Colibacter massiliensis gen. nov. sp. nov., a novel Gram-stain-positive anaerobic diplococcal bacterium, isolated from the human left colon
Authors: Morgane Mailhe
Pierre-Edouard Fournier
May Khoder
Rita Abou Abdallah
Davide Ricaboni
Didier Raoult
Hussein Anani
Anthony Fontanini
Journal: Scientific Reports (2019)
Discipline: Multidisciplinary
Keywords: gut microbiota; microbial diversity; taxonogenomic strategy; bacterium; strain Marseille-P2911
Affiliations: Aix Marseille Univ, Institut de Recherche pour le Développement (IRD), Service de Santé des Armées, AP-HM, UMR Vecteurs Infections Tropicales et Méditeranéennes (VITROME), Marseille, France., Institut Hospitalo-Universitaire Méditerranée-Infection, Marseille, France., Aix-Marseille Université, Institut de Recherche pour le Développement (IRD), UMR Microbes Evolution Phylogeny and Infections (MEPHI), Marseille, France., Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia., Aix Marseille Univ, Institut de Recherche pour le Développement (IRD), Service de Santé des Armées, AP-HM, UMR Vecteurs Infections Tropicales et Méditeranéennes (VITROME), Marseille, France. ., Institut Hospitalo-Universitaire Méditerranée-Infection, Marseille, France. .

Abstract:  The gut microbiota is considered to play a key role in human health. As a consequence, deciphering its microbial diversity is mandatory. A polyphasic taxonogenomic strategy based on the combination of phenotypic and genomic analyses was used to characterize a new bacterium, strain Marseille-P2911. This strain was isolated from a left colon sample of a 60-year old man who underwent a colonoscopy for an etiological investigation of iron-deficiency anemia in Marseille, France. On the basis of 16S rRNA sequence comparison, the closest phylogenetic neighbor was Anaeroglobus geminatus (94.59% 16S rRNA gene sequence similarity) within the family Veillonellaceae. Cells were anaerobic, Gram-stain-positive, non-spore-forming, catalase/oxidase negative cocci grouped in pairs. The bacterium was able to grow at 37 °C after 2 days of incubation. Strain Marseille-P2911 exhibited a genome size of 1,715,864-bp with a 50.2% G + C content, and digital DNA-DNA hybridization (dDDH) and OrthoANI values with A. geminatus of only 19.1 ± 4.5% and 74.42%, respectively. The latter value being lower than the threshold for genus delineation (80.5%), we propose the creation of the new genus Colibacter gen. nov., with strain Marseille-P2911T (=DSM 103304 = CSUR P2911) being the type strain of the new species Colibacter massiliensis gen. nov., sp. nov. 

Retracted: No

PubPeer comments:
 **In Materials and Methods section:** **1. u201cThe eligibility criteria were as follows: (1) patients with probable or confirmed EGFR-mutant LM from NSCLC according to ESMO-EANO guidelines; (2) aged 18 to 75 years; (3) multiline treatment failure(u22653), including systemic chemotherapy, tyrosine kinase inhibitor (TKI) treatment, intrathecal chemotherapy with MTX and cytarabine, or whole-brain radiation therapy.u201d** The topic of this study is efficacy and safety of intrathecal pemetrexed combined with dexamethasone for treating tyrosine kinase inhibitor-failed leptomeningeal metastases from EGFR-mutant NSCLC. However, failed TKI treatment was not necessary to enroll into this study. The inclusion criteria were leptomeningeal metastases from EGFR-mutant NSCLC, aged 18-75 years old, who had failed multiple lines of treatment in the past, including systemic chemotherapy, TKI treatment, intrathecal MTX and cytarabine, or radiotherapy. Any 3 or more treatments? If patients who had failed systemic chemotherapy, failed intrathecal chemotherapy, and failed radiotherapy were enrolled in the group, it may not be consistent with the subject of the study. In addition, what was the TKI-failed leptomeningeal metastases from EGFR-mutant NSCLC in the title? Did it refer to the failed leptomeningeal metastasis disease or the failed systemic disease? Or both? **2.u201cThere is no consensus on optimal dose, frequency of administration, or optimal duration of pemetrexed for intrathecal pharmacotherapy.u201d** Pan et al. had published a phase 1 dose-escalation study (Ref. 25) on intrathecal pemetrexed for human being in August 2019. In that study, a recommended dose of 10 mg for