BackgroundActivities of daily living (ADLs) and instrumental activities of daily living (IADLs) are essential for independent living and are predictors of morbidity and mortality in older populations. Older adults who are dependent in ADLs and IADLs are also more likely to have poor muscle measures defined as low muscle mass, muscle strength, and physical performance, which further limit their ability to perform activities. The aim of this systematic review and meta‐analysis was to determine if muscle measures are predictive of ADL and IADL in older populations.MethodsA systematic search was conducted using four databases  from date of inception to 7 June 2018. Longitudinal cohorts were included that reported baseline muscle measures defined by muscle mass, muscle strength, and physical performance in conjunction with prospective ADL or IADL in participants aged 65 years and older at follow‐up. Meta‐analyses were conducted using a random effect model.ResultsOf the 7760 articles screened, 83 articles were included for the systematic review and involved a total of 108 428  participants with a follow‐up duration ranging from 11 days to 25 years. Low muscle mass was positively associated with ADL dependency in 5/9 articles and 5/5 for IADL dependency. Low muscle strength was associated with ADL dependency in 22/34 articles and IADL dependency in 8/9 articles. Low physical performance was associated with ADL dependency in 37/49 articles and with IADL dependency in 9/11 articles. Forty‐five articles were pooled into the meta‐analyses, 36 reported ADL, 11 reported IADL, and 2 reported ADL and IADL as a composite outcome. Low muscle mass was associated with worsening ADL  3.19 (1.29–7.92)) and worsening IADL (1.28 (1.02–1.61)). Low handgrip strength was associated with both worsening ADL and IADL (1.51 (1.34–1.70); 1.59 (1.04–2.31) respectively). Low scores on the short physical performance battery and gait speed were associated with worsening ADL (3.49 (2.47–4.92); 2.33 (1.58–3.44) respectively) and IADL (3.09 (1.06–8.98); 1.93 (1.69–2.21) respectively). Low one leg balance (2.74 (1.31–5.72)), timed up and go (3.41 (1.86–6.28)), and chair stand test time (1.90 (1.63–2.21)) were associated with worsening ADL.ConclusionsMuscle measures at baseline are predictors of future ADL and IADL dependence in the older adult population. Dependence in activities of daily living (ADLs), the basic tasks required of an individual to maintain their independence at home, is associated with increased risk of morbidity and mortality.Older adults that develop ADL dependence are less likely to recover function, stressing the need for strategies that can prevent or delay the onset of ADL dependence.The aim of this systematic review and meta‐analysis was to determine if muscle mass, muscle strength, or physical performance are predictors of ADL and/or IADL at follow‐up in older populations.TableThis systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta‐analyses and registered with the PROSPERO International Prospective Register of Systematic Reviews CRD42019125666).125666.19Inclusion criteria consisted of prospective longitudinal cohorts of older adults with a reported mean/median age of 65 years and older at follow‐up and reporting at least one of the following measurements: muscle mass, muscle strength, or physical performance in conjunction with a follow‐up outcome of ADL or IADL.Exclusion criteria included cross‐sectional and case–control studies; anthropometric measurements as measures of muscle mass such as body mass index, hip‐waist ratio, waist or calf circumference measurements, and skin‐fold thickness; populations that suffered from cancer, muscular dystrophy, genetically inherited diseases, and HIV/AIDS; cohorts that received an intervention other than usual care or placebo; and non‐English articles. Articles including participants of the same cohort more than once were excluded in a hierarchical manner: (i) if there was no statistical analysis conducted regarding odds ratio (OR), hazards ratio, or relative risk and lacking data to calculate the OR; (ii) if their primary research question was not exploring the association between muscle measures with ADL or IADL; or (iii) an article had a smaller sample size.Articles obtained through the search strategy had their title and abstract screened followed by full‐text screening for eligibility independently by two authors  using Covidence . The decision made by authors was compared, and conflicts were settled by a third reviewer (E. M. R.).Data extraction was performed by two authors . The following data were extracted from included articles: author, year, study/cohort, setting, country/region, demographical information , and follow‐up duration. Muscle mass, muscle strength, physical performance, ADL, and IADL were extracted based on the measurement method, unit, and cut‐offs applied in analyses. Effect sizes were extracted from text, tables, or figures if not described elsewhere.The quality of the included articles was assessed by a modified version of the Newcastle–Ottawa Scale (NOS) for cohort studies.I‐squared (I2) test. Low heterogeneity was defined as an I2 ≤ 25%, moderate as 25–75%, and high as ≥75%.P‐value for significance was set at <0.05, and the P‐value for a trend was set at 0.05 < P < 0.01. Meta‐analyses were performed separately according to the unit of gait speed, ADL, and IADL. The meta‐analysis was performed using Comprehensive Meta‐analysis .Effect sizes were extracted and reported where associations were made between baseline muscle measures and follow‐up ADL and/or IADL. Inclusion into the meta‐analysis required articles to report an OR, hazards ratio, relative risk, or if the article provided sufficient information to calculate an OR for the association between baseline muscle measures and follow‐up ADL and/or IADL. Forest plots were generated for the graphical representation of the meta‐analysis. A random effect model was adopted to account for differences between articles.FigureTablen = 65/83). The mean or median age of the study ranged from 54 to 86 years at baseline. The number of participants in each study ranged from 41 to 8000 and included a total of 108 428  participants. Follow‐up duration ranged from 11 days to 25 years. ADL was measured in 73 articles that examined physical motor tasks, 30 of which had developed an adapted questionnaire to assess the motor component of ADL . IADL was measured in 35 articles with the Lawton–Brody IADL scale being used in 14 articles, three of which were modified. A total of 26 articles included measures of both ADL and IADL.TableMuscle mass was reported in 13 articles,TableMuscle strength was investigated in 41Tablen = 15), chair stand (n = 10), timed up and go (n = 8), and functional reach tests (n = 2) as well as tests that involve multiple assessments (n = 3). Poor physical performance was positively associated with worsening ADL in 37/49 articles and IADL in 9/11 at follow‐up: SPPB , gait speed , one leg balance , and chair stand test .A total of 62 articlesTableA complete breakdown of the NOS can be found in I2 = 68.8]. Four articlesI2 = 75.8)  . Four articlesI2 = 94.7)  FigureA. Ten ar) FigureB.I2 = 91.8) . Two articlesI2 = 57.6)  FigureA. Four a) FigureB.FigureI2 = 85.0) while a lower gait speed of 1.0 m/sI2 = 52.1; OR = 1.85, 95% CI: 1.44–2.36, I2 = 62.8). Six articlesI2 = 94.2)    with ADL FigureA. After I2 = 88.5) . Three articlesFigureI2 = 0.0). Two articlesI2 = 74.8)  FigureA. Two arAll meta‐analyses were ordered by study follow‐up duration from shortest to longest. No pattern was present based on follow‐up duration in all meta‐analyses.Muscle mass was associated with the development of IADL dependence. Muscle strength and physical performance were associated with the development of ADL and IADL dependence at follow‐up in older adults.Surprisingly, muscle mass was associated with the development of new IADL dependence, but not ADL dependence at follow‐up in this meta‐analysis. While the association between muscle mass and ADL was not statistically