immune response to single or joint treatments of A. attenuatus and matrine revealed the expression of 5, 54, and 0 immunity-related genes for BtA, BtM, and BtAM, respectively. Most immunity-related genes are also involved in NOD-like receptor signaling, Toll and Imd signaling, Fc gamma R-mediated phagocytosis, and antigen processing and presentation pathways. Our findings differ from those of Xia et al. [B. tabaci genes in response to infection with Beauveria bassiana. Their findings revealed that 654 and 1,681 genes were differentially expressed at 48 and 72 h post-infection, respectively, compared to the control. Chen et al. [Megalurothrips usitatus post-A. attenuatus infection when compared to infection with Beauveria brongniartii. The lower number of DEGs and zero expression of immunity-related genes by B. tabaci treated with combined treatments of A. attenuatus and matrine can be related to the disruption of the defense/immune system of the host [a et al. , who exan et al.  also obsA. attenuatus compared with 359 (324 upregulated and 35 downregulated) metabolic pathway DEGs in whiteflies treated with matrine and 12 metabolic pathway genes  in whiteflies treated with A. attenuatus + matrine. We observed an overall lower differential expression of metabolism-related genes (after treatment with A. attenuatus or matrine + A. attenuatus), which may play an important role in the complex interactions between B. tabaci and A. attenuatus. B. tabaci may reduce general metabolism and conserve energy to fight fungal infections with alternative immune responses or reproductive strategies [Insects respond to external stress by increasing carbohydrate metabolism and energy production . Major drategies –31; howeBta11879) in response to individual or combined treatments with pathogens and botanicals. The higher expression levels of the acyl-CoA desaturase gene in B. tabaci following different treatments suggest the acceleration of lipid metabolism to produce the necessary energy required for the induction of a strong immune response [Membrane-bound acyl-lipid desaturases and membrane-bound acyl-CoA desaturases are two types of membrane-bound fatty acid desaturases. Insect acyl-CoA desaturases play a critical role in semiochemical biosynthesis and sensing, cold tolerance, defensive fatty acid production, lipid metabolism, feeding behavior, and larval development –36. Our response .A. attenuatus + matrine, confirming the increased detoxification response of B. tabaci to the different treatments.Cytochrome P450 monooxygenases are enzymes found throughout the animal kingdom and are mainly involved in metabolism . Toxins A. attenuatus. In whiteflies treated with matrine, 123 DEGs (89 upregulated and 34 downregulated) were associated with signal transduction pathways, whereas only one upregulated DEG was annotated to signal transduction pathways.Among the genes annotated to signal transduction pathways, four upregulated DEGs were observed in whiteflies treated with Bta11879) in response to individual treatments with pathogens and botanicals. These findings suggest that fatty acid metabolism may be involved in the detoxification of pathogens and botanicals in whiteflies. Upregulated expression of fatty acyl-CoA reductase 1 gene may produce fatty alcohol and wax-ester in the insect epicuticle [In insects, FAR genes involved in sex pheromone production (pheromone-gland-specific fatty acyl reductase) form a distinct clade with significant changes in gene structure, fatty acyl substrate specificity, and selectivity . Fatty aicuticle .A. attenuatus might be related to their effort to cope with external stress through higher transcription or growth activities.Histone H1 is one of the important components of eukaryotic chromatin. This protein is known to play a key role in the chromatin folding into higher-order structure . It was shown to be a nonspecific repressor of gene expression . BesidesA. attenuatus and matrine  revealed some interesting findings. Among whiteflies treated with A. attenuatus, 5 immunity-related  DEGs were observed. These DEGs were mainly involved in the NOD-like receptor signaling pathway (map04621), the Toll and Imd signaling pathway (map04624), and the antigen processing and presentation pathway (04612). In whiteflies treated with matrine, 54 immunity-related DEGs (51 upregulated and three downregulated) were observed. The majority of immunity DEGs were annotated to the NOD-like receptor signaling pathway (map04621), Toll and Imd signaling pathway (map04624), Fc gamma R-mediated phagocytosis (map 04666), and antigen processing and presentation pathway (04612). In whiteflies treated with A. attenuatus + matrine, no immunity-related gene was expressed, which confirmed the complete disruption of the B. tabaci immune response by the combined application of A. attenuatus and matrine Vitellogenins are