a single dose, and the regimen of 1-2 times a week for 4-6 weeks were recommended based on pharmacokinetic studies. However, the authors in this study did not analyze any previous related studies. Instead, they referred to the schedule of intrathecal MTX. Surprisingly, the researchers designed the treatment plan did not based on the administration of intrathecal MTX or the previous study of rat intrathecal pemetrexed model establishing (Ref. 9). Regardless of the pharmacokinetic characteristics, the schedule was twice in the first week, once per 3 weeks, than once monthly. It is necessary for researchers to explain what theories or previous researches are the basis of the schedule in this study. **3. u201cTaking a value between 10 and 20 as the safety coefficient, the optimal starting dose of pemetrexed between 5 and 10 mg for human studies is reasonable. To achieve a good prognosis, we chose 15 mg as the starting dose.u201d** In reference 9, based on the study of animal pharmacokinetic models, an optimal starting dose of pemetrexed (5u201310 mg) was established for human studies. How could the researchers justify starting with 15 mg u201cto achieve a good prognosis  ? What is the objective of a phase 1 dose-escalation study? **4. u201cAn accelerated titration design was used in phase 1 of dose escalation[11]u201d** In a dose-escalation study, the drug dose should be steadily escalated within a certain range to observe the tolerability and safety. In the previous rat model research on intrathecal pemetrexed, the optimal starting dose of pemetrexed was only 5u201310mg for human studies. Furthermore, in previous phase 1 dose-escalation study (Ref.25), lethal hematological toxicity was observed in 15 mg group. In this study, the dose of pemetrexed started at 15 mg, followed by a large span dose of 30-50-80 mg escalation. What is the basis of this research design? It is suggested that the authors should declare the research plan and ethical review report. **5. u201cA single-patient cohort per dose level was enrolled in the accelerated dose-escalation phase, until one dose-limiting toxicity (DLT) was observed during one dose level or until dose level 4 was reached. u201d** The classical drug dose-escalation study protocol is 0 3, 1 6 model. A minimum of three patients and a maximum of six were enrolled in each cohort. It is calculated by medical statisticians on account of statistical model construction. The enrollment of a single patient cohort per dose level was improper. Because of the difference of individual tolerance, great bias can not be avoided. What is the evidence for only one patient distributed to a dose level in a dose-escalation study? Is there any publication applying this protocol? **6. u201cThe following treatment-related AEs were defined as DLTs: grade greater than or equal to 3 neurotoxicity, grade greater than or equal to 3 leukoencephalopathyu201d** Leukoencephalopathy is a radiographic findings caused by neuronal demyelination. It refers to a pathologic condition that affects the white matter of the brain. First of all, it is neither a specific complication related to intrathecal chemotherapy, nor a common indicator for the toxicity of intrathecal chemotherapy evaluation. Secondly, it is usually a chronic imaging change, which cannot be used as a toxicity evaluation standard in a dose escalation study. **7. u201c...and grade 4 for more than 7 days myelosuppression.u201d** What was grade 4 for more than 7 days myelosuppression? Was symptomatic treatment given to patients with grade 4 bone marrow suppression? Or no symptomatic treatment was given after grade 4 myelosuppression, only observation? **8. u201cA total of 131 doses of IP were given to the patients, with a median of 4.4 (2u201312) doses.u201d** A total of 131 doses of IP were given to the patients, with a median of 4.4 (2u201312) doses. Was it pre-designed or the actual treatment result? If it is the actual treatment result, this should be stated in the result section. Does  with a median of represent the median number? The u201cmedianu201d is not an u201caverageu201d number, and it should not be a number such as 4.4. **9. u201cPemetrexed (0.2 g, Hauson Pharma) was administered by intrathecal injection by means of lumbar puncture.u201d** Hauson Pharma, this pharmaceutical factory cannot be retrieved on the Internet. The researchers should give more information about the pharmaceuticals. Importantly, the pemetrexed disodium developed and produced by Eli Lilly is still in the patent protection period (until 2022) in the world. **10. u201cRecently, a study revealed that the RANO criteria are not useful, neither for current clinical practice nor for clinical trials.