significant, perhaps because of only two studies being included, a trend suggested that it was a clinically significant predictor of worsening ADL. The Concord Health and Ageing in Men Project reported that low muscle mass and sarcopenic obesity were significantly associated with worsening ADL and IADL.The predictive ability of muscle strength at baseline of ADL and IADL decline is consistent with a previous study consisting of 6089 participants, which suggested that having higher muscle strength was protective against the onset of future disability.It has previously been hypothesized that there is a minimal amount of muscle strength required to complete ADL The prevalence of ADL and IADL dependence increases with age, muscle strength amongst older adults has been shown to decrease.Muscle quality, which refers to the muscle strength or power per unit of muscle mass,Lower SPPB scores were associated with worsening ADL at follow‐up. The SPPB encompasses three assessments that require strength, balance, dexterity, and cognitive control, which captures functional elements that are required for the completion of ADL and IADL Gait speed measured by a 4 m walk test is a component of the SPPB and is often used in clinical settings to identify individuals at high risk of adverse health outcomesOne leg balance was significantly associated with worsening ADL in the follow‐up. The importance of balance is well described controlling both static and dynamic posture while performing a variety of daily activities,The chair stand test was significantly associated with worsening ADL but not IADL. Chair stand test has been used as a measure of lower body strength in older adults in community‐dwelling older adults and as part of the SPPB.This systematic review and meta‐analysis explored muscle mass, muscle strength, and physical performance as predictors for ADL and IADL rather than limiting to just a single measure. By evaluating the effects of all three measures, this review presented the most detailed assessment of this topic. To reduce the risk of reverse causation when interpreting the associations reported, only prospective studies were included. There are also limitations to this review. Not all studies were pooled into a meta‐analysis because of differences in measures and cut‐offs of muscle mass, muscle strength, and physical performance, the use of different statistical analyses, or the lack of data required to calculate an OR. Studies that were pooled were also reporting univariate and multivariate analyses, adjusting for different confounders that were inconsistent between studies that may have further led to an over or under estimation of the effect sizes. Studies that did not have a primary aim of exploring the association between baseline muscle measures with ADL and/or IADL may have used less standardized procedures. The follow‐up duration between studies varied significantly, which may have impacted the results.There were few pre‐existing studies that explored the association between baseline muscle mass and ADL and/or IADL. Studies should continue to investigate the association of muscle mass alone with ADL and/or IADL. Future studies should also explore the association of muscle quality with ADL and/or IADL. Interventions tailored for specifically increasing muscle measures should be developed to prevent individuals from having worsening ADL and/or IADL in their future. Interventions should be developed with the focus of preserving or increasing muscle measures as we age.This study quantified the current research available between muscle measures and (I)ADL. Muscle mass is predictive of ADL and IADL decline whereas muscle strength and physical performance are predictive of both ADL and/or IADL decline. Future studies should continue to develop and improve interventions to preserve and improve muscle measures to prevent ADL and IADL dependence.Daniel X. M. Wang, Jessica Yao, Yasar Zirek, Esmee M. Reijnierse, and Andrea B. Maier declare that they have no conflict of interest.Table S1. Search StrategyClick here for additional data file.Data S2. Newcastle–Ottawa ScaleClick here for additional data file.Table S3. Breakdown of the ADL components in articles that created their own questionnaireClick here for additional data file.


Telocytes (TCs) are recently described interstitial cells, present in almost all human organs. Among many other functions, TCs regulate gastrointestinal motility together with the interstitial cells of Cajal (ICCs). TCs and ICCs have close localization in the human myenteric plexus; however, the exact spatial relationship cannot be clearly examined by previously applied double immunofluorescence/confocal microscopy. Data on TCs and submucosal ganglia and their relationship to intestinal nerves are scarce. The aim of the study was to analyse the spatial relationship among these components in the normal human ileum and colon with double CD34/CD117 and CD34/S100 immunohistochemistry and high‐resolution light microscopy. TCs were found to almost completely encompass both myenteric and submucosal ganglia in ileum and colon. An incomplete monolayer of ICCs was localized between the TCs and the longitudinal muscle cells in ileum, whereas only scattered ICCs were present on both surfaces of the colonic myenteric ganglia. TC‐telopodes were observed within colonic myenteric ganglia. TCs, but no ICCs, were present within and around the interganglionic nerve fascicles, submucosal nerves and mesenterial nerves, but were only observed along small nerves intramuscularly. These anatomic differences probably reflect the various roles of TCs and ICCs in the bowel function. The other component of the enteric nervous system (ENS), the submucosal plexus, does not take part in the regulation of the bowel movement, but influences the secretory activity apart from that of the endocrine cells, mucosa and blood vessels.c‐kit (CD117) and positive for CD34 and platelet‐derived growth factor receptor α (PDGFRα).Immunohistochemical studies have since the 1990s revealed another fibroblast‐ or ICC‐like cell in similar localization as ICCs, but with different phenotype: negative for c‐kit CD1 and posiElectron microscopic studies showed that both ICCs and fibroblast‐like cells were present around the myenteric ganglia of the human small and large intestine.The exact spatial relationship between the different cellular components in the myenteric plexus is, however, better analysed in conventional microscope after double immunostainings. The few studies performed regarding ICCs and TC and their relation to the submucosal ganglia have shown varying results.2The study was performed according to the Declaration of Helsinki and approved by the Ethical Review Board at Lund University . Since the samples were unidentified samples used as controls, with only sex and age as known variables, the subjects did not have to leave informed consent, according to Swedish ethical rules. The study is not a part of a clinical trial.c‐Kit , mouse monoclonal antibody against to human CD34  and polyclonal antibody to bovine brain S100 . For detection of the immunostaining, Ultra View DAB Kit (brown colour) and Ultra View Fast Red Kit (red colour)  were used in Ventana's BenchMark ULTRA automated immunostainer system.Four specimens of normal human ileum and two specimens of normal colon were used for the analyses. The samples were taken from macroscopically normal ileum of right‐sided hemicolectomy resections due to carcinoma  and normal sigmoid colon also resected due to carcinoma . The specimens were fixed in buffered formalin overnight and embedded in paraffin. Serial sections of 4 μm thickness were deparaffinized and stained by haematoxylin & eosin. For immunohistochemistry, CC1 ph 8.5 buffer  was applied for the deparaffinized unstained sections before applying the antibodies. The following primary antibodies were used: rabbit monoclonal antiserum to human +) and mast cells (CD117). As negative controls, the primary antibodies were omitted and replaced by serum. Nuclear counterstain was Mayer's haematoxylin.With