Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. hyosynoviae are significant pathogens for the porcine industry worldwide. The aim of the present study was to determine the antimicrobial susceptibility of six key antimicrobials  routinely used for treating infections caused by these pathogens. Twenty-seven M. hyopneumoniae, 48 M. hyorhinis and 40 M. hyosynoviae field strains isolated from clinical samples from different Southern European countries between 2013 and 2018 using broth microdilution method were evaluated.90 values 4 to 5 two-fold dilutions lower than those of tylosin and tilmicosin. The pleuromutilin valnemulin showed one of the highest in vitro activities against the three mycoplasma species. On the contrary, lincomycin exhibited the highest MIC values of the antimicrobials tested.Tylvalosin exhibited the highest in vitro activity among the macrolides assayed, with MICThe data obtained in the present study supports the use of pleuromutilins and macrolides for the control of infections caused by porcine mycoplasmas. The use of lincomycin for the treatment of porcine mycoplasma infections should be carefully evaluated due to the presence of circulating field isolates with decreased susceptibility to this antimicrobial. Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. hyosynoviae are considered the most relevant Mollicutes to porcine health worldwide, and together with M. suis, a non-culturable haemotropic mycoplasma, represent the main pathogenic mycoplasmas of pigs [M. hyopneumoniae is a major porcine pathogen, due to its role as the aetiological agent of enzootic pneumonia and also, by interacting with other microorganisms, as a primary pathogen of the porcine respiratory disease complex [M. hyopneumoniae vaccines under field conditions has shown variable efficacy [M. hyopneumoniae infections in pigs are aminocyclitols, aminoglycosides, fluoroquinolones, florfenicol, lincosamides, macrolides, pleuromutilins and tetracyclines [M. hyosynoviae is one of the main bacterial pathogen involved in pig lameness [M. hyosynoviae infections [M. hyorhinis is a ubiquitous porcine pathogen, primarily associated with cases of polyserositis, arthritis and otitis in pigs. In some cases it has been linked to cases of pneumonia, acting as a secondary or opportunistic pathogen. Pneumonia caused by M. hyorhinis is clinically indistinguishable from cases produced by M. hyopneumoniae [M. hyorhinis, so additional control measures involving improved farm management strategies and the reduction of environmental stressors are required. As described above, the control of porcine mycoplasmosis still depends strongly on the use of antimicrobials. However, the historical overuse of antibiotics in human and animal medicine has led to a current situation of alert, due to the development of resistance that reduces the therapeutic options. In this scenario, the analysis and monitoring of antimicrobial susceptibility has become pivotal in animal health management [ of pigs , 2. M. h complex , a disea complex . Commercefficacy , leadingcyclines . On the lameness –9. Infecfections . Furthereumoniae –10. Tetreumoniae , 12. Comnagement . The aimM. hyopneumoniae isolates were obtained from cases of porcine respiratory disease. M. hyorhinis isolates (48) were divided into 18 isolates obtained from arthritic joints and 32 from respiratory diseases cases. The 40 M. hyosynoviae isolates were obtained from arthritis cases.A total of 27 50 and MIC90 for each antimicrobial tested against M. hyopneumoniae are presented in Table 50/90 values of 0.03/0.06 μg/ml. Results obtained for the other two macrolides tested, tylosin and tilmicosin, were higher than those described for tylvalosin, with an MIC range of 0.06–1 and 0.25–1 μg/ml respectively and MIC50/90 values at 0.5/1 μg/ml. Lincomycin MIC showed a wide range of dilutions, ranging from 0.06 to 16 μg/ml, including the highest MIC value of all M. hyopneumoniae strains tested. MIC50 value found for lincomycin was 0.25 μg/ml, while the MIC90 value observed was 4 μg/ml. Tiamulin MIC values were similar to those described for tylosin. For technical issues, valnemulin was only tested against 12 Spanish isolates, and it presented the lowest MIC range (0.008–0.03 μg/ml) of all antimicrobial assayed against M. hyopneumoniae. The distribution of MIC values per antimicrobials tested can be found in Table The MIC range, MIC50 and MIC90 for M. hyorhinis isolates and distribution of MIC values are summarised in Tables 50 and MIC90 were comparable for tylosin and tilmicosin, with just a 2-fold dilution difference in the lowest MIC value and MIC50 of tylosin. Tylvalosin showed a reproducible pattern of MIC distribution between the three countries of origin of the isolates, displaying one of the lowest MIC50/MIC90 of the antimicrobials tested (0.016/0.125 μg/ml). MIC values for lincomycin displayed the highest MIC values for all the countries and antimicrobials studied, with an MIC range of 0.125- > 64, and the highest MIC90, with a value of 16 μg/ml. MIC range for tiamulin was comparable to the range observed for tylosin (MIC range 0.06–8 μg/ml). Valnemulin MIC values were comparable to tylvalosin for this mycoplasma, apart from MIC90 value, that was the lowest observed for M. hyorhinis (0.03 μg/ml). The MIC distribution for all antimicrobials tested showed a broader distribution in comparison to those described for M. hyopneumoniae, with lincomycin presenting the widest distribution observed.MIC50 and MIC90 for M. hyosynoviae were fairly similar between countries, with just one 2-fold dilution difference between MIC values and the geographical origin of the samples (Tables 50/MIC90 for this pathogen (0.016/0.06 μg/ml). The highest MIC50/MIC90 for M. hyosynoviae were those observed for tilmicosin, with concentrations of 1 and 2 μg/ml respectively.MIC ranges, MICs Tables . ValnemuThis investigation was aimed at evaluating the antimicrobial susceptibility patterns of selected members of the macrolide, lincosamide and pleuromutilin antimicrobial families against isolates of the most clinically relevant porcine mycoplasmas collected from Southern European countries. Macrolides, lincosamides and pleuromutilins are extremely relevant for porcine health. For instance, the World Organisation for Animal Health (OIE) classifies these antimicrobial families as either critically (macrolides) or highly important antimicrobials (lincosamides and pleuromutilins) in veterinary medicine . Further90 values 16 to 32-times lower than those of tylosin and tilmicosin. Similar results were obtained by Tavío et al. [M. hyopneumoniae, as well as for the avian mycoplasma M. synoviae and Mycoplasma sp. 1220 [M. hyopneumoniae, M. hyorhinis, M. synoviae, Mycoplasma sp. 1220 and M. gallisepticum [M. hyorhinis and M. hyosynoviae, and the in vitro [M. hyopneumoniae infections, gives prominence to tylvalosin as one of the most effective macrolides against porcine mycoplasmosis, together with tulathromycin, a 15-membered macrolide. Up to date, there are limited comparative data between tylvalosin and tulathromycin MIC values for porcine mycoplasmas. Felde et al. [90 values of 1 and 4 μg/ml, 1–4 two-fold dilutions higher to those observed for tylvalosin after analysing Central European M. hyopneumoniae isolates. Conversely, Klein et al. [90 values for tulathromycin of 0.004 μg/ml, after analysing 50 European M. hyopneumoniae isolates, however, the authors did not include tylvalosin in their analysis. In addition, decreased susceptibility to tylosin and tilmicosin has been previously described in European M. hyosynoviae and M. hyopneumoniae isolates [M. hyosynoviae [M. hyorhinis [M. hyopneumoniae [M. hyopneumoniae strains [In our study tylvalosin exhibited the highest in vitro activity among the macrolides assayed against the three mycoplasma species, with MICo et al.  after evsp. 1220 , 19. Fresepticum –20. Howein vitro  and in vin vitro  demonstrn et al.  describeisolates , 24. Altisolates , describsynoviae , M. hyoreumoniae , 23. Bas strains , suggestM. hyorhinis presenting a MIC value of > 64 μg/ml. Lincomycin MIC values for M. hyopneumoniae displayed a decreased susceptibility pattern in comparison to Spanish isolates [90 values. However, similar values were observed in previous studies [M. hyopneumoniae include the respiratory exposure of gilts to lung homogenates containing viable strains of this pathogen [M. hyopneumoniae in the farm. Lincomycin has been previously described as effective in vitro against M. hyorhinis [M. hyorhinis against this antimicrobial in the population studied, in agreement with the data published by Bekö et al. [M. hyorhinis is a common commensal of the upper respiratory tract of pigs, acting as an opportunist pathogen of immunocompromised animal in a variety of clinical presentation [M. hyorhinis analysed showed a marked decrease in susceptibility for lincomycin. This antimicrobial has been extensively used as part of in-feed medication in porcine farming, administered orally in premix, oral powder and oral solution for years [M. hyorhinis strain population. This selective pressure may have enabled in the same way the lincomycin susceptibility and MIC value distribution observed in the M. hyosynoviae and M. hyopneumoniae population evaluated. M. hyosynoviae presented similar MIC values to previous reports against lincomycin, with little to no variation observed [With regards to lincomycin, this antimicrobial exhibited the highest MIC values of all the antimicrobial tested, with one strain of isolates , evidenc studies , 26. Curpathogen . Howeveryorhinis , 28. Conö et al.  in Hungaentation . In our or years . Therefoobserved .M. hyopneumoniae were in agreement with those previously reported by Tavío et al. [M. hyopneumoniae, with a difference in MIC90 found between studies of 8 to 16 times. Also, Hannan et al. [M. hyopneumoniae and M. hyosynoviae against both pleuromutilins. These data suggest a potential decreased activity against pleuromutilins for these two porcine pathogens. M. hyorhinis MIC data presented the broadest range of values for both pleuromutilins tested, in conjunction with the highest MIC values . Other authors have described lower MIC values for both pleuromutilins in M. hyorhinis for Hungarian isolates [M. hyopneumoniae in the same region after testing the same antimicrobials [M. hyorhinis, macrolide and lincomycin decreased susceptibility has been associated to multiple mutations in the same gene [M. hyorhinis population studied.Differences in effectivity between pleuromutilins were observed in the three mycoplasma species analysed as previously described , with vao et al. , and simo et al. . Howevero et al. , reporteisolates , with sicrobials . Reducedcrobials , mutatioame gene , suggestM. hyosynoviae, the high MIC values observed for lincomycin against M. hyorhinis and M. hyopneumoniae isolates, suggest the need for a more rational approach to the use of this antimicrobial in cases of porcine mycoplasmosis. The molecular basis of the potential cross-resistance for lincomycin, tilmicosin, tylosin and both pleuromutilins in M. hyorhinis should be analysed. The differences observed between isolates from different European regions draw special attention to the need for standardised antimicrobial susceptibility testing. Besides, coordinated monitoring schemes for these pathogens in Europe are essentialto effectively tackle the potential emergence of resistant mycoplasma strains in order to maintain an optimal level of health and welfare in the porcine industry.In conclusion, among the tested antimicrobials, valnemulin and tylvalosin