insect proteins that provide nutrients for yolk formation [A. attenuatus), botanicals (matrine), and their combination (A. attenuatus + matrine), thereby confirming the role of vitellogenins in B. tabaci defense against different treatments.The analysis of DEGs that may participate in the immune response to single or combined treatments of ormation , 49. Manormation , 50, 51.Manduca sexta. Our results showed the upregulation of cathepsin F-like protease gene (Bta05911) in response to individual or combined treatments of pathogen and botanical. The upregulated expression of cathepsin F-like protease genes in response to different treatments shows the importance of CathF in the immune defense of B. tabaci through antigen processing and presentation [In cysteine proteases, cysteine residues modulate catalytic processes . These eentation .Leptinotarsa decemlineata in response to stress, allowing the insects to adapt to new or changing settings [A. attenuatus) and botanical (matrine), whereas in the case of A. attenuatus + matrine, there was no or minimal expression of cuticle protein genes, which indicated disruption of the B. tabaci immune process.The insect cuticle is the first barrier against pathogen infection and maintains the form and movement of the insects . The cutsettings . Silva esettings  found thPolypedilum vanderplanki larvae. Our results showed the upregulation of the LEA protein-1 gene (Bta000818) in response to individual or combined treatments of pathogen and botanical. After different treatments, the upregulation of the LEA protein-1 gene by B. tabaci confirmed a strong immunological/detoxification response through trehalose accumulation to survive drying in response to infection [Late embryogenesis abundant (LEA) proteins are involved in insect response to environmental stress and thus act as water deficiency stress protectants . The LEAnfection .2+ [B. tabaci following different treatments could have resulted in a weaker immune response and increased melanization, implying susceptible whitefly individuals [C-type lectins (CTLs) are involved in the binding site of glycans in direct coordination with lectin-bound Ca2+ . CTLs ar2+ . The CTL2+ , 58. Ourividuals .Drosophila melanogaster. SVWC proteins are involved in response to environmental stressors, changes in nutritional status, and microbial infections [Bta01761) in response to individual or combined treatments of pathogens and botanicals. The lower expression levels of the von Willebrand factor type C protein-2 gene in B. tabaci following different treatments resulted in a reduced immune response during pathogen invasion through reduced phagocytosis and antimicrobial responses [Single von Willebrand factor C-domain proteins (SVWCs) are single-domain von Willebrand factor type C proteins known primarily from arthropods , 61. Klefections . Our resesponses , 56.A. attenuatus and matrine+A. attenuatus treatments ultimately led to the lower expression of immunity related genes. In short, reduced/disrupted metabolic response might have led to the lesser number of diferentially expressed genes in whiteflies exposed to matrine+A. attenuatus when compared to the remain treatments.A large amount of energy is normally required for a rapid and effective immune response . In inseA. attenuatus and matrine using RNA sequencing. Our data indicated that genes such as cytochrome P450, cuticle protein, vitellogenin, cysteine proteases, late embryogenesis abundant (LEA) proteins, desaturases, CTLMA2, and von Willebrand factor type C protein-2 were mainly involved in whitefly defense responses against A. attenuatus and matrine. The results also revealed that AMPK signaling, apoptosis, and drug metabolism pathways are likely involved in whitefly defense responses against A. attenuatus and matrine infection. To the best of our knowledge, this is the first report on the molecular interactions occurring between whiteflies and individual or combined treatments of A. attenuatus and matrine. These results will further improve our knowledge of the infection mechanism and complex biochemical processes involved in the synergistic action of A. attenuatus and matrine against B. tabaci.In summary, we investigated the complex interactions between the whitefly and individual or combined treatments of Whitefly cultures were reared on cotton plants according to the method described by Wang et al.  at the EAkanthomyces attenuatus isolate SCAUDCL53  was cultured according to the method described by Ali et al. [6 conidia/ml.i et al.  to calibMatrine (98% purity) was purchased from Guangdong New Scene Bioengineering Company Ltd., Yangjiang, P.R. China, and the basal concentration of matrine (1.0 mg/mL) was prepared following the methodology of Wu et al. .nd instar B. tabaci nymphs were individually dipped in A. attenuatus fungal suspension (1×06 conidia/ml), matrine (1.0 