double immunohistochemistry, either CD117 was detected first with Fast Red Kit followed by CD34 with DAB Kit, or CD34 was detected first with DAB Kit followed by S100 with Fast Red Kit. The double stainings were also performed in reverse order. As internal control served endothelium of blood vessels  were chosen. The results were given as mean of all individual counting.33.13.1.1The TCs and ICCs of ICC‐MP formed either an almost complete double layer at the inner surface of the ganglia, facing the circular muscle with the TCs always in between the ganglia and the ICCs, or there was only one very thin layer of TCs or telopodes, with or without a few ICCs/projections intermingling with TCs. On the outer surface of the ganglia towards the longitudinal muscle layer, only a few ICCs and their small projections were scattered partly intermingled with ICC, without the formation of a double layer Figure A‐D. Quan3.1.2The pattern of TCs and the localization of ICCs around the ganglia were similar in the colon to those of the ileum with two exceptions: (a) the number of ICCs was substantially less with only a few ICCs scattered around the ganglia and (b) telopodes were usually observed within the ganglia in close localization to neurons Figure A‐D. The The myenteric nerve fascicles, submucosal nerves and mesocolic nerves showed similar arrangements of TCs/telopodes as in the ileum. ICCs were not present within the interganglionic fascicles (data not shown).3.2Telocytes were present in a single ‘TC‐sheath’ encompassing the submucosal ganglia in both the ileum and colon, but ICCs never occurred in this localization Figure A‐C. Occa3.3On transversal section within the muscles, TCs formed a network with long telopodes Figure A‐C, wher3.4TCs and ICCs of the ICC‐IM were found in both muscle layers; similar observation was made in the interlamellar septa of the circular muscle Figure  A‐B. BotThe quantification of TCs and ICCs in the circular muscle layer showed 8 TCs per HPF in ileum and 6 TCs per HPF in colon, respectively, compared to 2 ICCs per HPF in both organs.4This study showed that TCs form an encompassing almost complete sheath around both myenteric and submucosal ganglia and interganglionic fascicles in both ileum and colon. Their telopodes entered the colonic myenteric ganglia. TCs with long telopodes were present along and within all the larger nerve fascicles and peripheral nerves of the bowel and the mesenterium. In contrast, ICCs formed a layer outside the TC layer of the myenteric ganglia in the ileum and were scattered in similar localization at the interganglionic fascicles. In the colon, ICCs were scarce and only scattered on both surfaces of the myenteric ganglia intermingled with TCs. These observations are in agreement with and support previous findings obtained in confocal microscopic analysis,the inner surface of the myenteric ganglia with TCs between the ganglia and the ICCs, or with TC and ICCs intermingling with each other, corroborating with previous observations by Vannucchi et althe outer surface of the ileal ganglia and of the colonic ganglia, there was only one almost complete TC layer with scattered ICCs, in agreement with previous reports.In the ileum, TCs and ICCs were either localized in parallel in two complete separate layers on Telocytes entered the myenteric ganglia at the beginning of the interganglionic nerve fascicles and ran on the surface of the fascicles in both ileum and colon, which has already been demonstrated.The almost complete TC layer around submucosal plexi confirmed the results from Milia et alOur findings of TCs and ICCs in the interlamellar septa of the circular muscle layer are well‐known data.Thus, the architecture of the two cell types differs markedly, which may reflect the functional role of these cells. Furthermore, the shape and distribution of TC and their location may vary in different wall layers of ileum, as previously described in a large study cohort.Differences between studies may be explained by small sample sizes of most studies. Differences in affinity and specificity of antibodies from different producers and batches may be another explanation to contradictory results. One hypothesis is that malignant diseases may affect tissues at a distance from the local tumour,In conclusion, regarding the localization and number of TCs and ICCs, the difference between human myenteric and submucosal ganglia, nerves and muscle layers on the one hand, and between ileum and colon on the other hand might probably reflect the role of these cells in the function of the bowel, that is the maintenance of homeostasis in the microenvironment and the motility of the bowel, respectively. Further investigations are necessary to prove or disprove this hypothesis.The authors confirm that there are no conflicts of interest.Béla Veress designed and performed the analyses and wrote the main part of the paper. Both authors contributed to the intellectual analysis of the result and approved the final version of the manuscript.


Antimicrobial compounds are used in a broad range of personal care, consumer and healthcare products and are frequently encountered in modern life. The use of these compounds is being reexamined as their safety, effectiveness and necessity are increasingly being questioned by regulators and consumers alike. Wastewater often contains significant amounts of these chemicals, much of which ends up being released into the environment as existing wastewater and sludge treatment processes are simply not designed to treat many of these contaminants. Furthermore, many biotic and abiotic processes during wastewater treatment can generate significant quantities of potentially toxic and persistent antimicrobial metabolites and byproducts, many of which may be even more concerning than their parent antimicrobials. This review article explores the occurrence and fate of two of the most common legacy antimicrobials, triclosan and triclocarban, their metabolites/byproducts during wastewater and sludge treatment and their potential impacts on the environment. This article also explores the fate and transformation of emerging alternative antimicrobials and addresses some of the growing concerns regarding these compounds. This is becoming increasingly important as consumers and regulators alike shift away from legacy antimicrobials to alternative chemicals which may have similar environmental and human health concerns. Emerging contaminants consist of wide-ranging and expanding classes of anthropogenic and naturally occurring unregulated substances, on the occurrence, behavior, environmental fate and toxicity of which there are limited data ,2,3. TheDepending on the physicochemical properties of the compounds and treatment technology used at the WWTP, wastewater treatment sludge is the endpoint of many hydrophobic and lipophilic compounds by sorption ,11. The Pharmaceutical and personal care products are a class of emerging contaminants that are produced and consumed at high volumes all over the world. PPCPs include a large group of prescription and over-the-counter medications that are widely used in human medicine, veterinary drugs, in animal husbandry as growth promoters and as active and inert ingredients for personal care purposes ,21. PPCPA review by Verlicchi and Zambello , based oIncreasing concerns have been raised regarding endocrine-disrupting antimicrobials because of their potential human and environmental health threats and their physicochemical characteristics . AntimicEven when used as they are intended, significant quantities of antimicrobials end up in wastewater, which are conveyed to wastewater treatment facilities in sewage . Antimicow). This typically results in substantially higher antimicrobial concentrations in wastewater sludge versus wastewater influent. Furthermore, many sludge stabilization treatments, including anaerobic digestion, degrade organic matter at a significantly higher rate than that of antimicrobials, resulting in higher antimicrobial concentrations in digested versus undigested sludge . Contaminants remaining in stabilized sludge may then enter the environment through land application of biosolids through soils [The fate of antimicrobials during wastewater and sludge treatment can be determined by investigating their concentrations in both aqueous  and solid  matrices. Significant antimicrobial removals from the aqueous phase have been observed during wastewater treatment. For example, TCS removals of 91 to 98% have been reported during conventional activated sludge treatments ,40,41,42gh soils ,46,47, ugh soils ,51,52,53gh soils ,55.A significant fraction of antimicrobials removed during wastewater treatment and/or sludge stabilization are often not completely mineralized ,56. RathEscherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) [Triclosan phenol), chemical abstract service (CAS) #3380-34-5, is incorporated into numerous consumer and healthcare products in many countries around the world. TCS is a synthetic, nonionic, lipid-soluble broad-spectrum antimicrobial which is effective against most Gram-negative and Gram-positive bacteria ,60, agais (MRSA) ,64,65. Ts (MRSA) ,67. Sincs (MRSA) ,69. For s (MRSA)  to over s (MRSA) . Howevers (MRSA) ,71,72,73ow value, is highly insoluble in water and is relatively non-volatile [As shown in volatile ,76. Due volatile ,77,78,794), several of which can have been shown to revert back into TCS [4 through sulfation during a batch simulated aerobic activated sludge process. This agrees with a later batch study by Trenholm et al. [4 under aerobic conditions and slower formation of TCS-SO4 under anoxic conditions using biological nutrient removal (BNR) sludge. Although Chen et al. [4 once it was formed over 10 days, Trenholm et al. [4 levels began to decrease after 5 days under aerobic conditions and observed the formation of a secondary TCS-SO4 metabolite, whose mass matched 3,5-dichloro-2-hydroxy-benzene sulfonate (or a closely related isomer thereof). TCS conjugates can also have significantly different physicochemical properties that those of its parent antimicrobial. For example, MeTCS is more persistent and bioaccumulative due to its even higher Log Kow walue (5.0–5.1) and lower water solubility (0.4 mg/L) than those of TCS [4 and TCS-gluc are less lipophilic , are far more polar and are more soluble in water  [Triclosan can be transformed into various other compounds by biotic and abiotic processes and mechanisms within organisms, WWTPs and/or the environment. These can include various conjugated forms, photolysis products, chlorinated derivatives and partial mineralization metabolites, as shown in into TCS ,80,81. Ainto TCS ,70. The into TCS ,82,83. Cinto TCS  observedm et al. , who obsn et al.  did not m et al.  observede of TCS ,85. Howectively) .Chlorella pyrenoidosa, Desmodesmus sp. and Scenedesmus sp. have been shown to remove between 77.2 to 99.7% of TCS through reductive dechlorination, ether bond cleavage, hydroxylation or the production of TCS conjugates through methylation and/or glucosylation [Sphingomonas-like strain isolated from an activated sludge basin. They observed the partial mineralization of the dichloro ring, with approximately 35% of 14C-TCS-labelled carbon being transformed to 14CO2 in a complex medium. The Sphingomonas sp. strain YL-JM2C, isolated from activated sludge, was shown to completely mineralize up to 99% of TCS within 72 h at an optimum pH of 7.0 and temperature of 30 °C and formed 2,4-dichlorophenol , 2-chlorohydroquinone and hydroquinone as intermediate decomposition products [Sphingomonas sp. PH-07, originally isolated from activated sludge by Kim et al. [Sphingopyxis strain KCY1, which was shown to rapidly degrade up to 90% of TCS within 24 h and showed 100% removal within 48 h [Mycobacterium vaccae JOB5 and Rhodococcus jostii RHA1 were able to degrade TCS through a meta-cleavage pathway when grown on propane and biphenyl, respectively [Triclosan can also form various intermediate metabolites, as shown in sylation ,88. Hay sylation  observedproducts . Mulla eproducts  proposedm et al. , was shom et al. . Similarhin 48 h . Two oxyectively . Biphenymeta-cleavage of the C-O bond between the two phenol rings. They also observed the production of monohydroxy-triclosan and dihydroxy-triclosan through the partial hydroxylation of the OH-containing TCS ring.Mixed microbial cultures under methanogenic, denitrifying (anoxic) and aerobic conditions have also been shown to be effective in degrading TCS. For example, Gangadharan et al.  observed2/L and at a contact time of less than 5 min, but observed a 50- to 100-fold increase in Vibrio fischeri toxicity compared to TCS prior to chlorination. Multiple TCS-containing personal care products have also been shown to react with free chlorine to form higher-chlorinated TCS intermediates, including dichloro-2-phenol and trichloro-2-phenol. This was through Cl− substitutions, various chlorophenols  through the meta-cleavage of the TCS C-O bond or its higher-chlorinated intermediates and chloroform formation from ring cleavage of TCS, TCS-intermediates, chlorophenols and Cl− substitutions [p-dioxins (DCDDs) and 2,4-DCP, when aqueous TCS is irradiated with sunlight/UV light, even at relatively low irradiation intensities [Other TCS transformation routes include abiotic processes, including chemical and photochemical reactions. However, many of these degradation products are far more toxic and biopersistant than the parent TCS. For example, Noutsopoulos et al.  found 92itutions ,99,100. itutions  when TCSitutions ,103. Othensities ,106,107.ensities . Furtherensities ,110.Wastewater undergoes a variety of different treatments and conditions  as it is processed in a WWTP. For example, wastewater undergoes a variety of oxidation-reduction potentials in BNR processes, during which different microorganisms are active and a wide range of metabolic processes are occurring. A portion of antimicrobials can be degraded and/or transformed into other compounds during treatment.2 per gram of material) and the highly hydrophobic and lipophilic nature of TCS [Various studies have reported TCS concentrations of 0.3 to 12.5 µg/L in domestic wastewater ,113,114.e of TCS ,75. Signe of TCS .4, which accumulated and persisted until the end of the experiment [Triclosan has been shown to degrade under aerobic conditions, and a wide range of biotransformation values during secondary  treatment have been observed. Guerra et al.  found thperiment . TCS remperiment ,42. Althperiment ,112.4 in undigested mixed sludge from a small- to medium-scale BNR facility. Relatively high concentrations of TCS and MeTCS have been reported in primary and secondary sludge as they are the primary sinks for TCS and TCS-related compounds. A survey of six Canadian WWTPs published in 2015 found primary sludge TCS concentrations of 6 to 14 µg/g dw, while a 2009 study of a large U.S. facility reported average primary sludge TCS concentrations of 20.3 ± 0.9 µg/g dw and 0.09 ± 0.002 µg/g dw of MeTCS ,121. TheMany emerging contaminants are somewhat resistant to degradation and typically degrade at much lower rates than that of organic matter during sludge stabilization processes. Due to this, effluent from many stabilization processes, such as anaerobic digestion, often contain far higher antimicrobial concentrations (in terms of µg/g dw) and appear to be concentrated as sludge volumes that are typically reduced by 50% or more. Depending on the method of calculation, many studies have found negative TCS removals, and some refer to this as “accumulation” during digestion. For example, McAvoy et al.  found th3/day), with clear correlations between increasing SRT and TCS removals (R2 = 0.89). This study also reported anaerobic digester effluent TCS-SO4 levels of 0.09 to 0.19 ng/g dw and significant removals during anaerobic digestion of up to a 72% removal during the longest 20-day SRT and trace amounts of the TCS metabolite 2,4-DCP.Little TCS removal was also observed in several Canadian WWTPs employing anaerobic digestion. Guerra et al.  found lo3/day). They also reported a clear