[15]u201d** Ref.15 did not state that the RANO criteria were not suitable for LM response evaluation, but that the imaging evaluation methods in RANO criteria were not suitable for clinical research applications. **11. u201cin the phase 2 cohort, we used the criteria for the evaluation of clinical response established in a previous study on the basis of improvement of neurologic symptoms or signs and changes in the Karnofsky performance status score (KPS) (Supplementary Table 1).[16]u201d** All patients included in Ref.16 had adverse prognostic factors. The clinical response evaluation criteria given by Ref.16 were applicable to the patients with LM-related neurological dysfunction or neurological symptoms, and may not be suitable for all patients in this study. **In Result section:** **12. u201cBetween January 12, 2018, and November 30, 2019, a total of 30 patients were enrolled in the phase 2 cohortu201d** This study was registered at the China Clinical Research Center on June 12, 2018. The research was pre-registered (ChiCTR1800016615). Study execute time was July 1, 2018. In this registration information, it was a phase 1 study. **13. u201cA total of 15 participants (50%) had positive CSF cytology result for the first time, and six more patients were positive for the second or third subsequent time.u201d** In table 1 and discussion section, 15 patients have positive CSF cytology. But here it showed 15+6 patients have positive CSF cytology. **14. u201ca total of 19 patients were MRI positive and 11 patients were negative.u201d** Of 30 patients with leptomeningeal metastasis from lung adenocarcinoma, 11 had negative neuroimaging finding, and the negative rate was 37%. This data obviously deviates from the common clinical characteristics. **15. u201cFour patients had a survival time of less than 4 weeks and could not be evaluated for efficacy.u201d** The survival time of the 4 patients was less than 4 weeks, which should not be the reason for failure to evaluate the effectiveness of the treatment. In Ref.6, there is no 4 weeks time interval for response evaluation. **16. u201cThese four patients were very ill with abnormal consciousness or behavior and could not undergo MRI scanning.u201d** This study lacks information about neuroimaging examination and findings. **17. u201cno significant difference in the clinical response rate was observed between IP plus EGFR TKI and IP alone (87.5% versus 80.0%, p=0.62)u201d** A statement on other antitumor therapy during the study is missing in the methods section. In this study, did patients receive any other antitumor therapy during the course of intrathecal chemotherapy with pemetrexed? Systemic chemotherapy, targeted therapy, immunotherapy, radiotherapy or other anti-tumor therapies? All treatment should also be described in the results section. In addition, the patients enrolled in this study were with failed TKI treatment. Why did these patients still received TKI? **18. Safety and Toxicity** There are obvious differences in toxicity and side effects compared with previously reported phase 1 clinical study on intrathecal pemetrexed (Ref.25), and a subsequent phase 1 2 clinical study on intrathecal pemetrexed (Pan, et al. Intrathecal pemetrexed combined with involved-field radiotherapy as a first-line intra-CSF therapy for leptomeningeal metastases from solid tumors: a phase I II study. Ther Adv Med Oncol. 