displayed the greatest in vitro activity against the three porcine mycoplasmas species collected from Southern European countries. Although active for Strains were isolated from clinical samples submitted by field veterinarians for routine diagnosis to the mycoplasma diagnostic service of the Instituto Universitario de Sanidad Animal y Seguridad Alimentaria  between 2013 and 2018. Samples were sourced from farrow-to-finish farms and obtained from fattening pigs, gilts and sows with no previous antimicrobial treatment. Clinical samples analysed included nasal swabs from those animals with respiratory disease symptoms compatible with mycoplasmal pneumonia and synovial fluid from cases of lameness.The isolation of the strains was performed as previously described , using mM. hyopneumoniae (J), M. hyorhinis (BTS-7) and M. hyosynoviae (S16) were used as a positive control. All control strains were sourced from the National Collection of Type Cultures (NCTC).The type strains of Tiamulin, tilmicosin, valnemulin and lincomycin were obtained from Fluka Analytical . Tylosin was obtained from Serva  and tylvalosin from Eco Animal Health . All antibiotic stock solutions were sterilised by filtration through 0.2 μm pore size membrane filters (Millipore).5 colour-changing units/ml of mycoplasma per well. Inoculated plates were then incubated at 37 °C with constant shaking at 150 rpm in a humidified atmosphere until growth in the drug-free control wells was evident. Bacterial growth was examined daily until a colour change was observed for a maximum of 21 days. MIC testing was performed on three different days, and duplicates of each strain were performed on each of the testing days. MIC was defined as the lowest concentration that completely inhibited growth, shown by a lack of colour change at the time that the drug-free growth control exhibited a colour change, while the negative control remained unchanged. MIC50 and MIC90 are defined as the lowest concentration that completely inhibited growth in 50 and 90% of the population studied respectively, and MIC ranges were also calculated. Due to the lack of official breakpoints for porcine mycoplasmas, the percentage of resistant isolates was not calculated. MIC values of the type strains can be found in Tables Minimum inhibitory concentration (MIC) testing was performed using a microbroth dilution method as previously described , using 9


In idiopathic sudden sensorineural hearing loss (ISSNHL), the relationship between the administration duration of vitamin B12 (vit B12) with adenosine triphosphate (ATP) and their therapeutic effect is not fully understood.To investigate the therapeutic effect of long-term 16 (≥weeks) administration of vit B12 with ATP on the prognosis of ISSNHL patients and compare it with those of short-term (<8 weeks) and middle-term  administration.We retrospectively reviewed the medical records of 117 patients with ISSNHL treated between 2015 and 2018.The overall recovery rate was 32.5%. Initial higher hearing threshold and initial higher grade of hearing loss (HL) were associated with a poor prognosis. However, the administration duration of vit B12 and ATP did not influence the overall hearing improvement. With regard to the time course of hearing recovery, there was no significant difference in hearing recovery among the long-, middle-, and short-term administration groups until 16 weeks after treatment. However, at 16–24 weeks after initial treatment, the short-term administration group exhibited significantly lower hearing recovery than did the long-term administration groups.The administration duration of vit B12 and ATP did not influence the overall hearing prognosis in ISSNHL, but long-term administration of vit B12 and ATP helped prevent the progression of HL after ISSNHL. Our results suggest that long-term administration of vit B12 and ATP is not necessarily required to treat ISSNHL patients, except for slowly progressing HL in the affected ears. Idiopathic sudden sensorineural hearing loss (ISSNHL) usually occurs as an acute unilateral hearing loss (HL) . The exaThe duration of vit B12 and ATP treatment depends on the period during which the treatment effect of these drugs is sustained. A patient would be willing to take vit B12 and ATP orally for the long term if these medications are likely to provide benefits for a longer period of time. However, the influence of the duration of vit B12 treatment with ATP on hearing prognosis is unclear. Thus, the present study aimed to investigate the influence of treatment duration of vit B12 with ATP on the hearing prognosis of ISSNHL. Towards this goal, we evaluated the therapeutic effect of long-term (≥16 weeks) administration of vit B12 with ATP in comparison to that of short-term (<8 weeks) and middle-term  administration.This retrospective study (B20-056) was approved by the Institutional Review Board of Kitasato University Hospital. The need for informed consent was waived owing to the retrospective nature of the study.The subjects were 117 patients with ISSNHL who underwent pure-tone audiometry at Kitasato University Hospital between 2015 and 2018. The inclusion criteria were as follows: (1) sudden sensorineural HL of 30 dB or greater in at least three consecutive frequencies; (2) underwent early therapeutic management, that is, treatment was started within 2 weeks after onset; (3) age over 18 years; and (4) follow-up duration longer than 6 months. The exclusion criteria were as follows: (1) age younger than 17 years; (2) middle ear or retro-cochlear pathology; (3) history of Meniere’s disease or autoimmune HL; (4) history of genetic or fluctuating HL; (5) history of hearing aid wearing or otologic surgery; and (6) history of intratympanic steroid injection for salvage treatment after systemic steroid administration.Pure-tone audiometry was performed using a conventional device  in a soundproof room. First, the hearing thresholds were obtained through air conduction (AC) and bone conduction (BC) in frequencies of 0.25, 0.5, 1, 2, and 4 kHz for both ears. To prevent cross-hearing phenomenon causing an erroneous measurement, masking noise was used to occupy the non-test ear while the other ear was tested, as necessary. Briefly, the necessity of masking during the measurement of the AC thresholds was identified based on the minimum interaural attenuation level of 40 dB for retesting through AC. Meanwhile, in the measurement of BC thresholds, a masking process was applied using ABC methods . ThreshoAll patients were treated with a 10-day course of systemic corticosteroids . Oral administration of vit B12 (1.5 mg daily) and ATP (300 mg daily) was started from initial day of treatment, concurrent with the corticosteroids. To investigate the therapeutic effects of long-term administration of vit B12 and ATP on hearing outcomes in ISSNHL, the patients were divided into three groups according to the administration duration as the short-term (<8 weeks), middle-term  and long-term (≥16 weeks) administration groups.p value of <0.05 was considered statistically significant.The Chi-squared test was used to evaluate clinical characteristics and possible prognostic factors. Student’s t-tests and non-parametric Mann–Whitney U tests were used to investigate continuous prognostic factors. For comparisons between more than two groups, a one-way analysis of variance was used followed by Dunn’s multiple comparisons for the post hoc test. The parameters that were statistically significant in the univariate analysis were entered into a binary logistic regression model for multivariate analysis. All statistical analyses were performed using GraphPad Prism 8  or JMP 14.2 . A The mean age was 58.2 ± 16.6 years, and there were 66 and 51 male and female patients, respectively. The patients’ clinicodemographic characteristics are presented in p <  0.0001). With regard to the associated symptoms, there was a significantly higher incidence of vertigo in the no recovery group than in the recovery group (p = 0.04). The other variables, including age, sex, days to initiation of treatment, and diabetes were not significantly different between the two groups. The duration of vit B12 and ATP administration was slightly longer in the no recovery group than that in the recovery group, but the difference was not significant. The significant variables in the univariate analysis were included in the multivariate analysis. The results showed that higher initial hearing threshold and higher initial grade of HL were associated with a poor prognosis in ISSNHL patients  were excluded because the vit B12 and ATP administration was terminated following the confirmation of complete recovery. Specifically, the duration of drug administration was shortened due to complete recovery, but the shortened administration did not contribute to complete recovery. Therefore, it was not appropriate to include the patients with complete recovery within 24 weeks after onset for the analysis of the effect of drug administration duration. In this study, the duration of administration was determined by the patients after consultation with their physician, even though they did not achieve complete recovery. The clinical features of each group are presented in p < 0.0001), but the clinical characteristics and hearing results were comparable (p > 0.05). This indicated that the duration of drug administration was not influenced by the patient’s clinical characteristics and did not affect the overall hearing outcome. Ultimately, these results show that long-term administration of vit B12 and ATP had no significant impact on the hearing prognosis within our observation period.The short-term, middle-term, and long-term administration groups involved 15, 41, and 38 patients, respectively. The patients with complete recovery within 24 weeks after onset (p = 0.76). The time course of hearing recovery in the overall population was 18.9 ± 1.7, 3.1 ± 0.7, and 0.04 ± 0.4 within 0–2, 2–4, and 4–6 months after symptom onset, respectively  and 4–6 months (p <  0.0001). Moreover, hearing recovery within 2–4 months after symptom onset was significantly higher than that within 4–6 months (p = 0.04). This result indicated that hearing recovery in the initial 2 months after onset is higher and that the probability of recovery decreases as the duration after symptom onset increases. The time course of hearing recovery of both the affected and contralateral ears in the three groups are shown in p = 0.03). Meanwhile, there was no significant difference in hearing recovery of the contralateral ears among the three groups throughout the study period. This indicated a slow progression of HL in the affected ear and that long-term administration of vit B12 and ATP might prevent progressive HL in the affected ear in ISSNHL. We performed an additional analysis of hearing recovery at each frequency. As shown in We also investigated the time course of hearing recovery in each group. As shown in The influence of the duration of vit B12 and ATP treatment on hearing prognosis has been unclear. In our study, the administration periods of vit B12 and ATP were not prognostic factors. However, this result does not necessarily indicate that long-term administration of vit B12 and ATP has no therapeutic effect on the overall prognosis of ISSNHL as hearing recovery was significantly lower in the short-term administration group than that in the middle and long-term administration groups within 4–6 months after onset. This indicates that vit B12 and ATP treatment might have gradual therapeutic effects on preventing the deterioration in hearing