correlation between increasing TCS removals and digester SRT (R2 = 0.88) and near-complete (86 to 95%) removals of TCS-SO4, even at the shortest (5-day) SRT (OLR = 7.61 kg VS/m3/day) and did not detect any other TCS metabolites.Aerobic sludge digestion has been shown to be more far effective than anaerobic digestion in degrading antimicrobials. The same study by McAvoy et al.  found th4 were removed across mesophilic anaerobic digesters when combined with mesophilic aerobic/anoxic digesters operated at total SRTs of 20 days. In this study, they did not detect any other TCS metabolites.Some studies have also shown that various types of sequential digestion can also be effective in degrading TCS and can improve TCS removals versus comparable single-stage digesters. Samaras et al.  found thTriclosan is commonly reported as one of the top trace contaminants in the environment ,31,125. Palaemon varians) have been shown to avoid TCS-contaminated areas at concentrations as low as 10 µg/L using both linear and heterogeneous multi-habitat assay systems [Oncorhynchus mykiss (rainbow trout), and a 48-h median effective concentration of 390 µg/L for Daphnia magna [Litopenaeus vannamei (shrimp) to 1.403 mg/L for Oreochromis niloticus (tilapia), 260 mg/L for Pimephales promelas (minnow) and 370 mg/L for Lepomis macrochirus (bluegill) [Oryzias latipes) and 170 µg/L for Crustacea (Ceriodaphnia sp.) [Triclosan can alter the function of aquatic environments and can have toxic effects on a variety of species, suggesting that aquatic ecosystems may be significantly altered by TCS contamination. For example, saltwater shrimp  ,138. Thrluegill) . The 25%nia sp.) .Danio rerio (zebrafish) showing a 5-week bioaccumulation factor of 4157 at aquatic system TCS concentrations of 3 µg/L. Adolfsson-Erici et al. [Rutilus rutilu (common roach fish) and concentrations of up to 47 mg/kg of fresh weight in caged Oncorhynchus mykiss immediately (0 km) downstream of a Swedish WWTP.Orvos et al.  observedi et al.  also obsTriclosan is also persistent in soils. Modeling of antimicrobials in aerobic soils suggests that TCS has a half-life of 18 days, while no appreciable degradation was found even after 70 days in anaerobic soils . AerobicAlthough many studies have been unable to find direct toxic effects of TCS at levels commonly found within humans, there are several other concerns . For exaIn vitro, TCS has been also shown to be a potent inhibitor of enzymes which are responsible for the detoxification of multiple industrial chemicals by blocking the genetic transcription of several key human liver enzymes. These enzymes are responsible for the “biotransformation of steroids, pharmaceuticals and other xenobiotics” ,72,153. n = 3041) [n = 8195) Canadian study found TCS urine concentrations with a geometric mean of 16 ng/mL of TCS, with 71% of children (3 to 19 years old) and 69% of adults (20 to 79 years old) having detectable concentrations [E. coli and MRSA in animal studies [In humans, TCS is primarily excreted in feces and in urine ,155. Uri = 3041) . A more trations . TCS is trations ,142,158,trations , donatedtrations  and urintrations ,157,159. studies . Other s studies . Studies studies .Vibrio fischeri, zebrafish (Danio rerio), sea urchins (Paracentrotus lividus) and European abalone  [While a large number of studies have been conducted which have explored potential human health risks and ecological impacts of TCS, far less is known about the potential impacts of its conjugates, transformation and degradation products ,163. Thirculata) ,166,167.rculata) ,169, whirculata) . AlthougStreptococcus [Triclocarban (3-(4-chlorophenyl)-1-urea; CAS #101-20-2) is a broad-spectrum antibacterial and antifungal agent that is mostly active against Gram-positive bacteria, such as MRSA, vancomycin-resistant enterococci and tococcus ,170,171.tococcus ,172,173.tococcus . TCC hastococcus . In 1999tococcus . More thow value and a dissociation constant (pKa), while it has low water-solubility [The antimicrobial TCC is a trichlorinated binuclear aromatic compound which has toxic, persistent and bioaccumulative characteristics ,175. Reglubility ,176. TCClubility ,178.Sphingomonas sp. strain YL-JM2C, which was isolated from activated sludge of a WWTP in Xiamen (China), was studied by Mulla et al. [Ochrobactrum sp. TCC-1 in wastewater sludge under anoxic nitrate respiration conditions. According to their results, enhanced biotransformation of TCC to chloroanilines could be supported by nitrate under anoxic conditions. TCC degradation and its metabolites’ production during TCC removal from wastewater were also evaluated using entrapped Pseudomonas fluorescens strain MC46 in the study by Taweetanawanit et al. [Pseudomonas fluorescens MC46, on TCC removal in contaminated soil. TCC was efficiently removed  from soil by transforming into 3,4-DCA and aniline. According to study by Sipahutar and Vangnai [Ochrobactrum sp. MC22 was found to be capable of degrading TCC and its chloroaniline metabolites under both aerobic and anaerobic conditions. In the study by Liang et al. [Ochrobactrum sp. TCC-2 and/or chloroanilines degrader Diaphorobacter sp. LD72. Their hydrolysis products (chloroanilines) were also further degraded by these functional degraders. Furthermore, functional characterization of a novel amidase involved in biotransformation of triclocarban in Ochrobactrum sp. TCC-2, isolated from a river sediment sample, was studied by Yun et al. [Partial elimination of TCC in WWTPs or in the environment has led to the formation of several transformation/degradation products and metabolites as a result of various abiotic and biotic mechanisms, such as microbial metabolism, hydrolysis, oxidation and photolysis. Among TCC’s potential degradative intermediates and/or metabolites detected in aquatic and terrestrial ecosystems, the most common compounds are chlorocarbanilide congeners (dichlorocarbanilide (DCC), monochlorocarbanilide (MCC) and carbanilide (NCC)), chloroanilines  and 4-chloroaniline (4-CA)), 4-CC, catechol and aniline ,184,185.a et al. . This mia et al. . Anothera et al.  showed ta et al.  also invt et al.  exploredg et al. , TCC andn et al. . A novelortho- or meta-cleavage pathways, respectively [Acinetobacter baylyi strain GFJ2 isolated from soil also has another degradation pathway that produces aniline from 4-CA. Aniline is then converted into catechol, which may further be degraded via an ortho-cleavage route, producing cis,cis-muconic acid ) into their lesser chlorinated congeners under anaerobic and anoxic conditions by the activity of microorganisms ,193,194.14C-labelled TCC to higher animals [N-glucuronides via urine.Regarding the metabolism of TCC by higher animals, TCC was significantly metabolized in rats, monkeys and humans and then excreted in bile, feces and/or urine. Previous studies identified five metabolites of TCC  after oral, intravenous or dermal administration of  animals ,197,198. animals  measuredn = 12) collected from three WWTPs located in different areas in China over one year. Average TCC concentrations in the influent and effluent ranged from 4.7 to 109 ng/L, whereas TCC levels in sludge were in the range of 1130–2180 ng/g dw with an average of 1700 ng/g dw. The occurrence of TCC in aqueous and solid waste streams of seven Chinese WWTPs was investigated in another study by Wang et al. [n = 49), with median concentrations of 968 and 1090 ng/g dw, respectively. On the other hand, among the 19 biocides monitored in eight municipal WWTPs in Thailand, TCC was determined as the predominant compound in the influent, effluent and excess sludge, with average concentrations of 838 ng/L, 95.6 ng/L and 4010 ng/g dw, respectively [As TCC is relatively non-volatile , photodeectively . These sn = 31) [n = 24) collected from six WWTPs in Canada and was one of the most abundant compounds, at concentrations from 1200 to 8900 ng/g dw, among the 62 PPCPs. Another study by Chu and Metcalfe [TCC has been identified as one of the top trace contaminants among wide-ranging PPCPs in sludge and biosolids assayed