2020; 12). In particular, no case with elevation of hepatic aminotransferases was observed. In two previous studies, the incidence of elevation of hepatic aminotransferases in 10-15 mg pemetrexed intrathecal chemotherapy was 15% and 29%; the incidence of grade 3 elevated transaminase was 8% and 9%, respectively. Elevation of hepatic aminotransferases is the most common toxicity besides bone marrow suppression for systemic administration of pemetrexed, with grade 1-2 elevated transaminases occurring in about 60-70% cases, and grade 3 elevated transaminases in about 12% cases. In this study, there were no patients with elevation of hepatic aminotransferases under 50 mg pemetrexed intrathecal administration. In this study, chemical meningitis was not observed. However, the symptoms and signs of headache, nausea and vomiting which conformed to the clinical manifestations of chemical meningitis were documented. Additionally, because of lacking cerebrospinal fluid biochemical examination and monitoring in this study, it was hard to make a accurate and objective judgment on chemical meningitis. **In Discussion section:** **19. u201cThis study is the first single-arm, prospective, openlabel, phase 1 2 clinical trial of IP for treating patients with LM from NSCLC worldwide.u201d** A phase 1 and a phase 1 2 studies on intrathecal pemetrexed for leptomeningeal metastasis had been reported in August, 2019 and May, 2020, respectively. This study is not the first single-arm, prospective, openlabel, phase 1 2 clinical trial of IP. **20. u201cUnlike many other phase 1 trials that use the traditional 3 plus 3 titration model, we chose an accelerated titration design in the phase 1 study. This was due to the specificity of this clinical trial, as the incidence of LM is relatively low in NSCLC (3%u20135%).u201d** Owing to the small number of patients with leptomeningeal metastases, the classical dose-escalation research protocol was changed, and researchers reduced 3 patients at each dose level to 1 patient. Such statement is indeed unconvincing. **21. u201cThe primary end point of clinical response rate in this trial was 84.6%, significantly higher than the clinical response rate for conventional intrathecal MTX, cytarabine, and thiotepa in patients with NSCLC-LM (53%u201379%).u201d** In the discussion section, the primary endpoint of the study was stated for the first time. First, for phase 1 studies, the feasibility and tolerance of the intervention drug are often the primary objective, not treatment response rate. Therefore, safety and tolerated dose are often the primary endpoints. Furthermore, in this statement, the relevant comparison of treatment response rate compared with other previous studies is not tenable, because the evaluation criteria of treatment response adopted in previous studies are not consistent with this study. **22. u201cThe response rate was 87.0% (20 of 23)u201d** What is the meaning of 20 of 23? **23. u201cIn our study, only 15 patients (50%) met this accepted standard owing to the low sensitivity of CSF cytology examination under...u201d** In this study, 15 patients were diagnosed as lemptomeningeal metastasis with positive CSF cytology. Another 15 patients with neurological symptoms and positive image findings were diagnosed as probable lemptomeningeal metastasis. In the result section, there were 19 patients with positive image findings. In other words, only 4 of the 30 patients had both positive CSF cytology and positive image findings. This ratio does not conform to the common clinical characteristics of leptomeningeal metastasis. The study has major problems in target drugs, research programs, medical ethics, and research results. , 
 **Third-party medical writing assistance, under the direction of the authors, was funded by AstraZeneca.** What were the benefits of AstraZeneca to this study? Why did AstraZeneca fund the study? , 
 There are other puzzling issues in the Figure 2. Does the horizontal axis  time of diagnoses 0 represent the time when LM was diagnosed? What is the treatment method represented by orange for No. 2,5,7,10,14, and 16 patients? We could not find the corresponding legend of orange. There were other potential incongruences between the figure 2 and the table 1 and the text. In the Figure 2, there were 7 patients and 24 patients who received previous WBRT and previous EGFR TKI-3rd respectively, which were not consistent with the numbers shown in the Table 1 (5 and 25 patients respectively). Whatu2019s even more unconvincing is that in the u201cPatient Characteristicsu201d of result section, the authors stated that u201cEight patients had received whole-brain radiotherapy treatment previouslyu201d. So which one is the real data? ,