rather than promoting the hearing recovery. Hearing recovery within the initial 2 months was also significantly higher than that in the later phase, indicating that early initial treatment within 2 months after symptom onset is extremely important.The optimal treatment strategy for ISSNHL has not been established to date because the cause and etiology of ISSNHL remain unclear. However, circulation and neurological disorders are thought to be one of the major causes for ISSNHL . BecauseVitamin B12 plays an important role in the maintenance of normal neural function . AccordiATPs are often used as vasodilators to increase cochlear blood flow in auditory diseases. The vasodilator effect of ATP is mediated by the endothelium and follows the release of NO. A previous study reported that treatment with ATP resulted in a large increase in cochlear blood flow and a decrease in blood pressure owing to the vasodilator actions of ATP . AnotherThe prognosis for recovery from ISSNHL can be influenced by various factors, including age, accompanying symptoms such as vertigo, degree of HL, audiometric configuration, and time to treatment initiation . The finThis study has some limitations. First, this was a retrospective study conducted in a single hospital, and the sample size was relatively small. Second, the duration of vit B12 and ATP treatment in this study was influenced by various factors, such as patient preferences and drug therapeutic effects. However, despite these limitations, we believe that our study is valuable because to the best of our knowledge, this is the first to focus on the relationship between the duration of vit B12 and ATP administration and prognosis in ISSNHL. Our findings have important clinical implications in the understanding and management of patients with ISSNHL. Although the underlying therapeutic mechanisms of vit B12 and ATP in ISSNHL are not well known, its effects need to be considered in the management of ISSNHL in the clinic. Further prospective studies with larger populations are needed to validate our findings.The administration duration of vit B12 and ATP did not influence the hearing prognosis in ISSNHL. Higher initial hearing threshold and higher initial grade of HL were associated with a poor prognosis. Compared to the contralateral ear, HL progressed gradually in the affected ear. Long-term administration of vit B12 and ATP might help prevent progressive HL in the affected ear.10.7717/peerj.10406/supp-1Supplemental Information 1Click here for additional data file.


The use of autologous hematopoietic stem cell transplantation (AHSCT) for autoimmune diseases has become the first indication for transplant in nonmalignant disease. Mucormycosis is a rare invasive infection with increasing incidence in patients treated with AHSCT. We report the first case of pulmonary mucormycosis following AHSCT for systemic sclerosis (SSc).A 24-year-old woman with rapidly progressive diffuse cutaneous SSc presented with an acute respiratory distress syndrome 6 days after AHSCT.The results of clinical and computed tomography scan were consistent with pulmonary mucormycosis and the diagnosis was confirmed by a positive Mucorales Polymerase Chain Reaction on a peripheral blood sample.Early antifungal therapy by intravenous amphotericin B provided rapid improvement within 4 days and sustained recovery after 2 years of follow-up.With the progressively increasing use of AHSCT and other stem cell therapy for treatment of severe SSc and other autoimmune diseases, the potential onset of rare post-transplant fungal infections, such as mucormycosis, requires careful patient monitoring and better awareness of early initiation of adequate therapy. Mucormycosis has never been reported following AHSCT for autoimmune diseases or in SSc patients.Over the past 20 years the use of autologous hematopoietic stem cell transplantation (AHSCT) to treat severe rapidly progressive systemic sclerosis (SSc) has expanded progressively and this procedure is now recommended as a first-line option,In this article we report on the first case of pulmonary mucormycosis after AHSCT for rapidly progressive diffuse cutaneous SSc.2A 24-year-old woman diagnosed with SSc 2 years ago and who had no other past medical history, was initially treated with low-dose corticosteroids and mycophenolate mofetil for 9 months then with methotrexate for 6 months and 2 courses of rituximab. She was then referred for AHSCT due to a rapidly progressive disease with extensive skin fibrosis (modified Rodnan skin score 25/51) and pulmonary involvement [decreased diffusing capacity of the lung for carbon monoxide at 56% of the theoretical values on lung function tests and the presence of ground-glass infiltrate and diffuse bronchial wall thickening on chest computerized tomography (CT) scan]. Right heart catheterization with fluid overload, echocardiography, cardiac magnetic imaging, and myocardial scintigraphy were all normal.2 administered over 2 consecutive days), and filgrastim (10 μ/kg/day for 7 days), allowing collection of 25 × 106 CD34+ cells/kg in 2 cytaphereses.Peripheral blood hematopoietic stem cells (PBSCs) were mobilized with intravenous (IV) cyclophosphamide (CPM)  was administered followed by a reinjection of non-selected PBSC (day 0).On day 0, the patient had leukopenia of less than 0.1 g/L and aplasia was sustained until day 12. She experienced her first febrile peak (39°C) the day after PBSC reinfusion while physical examination was otherwise normal and peripheral and port-a-cath blood cultures remained sterile. An empiric IV antibiotic therapy by piperacillin-tazobactam and amikacin was started. The patient remained afebrile until day 4 after PBSC reinjection, when she had a new onset of fever with a progressive increase in C-reactive protein level (278 mg/L). Repeated peripheral and port-a-cath blood cultures were sterile. The IV antibiotic therapy was switched to meropenem, ciprofloxacin, and vancomycin.The patient remained febrile (39°C) and 2 days later developed a dry cough. The chest CT scan revealed massive bilateral pleural effusion, upper right lobe condensation, and less extensive upper left lobe condensation Fig. .On day 6, an acute respiratory distress syndrome with hypoxemia at 60 mm Hg required endotracheal intubation and mechanical ventilation. Acyclovir (10 mg/kg/dose/8 h IV) and amphotericin B (5 mg/kg/dose/24 h IV) were added. Repeated CT scan showed bilateral parenchymal condensation with a focal ground-glass opacity surrounded by a consolidation ring in the right upper lobe, consistent with the reverse halo sign  by polymerase chain reaction (PCR) were all negative. Blood, Aspergillus antigenemia was negative, whereas Mucor sp. and/or Rhizopus sp PCR were positive.Bronchoalveolar lavage (BAL) was performed and tests for cytomegalovirus, herpes simplex virus 1, herpes simplex virus 2, varicella zoster virus, adenovirus, other respiratory viruses, as well as for The diagnosis of pulmonary mucormycosis was established on day 10 after AHSCT, and IV amphotericin B, increased to 7.5 mg/kg, with the addition of isavuconazole (200 mg/dose/8 h IV for 48 h and then 200 mg/24 h) were started. Antibiotics were discontinued and sulfamethoxazole-trimethoprim and valacyclovir were resumed at prophylactic doses. Rapid clinical improvement was observed, the patient was extubated on day 14 and oxygen therapy was stopped on day 17. Blood PCR for mucormycosis was negative on day 18.The follow-up chest CT scan (on day 20) showed almost complete regression of bilateral alveolar parenchymal condensation and resolution of the reverse halo sign Fig. B. IsavucAfter 1 year of follow-up, the patient is still in clinical and CT scan remission from mucormycosis.3 The clinical features of pulmonary mucormycosis are nonspecific and cannot be distinguished from pulmonary aspergillosis. High-grade fever and a nonproductive cough are the most common symptoms.Invasive mucormycosis has a high mortality rate of 76% and the lungs are the second most commonly reported site after the sinuses.Most of the signs of pulmonary mucormycosis on CT scan are indistinguishable from other invasive pulmonary fungal infections, with the presence of infiltrates, consolidation, nodules, cavitations, atelectasis, effusion, posterior tracheal band thickening and hilar or mediastinal lymphadenopathies. Only the reversed halo sign, a focal round area of ground-glass attenuation surrounded by a ring of consolidation, appears more specific and is highly suggestive of pulmonary mucormycosis. PCR detection of Mucorales DNA in blood samples is now recognized as the earliest biological indication of mucormycoses–17 and could even precede the diagnosis of proven mucormycosis by an average of 9 days.Evidence of mucormycosis on cultures or on histopathological examination is required for a definitive diagnosis. In this case, the patient presented with acute respiratory distress only 6 days after PBSC reinjection, which underscores the crucial role of prior immunosuppression, especially prolonged use of corticosteroids, which was the case for our patient.Mucormycosis tends to occur late in autologous stem cells recipients for various diseases . Even so, mortality rates are still high (61%).Management guidelines suggest amphotericin B as the first-line treatment (daily dose 5 mg/kg) combined with a surgical procedure, when necessary, and then a switch to posaconazole as a maintenance treatment. showed that delaying the administration of amphotericin B–based regimens by 5 days is associated with a 2-fold increase in mortality.Early antifungal therapy is essential, as soon as the diagnosis is suspected, without waiting for biological evidence. In fact, Chamilos et al which represents approximately 8% of the postallograft invasive fungal infections. Less data is available on the occurrence of mucormycosis after AHSCT, but the incidence appears to be lower with 7.8% of the post-autograft invasive fungal infections. In systemic autoimmune diseases, the onset of mucormycosis remains uncommon and appears to be related to the use of corticosteroids.,22 In a series of 24 cases of autoimmune disease patients with mucormycosis, 83% had systemic lupus erythematosus. All the subjects had been exposed to corticosteroids and 6 had received additional CPM. In autoimmune diseases, mucormycosis symptoms can mimic a flare of the underlying disease.In hematological patients, the risk of mucormycosis after stem cell transplantation is well-documented, with a postallograft incidence estimated at 0.3%, with no report of increased risk for post-transplantation invasive fungal infection. In the various other trials, which showed the benefit of AHSCT in SSc patients,–8 a higher proportion of viral infections were described but no increased risk for fungal infection has been noted.In France, between 1996 and 2018, 75 AHSCT for severe SSc were performed4In light of the increase in the availability of AHSCT for severe SSc clinicians should be aware of this rare post-transplant fungal infection and that early initiation of adequate therapy for patients with clinical and radiological features consistent with mucormycosis could change the outcome.Conceptualization: Grégory Pugnet.Supervision: Grégory Pugnet.Validation: Grégory Pugnet.Writing – original draft: Xavier Boumaza.Writing – review & editing: Xavier Boumaza, Lucie Lelievre, Sarah Guenounou, Cécile Borel, Anne Huynh, Guillaume Beziat, Karen Delavigne, Damien Guinault, Marie Garric, Marie Piel-Julian, Kim Paricaud, Guillaume Moulis, Leonardo Astudillo, Laurent Sailler, Dominique Farge, Grégory Pugnet.