in several countries, such as the U.S. ,201,204,n = 31) . TCC wasn = 31) . A studyn = 31)  reportedMetcalfe  reportedMetcalfe , TCC wasMetcalfe , TCC amoMetcalfe  also repAs the anaerobic digestion process is a commonly used sludge stabilization option in municipal WWTPs because of its capability by generating bioenergy and biosolids , severalow values  and low water solubilities  were reported for DCC and 4-Cl-TCC, respectively. Based on the quantitative structure–activity relationship analyses, DCC was found to be less particle-active and 34-times more water-soluble compared to 4-Cl-TCC [Although most studies have investigated the occurrence and fate of TCC in sludge and biosolids, only a few have reported its behavior coupled with its potential transformation products and metabolites ,201,206.4-Cl-TCC . In anot4-Cl-TCC  was the 4-Cl-TCC . Anaerob4-Cl-TCC , TCC wasThere are significant concerns that TCC released to the environment through wastewater sludge may have potential adverse effects on human health and on ecosystems. Due to its high chlorine content and aromatic nature, TCC exhibits a tendency for environmental persistence ,214,215.Daphnia magna (planktonic crustacean) with a 48-h LC50 of 0.006896 mg/L was the most sensitive species to TCC, followed by Rana chensinensis , Gobiocypris rarus  and Macrobrachium nipponense , respectively [Salmo gairdneri and Lepomis macrochirus (fish) were 0.12 and 0.097 mg/L, respectively [Several studies have explored potential endocrine-disrupting effects of TCC on animals  ,218,219 ustacean ). TCC alectively . Moreoveectively . TCC hasectively ,227,228.ectively ,232,233.ectively . The stuectively  demonstrectively ,53. TCC ectively ,53,234.Upon human exposure, TCC can be metabolized and excreted by the human body during its utilization in consumer and personal care products ,235,236.Allium cepa root-tip chromosomal aberration assay, root-tip cells exposed to TCC at various concentrations had more chromosomal abnormalities compared to root-tip cells exposed to TCC’s degradative intermediates. In addition to 4-chloroisocyanatobenzene and 4-chloronitrobenzene, 3,4-DCA and 4-CA were also identified as the main degradation products of TCC during photodegradation in aqueous systems under ultraviolet radiation [Lumbriculus variegatus (worm) [Regarding transformation/degradation products of TCC, their potential ecological and human health risks are still rarely known ,163,187.adiation  and simuadiation . Concernadiation ; thus, Tadiation . The higs (worm) ,230,247.s (worm)  reporteds (worm) ,195. Furs (worm) . Based oProducts containing emerging alternative antimicrobials are becoming increasingly common in the market place ,249,250.Over the last decade, benzyl-substituted quaternary ammonium chlorides (QACs) have become common in the marketplace as emerging alternative antimicrobials. The history of the use of QACs as a cleaning agent dates back to 1935, when Domagk reported the enhanced efficacy of the substituted long-chain alkyl groups . In the 8-C18) on the other side. BACs are a broad class of homologous n-alkylbenzyldimethylammonium chlorides with varying numbers of carbon atoms in the alkyl chain. Commercial formulations use either one or a mixture of homologues in order to meet the selective strength and activity of applications. The strength is influenced by the length of the alkyl chain [Benzalkonium chlorides are the most widely used compounds currently deferred to as an eligible active ingredient for consumer antiseptic products in the final rule of the 2019 U.S. FDA Federal Register . Due to yl chain . The mosyl chain ,268. Theyl chain ,269,270.N-nitrosodimethylamine (NDMA), has been well studied due to the carcinogenic characteristics of NDMA [The biodegradation pathways for BACs in wastewater and sludge have not yet been well established. Some research papers have shown degradation mechanisms along with their byproducts and metabolites in the wastewater treatment systems during chemical oxidation processes ,275,276. of NDMA ,280,281. of NDMA ,283,284.N-dealkylation at the active site of the quaternary nitrogen atom [β-oxidation metabolic pathway through the citric acid cycle [N-dealkylation cleavage at the quaternary ammonium active site [Microbial biodegradation of BACs has been reported to produce benzyldimethylamine as the primary metabolite through the initiation of gen atom . The alkid cycle ,286. Bacid cycle ,288,289.ive site . CYP-medBACs with positively-charged nitrogen atoms tend to accumulate in sludge due to their strong biosorption affinity ,292. BACow values and, hence, the binding affinities. The reported Log Kow values for BAC-12, BAC-14 and BAC-16 are 2.93, 3.91 and 4.89, respectively [The ultimate sink of BACs is wastewater treatment systems through which they can enter into different environmental compartments ,292,302.ectively ,271. Theectively . The diaectively ,302. Bioectively ,298. Theectively ,296,303.Chlorella vulgaris [Pseudokirchneriella subcapitata), 125 µg BAC/L (48-h) for rotifers  and IC50 values as low as 41 µg BAC/L (48-h) for daphnids (Daphnia magna). The potential for these compounds to have an impact will likely increase as environmental concentrations rise due to limited removals during wastewater treatment and increased use of these compounds as manufactures shift away from legacy antimicrobials.Benzalkonium chlorides may have adverse impacts on aquatic organisms in the environment. BAC-12, BAC-14 and BAC-16 demonstrated acute 96-h EC50 toxicity values of 0.597, 0.473 and 0.406 µmol/L, respectively, on the microalgae vulgaris . Kreuzinvulgaris  reportedThe unique physical and chemical properties of BACs allow them to be used for a variety of applications and they have become popular in a large number of industrial and domestic applications. Due to the recent outbreak of the novel coronavirus, massive hand sanitization and sterilization led to a drastic rise in the market of BACs in order to stop the spread of the disease and to protect public health . The heaThere are also increasing concerns regarding the development of antibiotic-resistant bacteria from the environmental release of BACs ,317,318.Legacy antimicrobials have been a fixture of modern life for over 40 years. Two of the most common legacy antimicrobials are TCS and TCC, which have toxic, persistent and bioaccumulative characteristics. These chemicals are widespread environmental contaminants due to large production volumes, heavy usage, limited degradation during wastewater treatment and their persistence in the environment. Although aqueous phase removals of up to 98% of TCS and 99% of TCC during wastewater treatment have been reported, the primary removal mechanism is partitioning to wastewater sludge. Although some TCS and TCC removals have been observed during various forms of biological sludge treatments , concentrations up to 19,000 ng/g dw of TCS and 63,000 ng/g dw of TCC have been reported. Furthermore, while a number of biotic and abiotic processes have been shown to be somewhat effective in degrading these chemicals, they often generate significant quantities of metabolites and byproducts, many of which may be even more toxic and environmentally persistent than their parent antimicrobials. Recent studies are increasingly suggesting that these chemicals and their byproducts may not be as innocuous as was first thought and may have adverse short- and long-term environmental and human health impacts.Several emerging alternative antimicrobials, including various benzyl-substituted quaternary ammonium chlorides, are rapidly replacing legacy antimicrobials due to bans on active ingredients and shifting consumer preferences. There are growing concerns that many of these replacements and their byproducts present similar environmental and human health concerns as legacy antimicrobials. Few studies to date have adequately explored these alternatives so further work is urgently needed to ensure that legacy antimicrobials are not replaced by chemicals which may present similar or even possibly even greater risks.