mg/L), and A. attenuatus and matrine (1×06 conidia/ml + 1.0 mg/L) for 10 seconds. The leaves were air dried before being placed in a 9 cm agar medium with 20% agar to maintain moisture. Bemisia tabaci dipped in 0.05% Tween 80 served as control. Each treatment consisted of four cotton leaves with 100 B. tabaci nymphs per leaf. The experimental setup was maintained at 25 °C, 85% RH, and a 16:8 h (light/dark photoperiod). The entire experiment was repeated three times . The mortality of the insects was tracked daily for 5 d after treatment.Cotton leaves having 2B. tabaci nymphs (200 nymphs pooled per sample and three samples for each treatment considered as three replicates) from different treatments were collected in 1.5 mL Eppendorf tubes after approximately 3 d of treatment, frozen in liquid nitrogen, and stored at -80 °C. Total RNA was extracted using TRIzol reagent , according to the manufacturer’s instructions (Invitrogen). The quality of the extracted RNA was determined using 1% agar-gel electrophoresis and a NanoDrop One ultraviolet spectrophotometer , and the RNA integrity number (IN) value was calculated using an Agilent 2100 bioanalyzer .The surviving The mRNAs (post RNA extraction) were enriched with Oligo (dT) having magnetic beads treated with DNase I. The mixture was then supplemented with fragmentation buffer to prepare the mRNA templates for the sysnthesis of first cDNA chain whereas RNA enzyme, DNA polymerase I, and dNTPs were added to synthesize the second cDNA chain. AMPure XP beads were used for cDNA (double-stranded) purification followed by the selection of cDNA library through PCR enrichment.. The library was sequenced with Illumina sequencing platform.http://cole-trapnell-lab.github.io/cufflinks/). The assembled sequences were removed from redundancy and spliced using TGICL to obtain the longest nonredundant unigene set. Further statistical analysis and quality control were performed using the unigene set. The unigene set obtained by de Novo and clustering was compared with the database using BLAST for functional annotation. The NR, Swiss-Prot, Pfam, EggNOG, GO, and KEGG databases were used for annotation. The annotation information of genes or transcripts could be comprehensively obtained, and the annotation of each database was determined. To understand the transcriptome database of the response of B. tabaci to A.attenuatus and matrine alone or in combination, 12 DGE marker libraries were constructed, including the transcription information of B. tabaci treated with A.attenuatus and matrine alone or in combination with a blank control group. The treatment groups were named as BtA-1, BtA-2, BtA-3, BtM-1, BtM-2, BtM-3, BtAM-1, BtAM-2, BtAM-3, Bt-1, Bt-2, and Bt-3. Table Raw reads were filtered and assembled using Cufflinks  with the above pathways  and GO f-ΔΔCt technique was used to calculate relative expression levels using β-actin as an internal control [Primer Premier 5 software  was used to create primers for the DEGs and a reference gene Table . To vali control , 69, 70.Additional file 1. a. The number of Illumina raw and processed reads produced per RNA-Seq library from whiteflies (Bemisia tabaci) treated with individual or combined treatments of Akanthomyces attenuatus and matrine. b. Length distribution of B. tabaci unigenes. c. Correlation matrix analysis for multiple biological replicates obtained from RNA-Seq libraries prepared from whiteflies (Bemisia tabaci) treated with individual or combined treatments of Akanthomyces attenuatus and matrine.Additional file 2. a. List of differentially expressed genes in whiteflies treated with Akanthomyces attenuatus. b. List of differentially expressed genes in whiteflies treated with matrine. c. List of differentially expressed genes in whiteflies treated with Akanthomyces attenuatus + matrine. d. Cluster analysis of differentially expressed genes in whiteflies treated with individual or combined treatments of Akanthomyces attenuatus and matrine. e. List of 66 common differentially expressed genes between whiteflies treated with individual treatments of Akanthomyces attenuatus and matrine (BtA and BtM). f. List of 66 common differentially expressed genes between whiteflies treated with individual and combined treatments of Akanthomyces attenuatus and matrine .Additional file 3. The main Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway in B. tabaci treated with Akanthomyces attenuatus matrine, Akanthomyces attenuatus + matrine, and controls.Additional file 4. Summary of Kyoto Encyclopedia of Genes and Genomes (KEGG) reference pathways of whiteflies associated with different treatments. (A) Akanthomyces attenuatus upregulated pathways; (B) matrine upregulated pathways; (C) matrine downregulated pathways; and (D) Akanthomyces attenuatus + matrine upregulated pathways.Additional file 5. Results of quantitative real time PCR.