Colorectal cancer remains one of the deadliest cancers worldwide. It has become evident that further efforts in colorectal cancer research are required, from providing a better understanding of the cellular and molecular mechanisms leading to colorectal neoplasm initiation and progression from adenoma to metastasis, to generating reliable non-invasive detection tests for identifying lesions at early stages, as well as refining the current therapeutic and personalized approaches and developing new ones. The aim of this Special Issue is to cover all aspects of colorectal cancer research, including basic, preclinical, and clinical approaches.The original articles of this Special Issue present innovative findings toward the design of new strategies that may contribute to the fight against colorectal cancer cells, such as treatments that enhance apoptosis-related mechanisms ,2, chemoReview articles have summarized the latest developments of clinical issues related to the management of improving chemotherapeutic approaches for metastatic colorectal cancer ,21,22, tThe variety of the content of this Special Issue devoted to colorectal cancer illustrates the need for investigating the cellular basis of this disease from every angle in order to develop efficient screening and treatment strategies that can significantly impact the quality of life of the patients. I would like to thank all of the authors that have contributed to the articles of this Special Issue for their work.


Hearing loss is a rarely reported complication of spinal anesthesia. The purpose of this study is to assess the effects of 0.5% Maracine (bupivacaine) on hearing threshold and auditory reaction time before and after spinal anesthesia among patients undergoing elective surgery.. After obtaining consent from the patients, audiometry and tympanometry tests were performed using AZ80 and Madsen otoflex tympanometer and related findings including the presence or absence of hearing loss at various frequencies, before and after the surgery, were noted in a form for each patients along with their demographic data. SPSS 21 was used for statistical analysis and the data were analyzed using descriptive statistics and chi-square inferential tests.This is a descriptive cross-sectional study performed on 60 patients undergoing elective surgery with ASA Class II and II anesthesia (0.5% bupivacaine) at Khorramabad Nursing HomeAt low frequencies of 250 and 500 Hz, no significant difference in pre- and postoperative hearing threshold in the right ear (P > 0.05) was seen, but at frequencies above 500 Hz, the hearing threshold was significantly decreased after surgery, (P < 0.05). In the left ear at 250, 1000, 3000, and 8000 Hz, there was no significant difference (P > 0.05) between pre- and postoperative hearing threshold. The results of this study showed that the preoperative hearing threshold for men and women did not differ and the auditory threshold and auditory response time after surgery did not differ between the two sexes (P > 0.05). Similarly, the difference was not correlated with the age and the levels of anesthesia (P > 0.05). The results also showed that changes in mean arterial blood pressure (MAP) and heart rate above 30% of baseline were also not correlated with hearing loss (P > 0.05).The results showed that at certain frequencies, hearing loss was observed in both ears after spinal anesthesia with 5% Marcaine, but this hearing loss was not related to age, sex, and spinal anesthesia level. The results also showed that changes in mean arterial blood pressure (MAP) and heart rate above 30% of baseline did not correlate with hearing loss. •Hearing loss is a rarely reported complication of spinal anesthesia.•Hearing loss in both ears is observed after spinal anesthesia with 5% Marcaine.•Changes in MAP and heart rate greater than 30% of baseline is not associated with the hearing loss. It is uBupivacaine is an amide that has 10-min slow onset and is more effective with isobaric forms . It is sThe variability of the solutions used for spinal anesthesia appears to cause significant differences in the distribution of anesthetic drugs within the spinal cord, which can have important effects on the extent of onset and duration of the anesthetic block, as well as the side effects ,21. StudThe aim of this study is to investigate the effects of spinal anesthesia, Marcaine 0.5% (bupivacaine) on hearing impairment among patients undergoing elective surgery.2In this descriptive cross-sectional study, all the patients referred for elective surgery meeting the requirements of American Anesthesiologists Association (ASA) Class I and II anesthesia I at Khorramabad Nursing Home were enrolled. The inclusion criteria of the study included: patients aged 19–45 years undergoing elective surgery with ASA Class I and II, patients with normal otoscopic examination before surgery, hearing threshold above 30 dB and type A tympanogram, without the history of otitis media, meningitis, systemic disease, mumps, ear surgery or the intake of agents that can lead to hearing intoxication, patients referred for back surgery, non-smokers, without drugs, alcohol abuse and psychotropic drugs and non-allergic to anesthesia. A written consent was obtained from all the patients for the participation in the study and details of study were provided to all the participants. Patients unwilling to participate, those with hearing intoxication medications during and after surgery, patients with serious complications after surgery and reduced level of consciousness along with those who did not meet inclusion criteria were excluded from the study.Prior to the surgery, the researcher completed a demographic questionnaire for all the participant. One audiologist was referred to the hospital admission ward who performed standard PTA and tympanometry tests using AZ80 and Madsen tofellax tympanometers and findings related to the presence or absence of hearing loss, its magnitude, frequencies, and tympanometric types were recorded in the questionnaire.Preoperative blood pressure and other vitals were recorded for all the patients. Intraoperative monitoring devices included pulse oximeter, non-invasive blood pressure and ECG (electrocardiography) and baseline values were recorded. 2 ml/kg of 0.9% saline was administered intravenously and 27-gauge needle was used to administer 2.5 ml bupivacaine 0.5% hyperbaric using lateral approach at L2-L3 or L4-L5 slowly for 10 s at minimum.•125–2000 Hz = (Low) low frequency•2000–4000 Hz = (Mid) Medium frequency•4000–8000 Hz = (High) High FrequencyAfter anesthesia, the patient was turned into supine position. Twenty minutes after anesthesia, surgery was commenced, before which sensory and motor blockade was accessed using pinprick test and Bromage scale, respectively. Blood pressure, heart rate, and pulse oximetry were measured every 15 min during the surgery. If systolic blood pressure was above 90 mmHg, 5–10 mg of intravenous ephedrine was administered to the patient. Twelve hours after surgery, the audiometry was performed again by the same audiologist. Hearing frequencies were classified as follows:In this study, a data entry form was used for data collection, which consisted of two parts. The first part comprised of demographic data and the second part was for recording audiometric data. This section included 7 questions including age, sex, type of surgery, MAP , PR (pulse rate) changes above 30% baseline, amount of cerebrospinal fluid injected, and fasting time. Part II comprised of information related to the presence or absence of hearing loss and its magnitude, frequencies, and tympanometric types.The research was approved by Ethical Committee of Lorestan University of Medical Sciences. The research was conducted in accordance with the Declaration of Helsinki (1964) and subsequent revisions. The work has been reported in line with the STROCSS criteria .The collected data were analyzed by the means of descriptive statistics and analysis of variance for factorial design using SPSS 21 software. Descriptive statistics were used to summarize demographic data of the participants. Chi-square test was used to compare baseline and demographic data. Relative frequency of each case was obtained. P < 0.05 was considered significant. This study was approved by the Research Ethics Board of (XX) University of Medical Sciences (XX).3In this study, 60 patients were evaluated for the auditory function who underwent elective surgery under bupivacaine (5% marcain). The mean age of patients was 37.78 ± 8.2 years (minimum 20 years and maximum 45 years). Thirty (50%) patients were male and 30 (50%) were female. None of the patients had a history of previous gastrointestinal illness or history of substance abuse. Anesthesia was performed in 26 patients (43.30%) at L3-L4 level and in 34 patients (56.7%) at L4-L5.t-test results showed that difference in mean left ear threshold of patients before and after surgery at frequencies 250 Hz (p = 0.085), 1000 Hz (p = 0.096), 3000 Hz (p = 0.704), 6000 Hz (p = 0.38) and 8000 Hz (p = 0.349) were not statistically significant. But based on paired t-test results, the difference in mean values of left ear threshold of patients before and after surgery at 500 Hz (p = 0.033), 2000 Hz (p = 0.018) and 4000 Hz (p = 0.002) was statistically significant, showing that the threshold at these frequencies decreased significantly after the surgery , respectively.The difference in left auditory threshold was not statistically significant in any of the thresholds before surgery.According to the results from Friedman test, the difference in mean MAP values > 30% of baseline in was statistically significant (p = 0.009) . SimilarAccording to Pearson correlation coefficients, no correlation was found between the age of patients and their hearing threshold before and after the surgery at different frequencies (p > 0.05), respectively.Based on Spearman correlation coefficient values, no significant correlation was found between MAP values > 30% at different time interval during surgery and hearing threshold at different frequencies (p > 0.05). No significant relationship was found between values of PR changes above 30% baseline at different time intervals during surgery and patients' hearing threshold at different frequencies (p > 0.05).t-test results, the difference between the preoperative and postoperative hearing response time was not statistically significant (p = 0.702).According to the paired t-test, the difference in the mean of auditory reaction time of patients before the surgery was also not statistically significant (p = 0.945). This was also not significantly different in the two gender groups (p = 0.835).Based on the results of independent t-test, the difference in the mean of auditory reaction time before anesthesia was not statistically significant (p = 0.904). Furthermore, age and auditory response time before surgery were not correlated significantly (p = 0.447). But according to Pearson correlation coefficients, there was a significant direct and linear relationship between age and auditory response time after surgery (p = 0.012) (Based on the results of independent = 0.012) .Table 5SAccording to Spearman correlation coefficient, preoperative auditory reaction time was not correlated with PR changes above 30% at different time intervals during the surgery.Similarly, preoperative auditory reaction time and MAP changes> 30% of baseline at different time intervals during the surgery was also not significantly related.4The overall results of this study show that at certain frequencies, hearing loss occurs in both the ears after spinal anesthesia, which is not related to age, sex, or level of spinal anesthesia. The results also showed that changes in mean arterial blood pressure (MAP) and heart rate above 30% of the baseline levels is not associated with the hearing loss. At low frequencies of 250 Hz and 500 Hz, hearing thresholds did not differ in the right ear, but at frequencies above 500 Hz, the hearing threshold was significantly reduced after surgery.In a study conducted at Freeman Hospital in the Department of Otolaryngology, low frequency hearing loss was reported in patients with spinal anesthesia . In the In the left ear at 250, 1000, 3000, and 8000 Hz, there was no difference in mean pre-operative hearing threshold in the right ear, and the hearing loss did not follow a specific pattern. A study reported in the anesthesiology department of Tollohelsinki Hospital, Finland, reported a reduction in the hearing ability as the risk of spinal anesthesia . Acute cIn a study by Schaffartzik et al. comparing hearing loss after spinal and general anesthesia, it was concluded that hearing loss