Beauveria bassiana is one of the most widespread insect pathogens and can be used in the biological control of agricultural, forestry and medical pests. The mechanisms by which B. bassiana leads to mortality in different host insects are also different. For house flies, B. bassiana has strong virulence, but its microecological mechanism is not clear. In this paper, the virulence of three strains of B. bassiana  isolated from different hosts to house flies was studied. The results showed that the three strains of B. bassiana had strong pathogenicity to house fly adults. Specifically, TB was the strongest, CB was the second strongest, and BB was the weakest, with maximum lethal effects on house fly populations 5, 6, and 7 days after infection, respectively. Further study showed that the intestinal flora of house flies was disordered 3, 4, and 5 days after B. bassiana TB, CB and BB strain infection, respectively. Intestinal flora dysbacteriosis may be an important reason for the death of house flies caused by B. bassiana. After infection, the negative interaction ratio of bacteria in the house fly intestine decreased, and the stronger the virulence was, the lower the negative interaction ratio was. The time from B. bassiana infection to intestinal flora dysbacteriosis was not fixed. We named this period the “spring stage”. The stronger the virulence of the B. bassiana strain was, the shorter the “spring stage” was. Therefore, the “spring stage” can be used as a virulence marker for evaluating the pathogenicity of different B. bassiana strains. Musca domestica L. (Diptera: Muscidae), is an important medical insect that interferes with human life by causing annoyance, irritation, and food spoilage. It can also mechanically transport pathogenic disease in both people and animals. Over 130 pathogens  have been identified in house flies, causing intestinal infections, eye infection, certain skin infections, polio, bird flu and other illnesses . Biological control is a green and environmentally friendly pest control method that is also widely used in IPM . BiologiBeauveria bassiana is one of the most common fungal parasites. The first reported natural occurrence of B. bassiana in the house fly was in 1990  penetrate into the cuticle. After penetration, fungi inhibit the host immune system and grow in the hemocoel, taking up nutrients, producing toxins, destroying host cells and eventually killing the insect  with mesh screens on opposite sides and a cloth sleeve opening at the front. The adult flies were provided with brown sugar in Petri dishes as a diet and allowed water ad libitum. After 2 to 3 days of feeding, clear plastic cups containing the larval diet were placed in the cages as an egg laying substrate. The diet was composited with sterilized wheat bran, dry milk powder and sterilized water (45.5:4.5:40), mixed to paste in proportion. When eggs became visible on the sides of cups or attached to the food, the cups were removed and kept separated for larval development. The larval food was changed every day. The insects were kept in an artificial climate chest at 28 ± 1°C, a 45% to 55% relative humidity (RH) and a photoperiod of LD 16:8 h.The house fly strain was reared in the vector biological laboratory of Shandong First Medical University for approximately 15 years without exposure to pesticides or entomopathogenic fungi. Adult B. bassiana were collected from naturally infected insects. B. bassiana BB was isolated from naturally infected cicada Cryptotympana atrata Bsg in Bashangou of Mount Tai, Shandong Province ; B. bassiana CB was isolated from naturally infected stink bug Cyclopelta parva Sddy on the campus of Shandong First Medical University, Taian city, Shandong Province ; and B. bassiana TB was isolated from naturally infected longicorn larvae of Anoplophora glabripennis Pzs at Puzhao Temple, Taian city, Shandong Province   against M. domestica adults was determined independently using the bait method (9 spores/ml) was mixed with 50 g wheat bran (containing 4.5 g powdered milk) as bioassay baits for house flies. For each group, the baits were put into gauze cages (30 cm × 30 cm × 30 cm) containing approximately 150 of 12 h-emerged house flies. Sterile water was provided as drinking water. The baits and water were changed every day. The experiment was carried out in three cages independently, and sampled in each cage every day, respectively, as replications. The house flies fed baits without B. bassiana spores were used as controls. The dead house flies in different cages were identified and collected daily until 9 days after treatment. The collected dead house flies were maintained at over 90% RH in an incubator at 28 ± 1°C. Dead house flies with visible fungal growth on their body surface were considered to have died of fungal infection.The activity of the three strains of t method . The 40-®, China) as bioassay baits for house flies. For each group, the baits were put into gauze cages (30 cm × 30 cm × 30 cm) containing approximately 50 female and 50 male 12 h-emerged house flies. Sterile water containing 2% RNEP  was provided as drinking water. The baits and water were changed every day. The experiment was carried out in three cages independently, and sampled in each cage every day, respectively, as replications. The house flies fed baits without B. bassiana spores were used as controls. Every day, 10 female and male house flies were randomly removed from each cage and placed in a Petri dish with clean filter paper on the bottom. They were allowed to defecate for 2 h, and the amount of red feces produced by house flies on the filter paper was assessed. After the test, the house fly was put back into the original cage. The eggs oviposited in the bait were counted daily when baits were changed.Furthermore, in order to analyze the amount of feces excreted by male and female house flies, and the amount of eggs oviposited by female house flies fed on different baits, the 40-ml spore suspension (prepared by sterile Tween 80 solution to a concentration of approximately 109 spores/ml) was mixed with 48 g wheat bran (containing 4.5 g powdered milk) and 2 g red natural edible pigment (RNEP)  followed by genomic DNA isolation using the Wizard Genomic DNA Purification Kit  according to the manufacturer’s instructions, with DNA suspended in 30 μl nuclease-free water. The concentration and quality of extracted DNA were assessed using a NanoDrop 2000 spectrophotometer  and 2% agarose gel electrophoresis, respectively. Extracted DNA was stored at −20°C until further processing.The hypervariable V3-V4 region of the bacterial 16S rRNA gene was amplified with the primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′). Twenty-microliter PCR mixtures were set up with 4 μl 5 × FastPfu buffer, 2 μl deoxynucleoside triphosphates (dNTPs) (2.5 mM), 0.8 μl each primer, 0.4 μl FastPfu polymerase, and 10 ng template DNA. Reactions proceeded in a GeneAmp 9700 (ABI) thermocycler with 95°C for 5 min; 27 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 45 s, followed by additional elongation