after spinal anesthesia is greater. In this study, a specific relationship was found between intravascular volume changes and low frequency of hearing loss after spinal anesthesia . In anotThe results of this study showed that pre-operative hearing threshold was not different between men and women and post-operative hearing threshold was not different between the two genders. The results of this study also showed that the preoperative hearing reaction time of patients was similar among the two genders and there was no significant difference in post-operative hearing time after the surgery. This showed that gender did not affect the hearing response time of patients after spinal anesthesia with marcaine 5%. In the study of Maleki et al. there was a significant difference between the two genders in terms of word recognition score in silence and in 3 signal-to-noise ratios. In this comparison, the mean breakthrough thresholds at the four severity levels were similar for the two groups. This means that gender is unaffected by the temporal resolution ability of individuals. Similar outcomes have been obtained from the studies by Lotze et al. . and KelFitzgibbons and his colleagues studied the effect of age on long-term auditory differentiation. The results concluded that short-distance differentiation was influenced by age and hearing . In geneThe results of this study showed that there was no difference in right and left ear threshold in two levels of L3-L4 and L4-L5 anesthesia before surgery, indicating that the level of anesthesia has no effect on reduction of postoperative hearing level. The results showed that auditory reaction time was similar in the patients before and after anesthesia and there was no significant difference after surgery. We did not find any study addressing the level of anesthesia and its effect on hearing loss, but various studies have shown that spine surgery in different lumbar vertebrae can cause hearing loss in both right and left ears. These include the use of nitrous oxide and CSF leakage. CSF leakage is an important factor in hearing loss following spinal anesthesia because it causes brain tissue to stretch and impacts cochlear vestibular nerve and traction inside the internal acoustic crater, leading to hearing impairment.Our study does not compare the effects of different doses and types of spinal anesthesia on hearing loss. Additionally, the data of the study is restricted to a relatively smaller sample size and is generalized for all elective surgeries (excluding back and ear). Future multicenter studies with more variables and greater sample size can give better conclusions.5The results show that at certain frequencies, hearing loss in both ears is observed after spinal anesthesia with 5% Marcaine, but this hearing loss is not related to age, sex and the level of spinal anesthesia. The results also showed that changes in mean arterial blood pressure (MAP) and heart rate greater than 30% of baseline is not associated with the hearing loss.All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.No funding was secured for this study.Sepideh Vahabi: Conceptualization, Writing - original draft, Writing - review & editing, Conceptualized and designed the study, drafted the initial manuscript, and reviewed and revised the manuscript, Parvin Veiskarami: Writing - original draft, Writing - review & editing, Designed the data collection instruments, collected data, carried out the initial analyses, and reviewed and revised the manuscript, Mehdi Roozbahani: Writing - original draft, Writing - review & editing, Supervision, Coordinated and supervised data collection, and critically reviewed the manuscript for important intellectual content, Shahrzad Lashani: Writing - original draft, Writing - review & editing, Designed the data collection instruments, collected data, carried out the initial analyses, and reviewed and revised the manuscript, Behrouz Farzan: Conceptualization, Writing - original draft, Writing - review & editing, conceptualized and designed the study, drafted the initial manuscript, and reviewed and revised the manuscript.Name of the registry: Lorestan University of Medical SciencesUnique Identifying number or registration ID: IR.LUMS.REC.1398.046.https://ethics.research.ac.ir/ProposalCertificate.php?id=65108&Print=true&NoPrintHeader=true&NoPrintFooter=true&NoPrintPageBorder=true&LetterPrint=trueHyperlink to the registration (must be publicly accessible): Sepideh VahabiNot applicable.Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.Not commissioned, externally peer reviewed.The authors deny any conflict of interest in any terms or by any means during the study.


Age-related hearing loss reduces the quality of life in older adults. Low socioeconomic status (SES) has been reported as a risk factor for hearing loss, although this has not been verified in Japan. This study aimed to assess the association between low SES and hearing loss, excluding people with dementia, in Japan.Data from the Toyama Dementia Survey, Japan, were used. Overall, 126 patients with hearing loss and 913 unimpaired controls were identified. Participants’ presentation of dementia, self-reported hearing loss, history of medically diagnosed disease , lifestyle factors , and SES  were assessed. Any association between low SES and hearing loss was investigated using logistic regression analysis.The odds ratio (OR) for hearing loss was higher for participants with low educational attainment than for those with high educational attainment . After adjusting the models for SES, lifestyle factors, and medical history, the OR increased from 2.90  to 3.43 . The OR for hearing loss for participants with blue-collar jobs compared with that of participants with white-collar jobs was not significant . Older age and a history of angina pectoris or cardiovascular disease were found to increase the risk of hearing loss.Low educational attainment was independently associated with hearing loss in older adults without dementia in Japan. The onset of age-related hearing loss generally occurs around 50 years of age; there are reports of people who identify themselves as having trouble hearing in one out of three individuals aged ≥65 years and more than 70% of those were aged ≥75 years . The preTo prevent the development of hearing loss, it is necessary to identify its causes. Previous studies have revealed that diseases such as hypertension, dyslipidemia, and diabetes are involved in the development of hearing loss; other factors include noise, ear infections, and stress , 8. MoreIn this study, we aimed to evaluate associations between socioeconomic factors and age-related hearing loss, with respect to dementia, in Japan. Determination of these associations may enable the prevention of age-related hearing loss at an earlier age.We used the data from Toyama Dementia Survey , 18. OurParticipation in the Toyama Dementia Study was voluntary. All participants or their family members provided written informed consent prior to participation. The Ethics Committee of the University of Toyama approved the study protocol.Participants’ self-reported current level of hearing loss was classified as either “yes” or “no”. Those with hearing loss further classified their impairment (including the use of hearing aids) as being either inconvenient or barely able to hear.We surveyed participants with respect to their sex, age, educational attainment, occupation, lifestyle factors, and medical history. Educational attainment was stratified as follows: ≤6 years, 7–9 years, and ≥ 10 years.According to the Japan Standard Occupational Classification , occupatFor lifestyle factors, alcohol consumption and smoking habits were surveyed. Regarding alcohol consumption, participants were categorized as current drinkers, former drinkers, or non-drinkers. Current drinkers were those who consumed alcohol daily, occasionally, or within the preceding year; former drinkers were those who had ceased alcohol consumption for ≥1 year; and non-drinkers were those who had never habitually consumed alcohol. For smoking, participants were categorized as current smokers, former smokers, or non-smokers. Current smokers were those who smoked daily or within the preceding year; former smokers were those who had stopped smoking for ≥1 year; and non-smokers were those who had never habitually smoked.Participants’ medical history was assessed in accordance with International Classification of Diseases, Tenth Revision (ICD-10) criteria . The conA detailed procedure for the diagnosis of dementia has been published elsewhere . In brieThe chi-squared test was used to determine whether socioeconomic variables, lifestyle variables, and medical history differed between participants with and without hearing loss among those without dementia. A logistic regression analysis was used to test whether SES was associated with hearing loss; hearing loss was the dependent variable, and sex, age, lifestyle factors, and medical history were the independent variables. We created several multivariate models to inspect whether lifestyle factors, or medical history explained any observed associations between socioeconomic factors and hearing loss . First, Table Table The current study found that ≤6 years of educational attainment was associated with hearing loss, after excluding people with dementia among those aged ≥65 years and adjusting for lifestyle and medical history associated with SES.The risk of hearing loss was approximately 3.4 times greater for participants with ≤6 years of education than for those with ≥10 years of education. This finding suggests that low educational attainment is an important risk factor for hearing loss. The risk of hearing loss for low educational attainment of ≤6 years fell after adjusting for age, sex, and work history, suggesting that age, sex, and work history are confounding factors of hearing loss for low educational attainment. On the other hand, the risk of hearing loss in low educational attainment was increased after adjusting for lifestyle and medical history, suggesting that lifestyle and medical history are protective factors for hearing loss in low educational attainment. Low SES has been reported to be associated with unfavorable habits such as smoking . In EuroIn the current study, blue-collar versus white-collar in terms of job history was used as an indicator of low SES. Blue collar workers are likely to be exposed to higher noise exposure than white collar workers. However, the results of the present study did not show a significant difference in the risk of hearing loss in those with blue-collar jobs when compared with those with white-collar jobs. Adjusting for age and sex, educational history, lifestyle, and medical history lowered the risk of hearing loss, suggesting that these adjustment factors are confounding factors for blue-collar hearing loss. As reported in previous studies –30, lifeAmong the factors adjusted for hearing loss, previous angina pectoris or myocardial infarction was strongly associated with hearing loss. It has been reported that blood flow in the inner ear and brain is impaired in the presence of lifestyle-related diseases, negatively affecting hearing function , 32, andThe results of this study suggest that the low SES status  may be associated with a blue-collar work history, undesirable lifestyle habits such as drinking and smoking, and the development of lifestyle diseases, which may worsen systemic atherosclerosis, which in turn may lead to hearing loss. In the future, reducing the risk of low SES environments, in terms of disease incidence, may lead to the prevention of hearing loss.Several limitations of this study should be noted. First, this study does not consider occupational and recreational noise exposure, which is one of the risk factors of hearing loss. However, most blue-collar occupations with high noise exposure were included in the blue-collar population, which could have been an alternative indicator. Second, the duration of hearing loss was not examined in this study and hearing tests were not conducted. The percentage of participants who reported hearing loss was less than the prevalence of hearing loss reported in the literature. In this study, hearing ability in everyday life was assessed, rather than hearing ability measured by pure tone audiometry. Previous studies have reported that more than half of individuals examined exhibited hearing loss when using pure tone audiometry, but they were unaware of this loss . TherefoAfter adjusting for lifestyle and history of medically diagnosed disease, low educational attainment (≤6 years) was independently associated with self-reported hearing loss in older adults without dementia in Japan.


Mycoplasma hyopneumoniae is the major pathogenic microorganism causing enzootic pneumonia in pigs. With increasing resistance of M. hyopneumoniae to conventional antibiotics, treatment is becoming complicated. Herein, we investigated the mutant selection window (MSW) of doxycycline, tylosin, danofloxacin, tiamulin, and valnemulin for treating the M. hyopneumoniae type strain (ATCC 25934) to determine the likelihood of promoting resistance with continued use of these antibiotics. Minimum inhibitory concentration (MIC) values against M. hyopneumoniae were determined for each antimicrobial agent based on microdilution broth and agar dilution methods /mL to 109 CFU/mL). The minimal concentration inhibiting colony formation by 99% (MIC99) and the mutant prevention concentration (MPC) were determined by the agar dilution method with three inoculum sizes. Antimicrobial killing was determined based on MIC99 and MPC values for all five agents. MIC values ranged from 0.001 to 0.25 μg/mL based on the microdilution broth method, and from 0.008 to 1.0 μg/mL based on the agar dilution method. MPC values ranged from 0.0016 to 10.24 μg/mL. MPC/MIC99 values were ordered tylosin > doxycycline > danofloxacin > tiamulin > valnemulin. MPC achieved better bactericidal action than MIC99. Based on pharmacodynamic analyses, danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin. Mycoplasma hyopneumoniae is the primary pathogen causing enzootic pneumonia, an important chronic respiratory disease in pigs resulting in high morbidity, low feed conversion rate, and considerable economic losses in the swine breeding industry . Additionally, the disease makes pigs more susceptible to infection by secondary bacterial pathogens such as Pasteurella multocida and Actinobacillus pleuropneumoniae. There are several classes of antimicrobial agents that exhibit in vitro activity against M. hyopneumoniae, such as pleuromutilins, fluoroquinolones, macrolides, and tetracyclines , 4. However, widespread use of these agents has resulted in acquired resistance of M. hyopneumoniae to fluoroquinolones, lincosamides, and macrolides –7. Thus, in order to reduce the risk of drug resistance, it is necessary to develop novel drugs. However, even if new drugs are discovered, re-evaluation of antimicrobial dosing is essential, and is the main method aimed at preventing the emergence and expansion of drug-resistant strains.. The upper and lower boundaries of the MSW are the mutant prevention concentration (MPC) and the minimal concentration that inhibits colony formation by 99% (MIC99), respectively. The MPC is the minimum concentration that inhibits colony formation of the least antibacterial drug-susceptible mutant subpopulation. Therefore, when antimicrobial concentrations fall within the range of the MSW, this tends to lead to the enrichment of drug-resistant bacteria. Keeping drug concentrations above the MPC is likely to restrict the emergence of resistance . This hypothesis has been verified by in vitro and in vivo experiments –13.The mutant selection window (MSW) hypothesis postulates that, for each antimicrobial-pathogen combination, an antimicrobial concentration range exists in which selective amplification of single-step, drug-resistant mutants occurs M. hyopneumoniae lacks a cell wall and is a small bacterium (0.4–1.2 μm), and it is fastidious and generates small colonies, culture isolation conditions in vitro are a technical challenge. In particular, quantification by the viable count method to determine colony-forming units (CFU) is arduous. Therefore, studies on the pharmacodynamics of M. hyopneumoniae are scarce. In the current study we determined for the first time the MPC and identified the MSW for doxycycline, tylosin, danofloxacin, tiamulin and valnemulin against M. hyopneumoniae in vitro. We also used time-kill tests to determine the relative antibacterial effects at the MIC99 and the MPC. MSW and MPC are useful parameters for optimizing dosing regimens, reducing the emergence of resistant mutants, and analyzing treatment failure . In addition, using the CFU counting method, changes in the amount of M. hyopneumoniae after antibiotic action can be determined.Because Mycoplasma hyopneumoniae type strain ATCC 25934 was obtained as a freeze-dried powder from the China Institute of Veterinary Drug Control  and stored at -80°C. Pig Mycoplasma Base , cysteine, and NADH were purchased from Qingdao Hope Biological Technology . Sterile swine serum was bought from Guangzhou Ruite Biological Technology . The initial pH of the medium was 7.7 ± 0.1, and 1% agar was added to solid media.Doxycycline (85.8%), danofloxacin (100%), tiamulin (99%), tylosin (82.6%), and valnemulin (98.3%) were obtained from Guangdong Dahuanong Animal Health Products . The percentages refer to the active ingredients of the drug. These five antibacterial agents were dissolved in Milli-Q water and sterilized by filtration. A 1280 μg/mL fresh stock solution of each antibacterial agent was prepared for each experiment.. Briefly, MIC values were calculated for 105, 106, and 107 CFU/mL M. hyopneumoniae cultures in the exponential phase. A 100 μL sample of exponential phase culture was added to an equal volume of drug-containing medium culture in a 96-well plate. A growth control , sterility control (sterile broth at pH 7.8), and end-point control (blank medium at pH 6.8) were included. Plates were cultured at 37°C with 5% CO2 in an incubator after being sealed. When the color of the growth control was the same as the end-point control, the MIC was determined as the minimal concentration of antibacterial agent that resulted in no color change.MIC values were determined as described previously . Ten drops of a 10-μL sample of M. hyopneumoniae culture  were evenly placed on the surface of a plate containing 1.25−20 μg/mL danofloxacin, 2−32 μg/mL tiamulin, 4−64 μg/mL tylosin, 5−80 μg/mL doxycycline, or 0.16−2.56 μg/mL for two-fold agar dilution analysis. All plates were incubated for at least 8 days. Meanwhile, growth control plates without antimicrobials were set up for each test, and all experiments were repeated three times. The lowest concentration without M. hyopneumoniae growth on agar plates was taken as the MIC value. Each test was repeated three times.MIC values were determined by the agar dilution method as described previously 99 values were measured as reported previously  with modifications. MIC99 drug concentrations were based on linear decreasing dilutions of MIC values. The antibiotic concentration ranged from 1 × MIC to 0.5 × MIC in sequential 10% dilution decreases. The quantity of bacteria in the logarithmic growth phase reached 107 CFU/mL. Three 10 μL drops of each diluted suspension were inoculated onto agar plates and cultured for at least 8 days as described above. Colony numbers between 30 and 300 were counted.MIC9 CFU/mL bacteria . We attempted different centrifugal methods for enriching M. hyopneumoniae. Ultimately, an 800 mL stationary growth phase culture was transferred into 20 tubes (each 50 mL), tubes were centrifuged (5000 × g for 20 min), and each bacterial solution was resuspended in 1 mL fresh medium. All 20 enriched cultures were combined into two 15 mL tubes, centrifuged (5000 × g for 20 min), and resuspended in 1 mL fresh medium for counting. The final concentration of M. hyopneumoniae was 8.8×109 CFU/mL.The MPC is defined as the lowest drug concentration that prevents bacterial colony formation from a culture containing ≥10. Briefly, 200 μL samples of each enriched culture were inoculated onto agar plates containing various concentrations of antibiotic . These plates were incubated at 37°C with 5% CO2 in a humidified incubator for 8−10 days. The lowest antibiotic concentration that resulted in no colony formation was considered the primary MPC (MPCpr). After a 20% linear drug concentration decrease in MPCpr, the MPC was tested again, and recorded as the lowest drug concentration preventing bacterial growth. Each test was repeated three times.MPC values were measured by the agar method as described previously In vitro time-killing assays were performed as described previously . Briefly, MIC99 and MPC values for all five agents were tested. After adding 3.5 mL blank medium and 0.1 mL drug solution (40 times the target concentration) to each penicillin bottle (a glass bottle of 10 ml with a rubber stopper), 0.4 mL exponential M. hyopneumoniae suspension with an inoculum size between 105 CFU/mL and 109 CFU/mL was added. Cultures were incubated at 37°C with 5% CO2 for 48 h. Aliquots of 100 μL were collected from each culture at 0, 3, 6, 9, 12, 24, 36, and 48 h. The viable cell number was determined via 10-fold serial dilutions and plating 10 μL of each diluted culture on drug-free agar. Growth controls (M. hyopneumoniae cultures without drugs) and sterility controls (5 mL medium at pH 7.8) were also included. Plates were incubated for at least 8 days at 37°C with 5% CO2 in a humidified incubator. Each test was repeated three times.M. hyopneumoniae determined by the microdilution and agar dilution methods are shown in  Values determined using the solid MIC