at 72°C for 10 min; and a dissociation stage at the end of the run.PCR products were detected by 2% agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit (Qiagen). Library pools were constructed with equal amounts of each PCR product by using the TruSeq Nano DNA LT Sample Prep Kit (Illumina), which was amplified through paired-end sequencing on the Illumina MiSeq PE300 platform.http://www.usadellab.org/cms/index.php?page=trimmomatic). Based on the overlap (minimum: 10 bp) between PE reads after quality control, PE reads were assembled using FLASH v1.2.11 software (FLASH: fast length adjustment of short reads to improve genome assemblies). QIIME v1.9.1 software  was adopted for processing, and VSEARCH v2.14.1 software (VSEARCH: a versatile open source tool for metagenomics) was used for detecting chimeric sequences. Based on a sequence similarity level of 97%, the UCLUST method in QIIME software was employed to perform OTU clustering analysis. On the basis of the Silva reference database (Release 138), taxonomic annotations were made for the OTUs in each sample. The Shannon, Simpson, Chao1, and Ace indexes of microbial communities were calculated by mothur (https://mothur.org/). LEfSe software (https://bitbucket.org/nsegata/lefse/src/default/) was used to estimate the abundance differences among microbial species in samples.Quality control of the original data was carried out using Trimmomatic v0.39 software  based on Bray-Curtis dissimilarity and an unweighted pair group method with arithmetic mean (UPGMA) tree based on unweighted UniFrac phylogenetic distances were used to determine the difference in bacterial community beta diversity in different samples. Bray-Curtis ordination is an effective strategy for the analysis of multivariate ecological data. Fast UniFrac facilitates high-throughput phylogenetic analyses of microbial communities, including analysis of pyrosequencing and PhyloChip data.B. bassiana infection on the Ace, Chao1, Simpson and Shannon indexes using SAS software . Fisher’s least significant difference approach was used to assess the mean differences among treatments and their interactions at p< 0.05.Paired-end reads were assigned to samples based on their unique barcodes and truncated by cutting off the barcode and primer sequence. Then, the paired-end reads were merged into longer single sequences using FLASH (v1.2.11) . Quality filtering was performed on the raw tags under specific filtering conditions to obtain high-quality clean tags  accordinB. bassiana strains BB, CB, and TB and the control house flies were referred to as groups B, C, T and K, respectively. The three B. bassiana strains had different virulence to house flies, and B. bassiana strain TB had the strongest virulence, followed by strains CB and BB. The peak death of house flies caused by TB, CB and BB occurred on the 5th, 6th, and 7th days after exposure, respectively. After treatment for 6 and 7 days, all the house flies in the T and C groups died, but the cumulative mortalities in the B and K groups were 97.25% and 6.29% until 9 days after treatment, respectively  for 5 days, representing approximately 76.05 ± 1.89% of the total bacteria. Cyanobacteria_c_norank was the dominant taxon in the intestines of adult house flies after infection by B. bassiana CB (group C) for 4 days and B. bassiana TB (group T) for 3 days, accounting for approximately 93.65 ± 1.40% and 94.68 ± 2.12% of the total bacteria, respectively. Therefore, after infection with B. bassiana BB, CB and TB for 5, 4 and 3 days, the intestinal flora in the house flies was disturbed. The death peak of house flies infected by B. bassiana strains BB, CB and TB occurred 7, 6 and 5 days after infection, respectively. Therefore, we can conclude that the time interval between intestinal flora disorder induced by B. bassiana infection and the population death peak was 2 days, and the phenomenon of intestinal flora disorder occurred first. However, for different strains of B. bassiana, the time from exposure to intestinal flora disturbance was variable. We named the stage from B. bassiana infection to house fly intestinal flora disturbance the “spring stage”. The “spring stage” can be used as a marker of B. bassiana virulence. The length of “the spring stage” was inversely proportional to the virulence of the different B. bassiana strains. The longer the “spring stage” was, the weaker the virulence of B. bassiana was.B. bassiana CB infection (the day before the peak of population death), Pseudomonas (22.54% ± 0.27%), Cyanobacteria_c_norank (21.96% ± 0.22%), Aquabacterium (12.26% ± 0.70%), Bacillus (8.73% ± 0.06%) and Sphingobium (6.42% ± 0.40%) were the top five most abundant bacteria in the intestines of house flies.At the same time, on the 5th day after B. bassiana TB infection (the day before the peak of population death), the dominant bacteria in the house fly intestine were Weissella (15.98% ± 1.49%), Cyanobacteria_c_norank (14.85% ± 0.28%), Lactococcus (13.17% ± 0.07%), Rheinheimera (27.49% ± 13.78%), Vagococcus (5.98% ± 2.01%) and Escherichia shigella (4.61%) ± 1.50%) ; the bacterial group with the largest proportion at 1 day and 2 days was B. bassiana infects house flies, it will first cause disturbance of the intestinal flora of the house flies, and some kinds of bacteria will multiply to large numbers (such as Pantoea in group B or Cyanobacteria_c_norank in groups C and T), while disturbance of the intestinal flora generally occurs two days before the population death peak of house flies.Therefore, when Cyanobacteria to the drinking water of the house fly groups with B. bassiana CB and TB infection or Pantoea was added to the drinking water of the house fly group infected with B. bassiana BB, the peak time of death was one day earlier than that of the groups without such addition  and Ace (p = 4.63E−07) indexes of intestinal bacteria from group K house flies were higher than those from house flies infected by B. bassiana, but the two indexes were not significantly different among the three infected groups. Therefore, the bacterial community richness of the house fly intestine decreased after B. bassiana infection. The Shannon index (p = 2.12E−06) and Simpson index (p = 1.88E−05) differed significantly among the four groups. The Shannon index of group K was significantly higher than those of groups B and C but not significantly different from that of group T, and the index was not significantly different among groups B, C and T. The Simpson indexes of groups K, B and T were significantly higher than that of group C  were significantly lower, but the Simpson index was higher, than those in the control group  that endanger human health , and could be influenced by several virulence factors during cuticle adhesion, penetration, and insect body invasion. The longer the “spring stage” was, the weaker the gicornis .Staphylococcus and Weissella were the dominant bacterial genera in the intestine of house flies in a previous study . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.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.