method were 8-fold higher (tylosin), 4-fold higher , and 2-fold higher (tiamulin) than those determined by the liquid method at an identical inoculum size of 105 CFU/mL. In addition, as the inoculum size (106 and 107 CFU/mL) used in these assays was increased, the MIC values also increased, except for tiamulin and valnemulin. M. hyopneumoniae displayed its greatest sensitivity to valnemulin and its least to doxycycline.MICs of danofloxacin, tiamulin, tylosin, doxycycline, and valnemulin against 99, MPC, and SI values are shown in  The SI, the ratio of MPC to MIC99, reflects the ability of an antibacterial agent to induce resistant mutants. MIC99 values for all antibacterial agents ranged from 0.0122 to 0.343 μg/mL, and MPC values ranged from 0.016 to 10.24 μg/mL. SI values ranged from 1.31 to 30.12, and were ranked valnemulin <tiamulin <danofloxacin <doxycycline <tylosin (low to high). Thus, valnemulin displayed the lowest SI value, while tylosin exhibited the highest SI value.MICM. hyopneumoniae were obtained using three different inoculum sizes. Reductions in M. hyopneumoniae count with different inoculum sizes for MIC99 and MPC are listed in Table 3. Danofloxacin, tiamulin, tylosin, and valnemulin achieved bactericidal activity against 105 CFU/mL M. hyopneumoniae with MIC99 dosage, while doxycycline achieved bacteriostatic activity only. Colony count reductions recorded at the 48-h time point 3.63, 3.68, 3.75, and 3.61 log10 CFU/mL for danofloxacin, tiamulin, tylosin, and valnemulin, respectively, but only 1.4 log10 CFU/mL for doxycycline. All five compounds achieved bactericidal activity against 105 CFU/mL M. hyopneumoniae with MPC dosage .Time-kill curves of compounds against 7 CFU/mL), danofloxacin, tiamulin, tylosin and valnemulin were bactericidal at the MIC99 while doxycycline was bacteriostatic only. Colony count reductions recorded at the 48-h time point were 5.15 log10 CFU/mL for danofloxacin, 5.09 log10 CFU/mL for valnemulin, 3.51 log10 CFU/mL for tiamulin, 4.13 log10 CFU/mL for tylosin, and 1.16 log10 CFU/mL for doxycycline. All five compounds achieved bactericidal activity against 107 CFU/mL M. hyopneumoniae with MPC dosage .At a greater inoculum size  for M. hyopneumoniae, systematic in vitro pharmacodynamic evaluation of antibacterial agents against M. hyopneumoniae is scarce. Thus, in the present study, in vitro pharmacodynamic indices of several representative antimicrobial agents against M. hyopneumoniae were determined, and killing curves were plotted. Based on the study of MSW of Mycoplasma gallisepticum, we conducted a related study on MSW of M. hyopneumoniae. To the best of our knowledge, this is the first study to explore the risk of M. hyopneumoniae resistance using MPC and MSW parameters., 5. Using both liquid and solid agar methods, MIC values increased with increasing inoculum size; values obtained with a large inoculum were two to four times higher than those obtained with small inoculum. It has been reported previously that MIC values increase with increasing bacterial load , 25. Among the five antibiotics tested, danofloxacin and tylosin were more sensitive to inoculum size for MIC determination. In an earlier report , a larger inoculum of Staphylococcus aureus had a more significant effect on the antibacterial activity of nafcillin and vancomycin than a smaller inoculum. Therefore, in cases of high bacterial inoculation, more careful consideration is required when selecting the MIC reference value for the relevant experiment. In clinical treatment, the MIC value should be determined for different bacterial counts according to the severity of animal infection to establish a better treatment plan.MIC values determined by the liquid method were similar to those measured in previous studies M. hyopneumoniae is difficult in vitro, determination of MPC values is challenging; after much effort, M. hyopneumoniae was cultured to a cell density of 107−108 CFU/mL. We tried a variety of enrichment methods to generate quantities of bacteria sufficient for determining MPCs, and eventually managed to measure MPCs for all five antibiotics. Relationships between antimicrobial exposure, MPC/MSW values and antimicrobial resistance selection have been explored previously. For example, in S. aureus, cefquinome concentrations below the MIC99, intermediate between the MIC99 and MPC, and above the MPC resulted in the selection of mutants that differed in terms of the proportion of resistant and susceptible bacteria . When the concentration of levofloxacin fell within the MSW, the sensitivity of S. aureus decreased and mutant subpopulations emerged . For M. hyopneumoniae, the reported danofloxacin, tylosin, and doxycycline concentrations in pigs fell within the MSW completely after an intramuscular dose of 2.5 or 10 mg/kg (body weight); after oral administration at a dose of 20 mg/kg, Cmax values for danofloxacin, tylosin, and doxycycline were 0.45 ± 0.09, 2.71 ± 1.09, and 2.44 ± 0.51 μg/mL –30. Correlative MIC99 and MPC values were 0.101 and 1.0, 0.34 and 10.24, and 0.343 and 4.0 μg/mL, respectively. The reported tiamulin and valnemulin concentrations were comfortably above the MSW values only after oral administration at a dose of 10 or 25 mg/kg , 32. Consequently, by combining the in vivo pharmacokinetic parameters and the in vitro pharmacodynamic results, we speculate that danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin. Continued use of first-line antibacterial agents against M. hyopneumoniae according to current dosing regimens may therefore promote drug resistance selection, and hence limit their long-term efficacy in the treatment of endemic pneumonia in pigs.The MPC is defined as the concentration of antibacterial drug that prevents the growth of large quantities of resistant sub-populations. Because culturing of M. hyopneumoniae at various bacterial densities and drug concentrations. At three different inoculation amounts, doxycycline displayed bacteriostatic activity at MIC99 dosage and bactericidal action at MPC dosage. At the highest inoculation amount, tiamulin and valnemulin acted as bacteriostatic agents at MIC99 dosage and as bactericidal agents at MPC dosage. Danofloxacin exhibited the fastest sterilization rate. Moreover, M. hyopneumoniae was highly sensitive to valnemulin, and exerted an obvious bactericidal effect. These results showed that when the concentration of antibiotic equaled or exceeded the MPC, M. hyopneumoniae was rapidly killed. Drug concentrations at the MPC also reduced the chances of bacteria re-growing during drug exposure. These results are similar to those of an earlier report . In that previously study, the bactericidal effect at MIC was slow and incomplete. However, at MPC and maximum serum or tissue drug concentrations, killing was more pronounced than at MIC, and increased with increasing duration of drug exposure. It is worth noting that macrolides and tetracyclines are typical bacteriostatic agents. However, in this study, when the drug concentration reached MPC, it had a bactericidal effect on M. hyopneumoniae. At the dose of MPC, macrolides and tetracyclines have a bactericidal effect, which has also been reported in other studies. According to the study reported by Joseph et al. , tilmicosin and tulathromycin had bacteriostatic effect on Mannheimia haemolytica at MIC dose and bactericidal effect at MPC dose. The research reported by Zhang et al. , with the increase of the concentration of doxycycline, the antibacterial effect of Mycoplasma gallisepticum was more obvious. The antibacterial type was concentration-dependent. It can also be proved that when the concentration is continuously increased to reach or exceed MPC, it will have a bactericidal effect.We determined the bactericidal effects of danofloxacin, tylosin, doxycycline, tiamulin, and valnemulin against in vitro pharmacodynamic determination of M. hyopneumoniae was carried out for the type strain, and clinical isolates should be assessed to confirm our findings. Nevertheless, the present work represents a meaningful pilot study in this area. A second limitation is that all experiments were performed under ideal conditions in vitro, without considering the complexity of factors in vivo. Thus, in vivo experiments are currently being explored. The third limitation is that this study is only a proof of concept for the MSW hypothesis and does not provide proactive validation to prevent drug resistance.The main limitation of the present study was that the M. hyopneumoniae. We determined MPC and MSW parameters to explore the risk of M. hyopneumoniae resistance. The results showed that the bactericidal action of MPC was better than MIC99, and the antibacterial effects of these drugs against M. hyopneumoniae are significantly different. These pharmacodynamic results are meaningful in choosing antimicrobials for therapy. Danofloxacin, tylosin, and doxycycline are more likely to select resistant mutants than tiamulin and valnemulin.In conclusion, the present study was the first to establish pharmacodynamic analyses of five antimicrobial agents against S1 Data(DOCX)Click here for additional data file.


Drosophila melanogaster Genetic Reference Panel and observed significant genetic variation in spontaneous grooming, with broad-sense heritabilities of 0.25 and 0.24 in females and males, respectively. Although grooming behavior is highly correlated between males and females, we observed significant sex by genotype interactions, indicating that the genetic basis of spontaneous grooming is partially distinct in the two sexes. We performed genome-wide association analyses of grooming behavior, and mapped 107 molecular polymorphisms associated with spontaneous grooming behavior, of which 73 were in or near 70 genes and 34 were over 1 kilobase from the nearest gene. The candidate genes were associated with a wide variety of gene ontology terms, and several of the candidate genes were significantly enriched in a genetic interaction network. We performed functional assessments of 29 candidate genes using RNA interference, and found that 11 affected spontaneous grooming behavior. The genes associated with natural variation in Drosophila grooming are involved with glutamate metabolism (Gdh) and transport (Eaat); interact genetically with (CCKLR-17D1) or are in the same gene family as (PGRP-LA) genes previously implicated in grooming behavior; are involved in the development of the nervous system and other tissues; or regulate the Notch and Epidermal growth factor receptor signaling pathways. Several DGRP lines exhibited extreme grooming behavior. Excessive grooming behavior can serve as a model for repetitive behaviors diagnostic of several human neuropsychiatric diseases.Spontaneous grooming behavior is a component of insect fitness. We quantified spontaneous grooming behavior in 201 sequenced lines of the  Drosophila melanogaster ; Nf1, which encodes Neurofibromin 1, a Ras GTPase activating protein  is a population of wild-derived inbred fly lines with fully sequenced genomes, and is a community resource for genome wide association (GWA) mapping of variants associated with quantitative traits when all variants are known : CCKLR-17D3 ; CG13229 ; CG17839 ; CG31221 ; CG33181 ; CG33926 ; CG34401 ; CG6767 ; Cht6 ; Eaat1 ; for ; Gdh ; GEFmeso , kirre ; Lrch ; mtt ; Nrg ; osp , PGRP-LA ; pnt ; PsGEF ; Ptp10D ; Ptp99A ; pxn ; scat ; SdhA ; Smg5 ; sty ; Trf2 . The P{KK} RNAi lines are from the same genetic background and contain UAS-RNAi constructs for each candidate gene at the same locus on the second chromosome; the P{GD} RNAi lines are from the same genetic background, and contain P-element based transgenes in a random insertion site  and 1118w (v60000), also obtained from the VDRC. We initially crossed all UAS-RNAi lines to an ubiquitously expressed GAL4 driver, Ubi-GAL4 (P{Ubi-GAL4}2). Lines without significant effects on grooming behavior using this driver were additionally crossed to two other ubiquitously expressed GAL4 driver lines, Act-GAL4 (P{Act5C-GAL4}25FO1) and Ubi[156]-GAL4. Ubi-GAL4 and Act-GAL4 were obtained from the Bloomington Drosophila Stock Center. Ubi[156]-GAL4 was created in-house by introducing the original Ubi-GAL4 transgene onto the third chromosome of the Canton-S B wild type strain by Δ2-3 transposase-mediated hopping . Thirty minutes after the transfer, the flies were video-recorded for three minutes. The duration of spontaneous grooming was quantified during the three minutes, separately for foreleg and hindleg grooming. The fly uses its forelegs to groom the eyes, head, thorax, forelegs and midlegs, and it uses the hindlegs to groom the hindlegs, genitalia and wings. The midlegs serve to groom the forelegs and hindlegs, so midleg grooming was included in foreleg grooming and hindleg grooming, respectively.Spontaneous grooming of single-sex groups of 20 flies per sex and genotype was measured for each line for 3 min. All flies were mated and 3-5 days old at the time of the assays. The genotypes included the 201 DGRP lines, and the F1 offspring of Y = μ + Sex + Line + Sex × Line + e, where μ is the overall mean, Sex is the fixed effect of sex, Line is the random effect of DGRP line, Sex × Line is the interaction between sex and genotype, and e is the residual. Reduced models were also fitted for the two sexes separately. We estimated the broad-sense heritability, We performed quantitative genetic analysis of total grooming time across sexes using the factorial mixed ANOVA model http://dgrp2.gnets.ncsu.edu. Briefly, we adjusted the phenotypic data for the effects of Wolbachia infection and major polymorphic inversions, and used the adjusted line means to fit a linear mixed model in the form of y = Xb + Zu + e, where y is the adjusted phenotypic values, X is the design matrix for the fixed SNP effect b, Z is the incidence matrix for the random polygenic effect u, and e is the residual. The vector of polygenic effects u has a covariance matrix in the form of Aσ2, where σ2 is the polygenic variance component.We performed the GWA analysis of total grooming time using line means for each sex as described in P < 10−5) to the network graph as described previously   under accession numbers listed in Supplemental Data File S1 of http://dgrp2.gnets.ncsu.edu. Supplemental material available at figshare: https://doi.org/10.25387/g3.12252719The DGRP lines and the To understand the genetic basis of natural variation in spontaneous grooming behavior, we manually annotated video recordings of 20 females and 20 males from each of 201 DGRP lines, and quantified the total time spent grooming during a 3-minute period. There was substantial phenotypic variation both at the individual fly level  and at tH2 = 0.87, by reducing the residual variance by n is the number of individuals measured per sex and line. Only two variants were significant using a strict Bonferroni correction for nearly 2 million variants: X_8312234_INS in an intron of Trf2  and the intergenic polymorphism 3L_3396966_SNP (P = 2.18 × 10−8). At the more lenient significance threshold of P < 10−5, we identified 107 DNA variants associated with spontaneous grooming in either males or females, and/or the average or difference of the two sexes (Table S2). Of these variants, 34 were intergenic (> 1 kb from gene boundaries) while the remaining fell within 1 kb of 70 annotated genes (Table S2).Because there was significant genetic variation for grooming behavior . However, allowing genes to be connected by at most one gene not in the GWA gene list captured a cluster of 24 genes that was significantly larger than a randomly seeded network, centered on three highly connected genes: pnt, which encodes a transcriptional regulator; scrib, which encodes a synaptic scaffold protein; and the morphogenetic gene sty  categories, including 20 genes with GO terms associated with nervous system development and function; 11 genes with GO terms associated with organism or tissue development; seven genes associated with GO terms associated with transcriptional regulation; and seven genes with GO terms associated with epidermal growth factor, fibroblast growth factor, Notch, BMP or other signaling pathways (Table S3). We performed a GO enrichment analysis, accounting for difference in gene length as this affects the differential baseline probability of being identified in the GWA analysis. None of the tested GO terms were enriched for the grooming-associated genes, consistent with a highly polygenic genetic architecture. Next, we mapped the significant genes detected in the GWA analysis to known genetic interaction networks. We extracted subnetworks from the global network whose edges were either a direct connection between candidate genes or bridged by only one gene not among the candidate gene list, and evaluated the significance of the size of the largest cluster among the subnetworks by a randomization test in which we randomly extracted subnetworks with the same number of input genes. The subnetwork with no missing genes resulted in only two connected genes  and/or a strong ubiquitous driver (Act-GAL4). A total of 20 candidate genes had significant effects on spontaneous grooming behavior in at least one sex and driver combination at a nominal P-value of P < 0.05, and 11 genes had significant effects in the same direction in both sexes and/or for more than one driver  (Table S4). A total of 11 genes remained significant (P < 0.05) when we used Dunnett’s test to account for multiple tests when several treatments are compared to a control group   .D. melanogaster, and that the genetic architecture of grooming behavior is partially distinct between males and females, as has been observed for most quantitative traits in the DGRP ; and Eaat, which encodes Excitatory amino acid transporter 1 (Eaat1), were candidate genes in the GWA analysis, and RNAi knockdown of Gdh affected grooming behavior. Gdh is involved in glutamate metabolism, and is the enzyme that catalyzes the reversible conversion of L-glutamate to α-ketoglutarate .None of the candidate genes identified in our GWA analysis has been associated previously with spontaneous grooming behavior, highlighting the complementary information to be gained from mutational analyses and naturally occurring variation due to common alleles. However, many of the candidate genes identified in this study are plausible given the trait assessed, and some are components of pathways implicated previously in grooming behavior. For example, glutamate is the primary excitatory neurotransmitter at the neuromuscular junction in insects . Gdh, whNf1 is known to affect grooming behavior; Nf1 genetically interacts with CCKLR-17D1, one of the candidate genes from the GWA analysis  and Epidermal growth factor  pathway signaling (Table S3).The candidate genes associated with grooming behavior span a wide variety of gene ontology categories, suggesting novel genetic contributions to this complex behavior. Many of the genes are involved in nervous system development and/or function in organism or tissue development  (Table S3). However, transcriptional regulation and post-transcriptional modification are also implicated. Finally, the Notch and Epidermal growth factor signaling pathways are implicated directly by the inclusion of CG13229, CG34401, Gdh, GEFmeso, kirre, Lrch, mtt, osp, PGRP-LA, sty, Trf2) affect spontaneous grooming behavior. However, the outcome of RNAi depends on many factors. RNAi can have off-target effects on assessed phenotypes , and ubiquitous RNAi affects all development times and tissues/cell types in which the target gene is expressed. It is possible that utilizing neuronal, glial or more specific GAL4 drivers, or GAL4 drivers with expression restricted to particular developmental stages, may have implicated more candidate genes, and future studies are needed to assess in which tissues/cell types and stages of development knockdown of expression affects the phenotype. Furthermore, RNAi cannot capture the potentially subtle effects of the naturally occurring variants; future variant-specific tests are needed for functional confirmation.Functional assessment of 29 candidate genes using RNAi showed that 11 (Grooming is an important fitness trait in insects . Further