Antibiotic resistance mechanisms of Myroides sp.

Antibiotic resistance mechanisms of Myroides sp. 芳香黄杆菌的耐药机制 1 first-author 2 3 1 1 1 4 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:mxwang@hqu.edu.cnmds6430@126.com †E-mail:mxwang@hqu.edu.cnmds6430@126.com mxwang@hqu.edu.cn 5 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:mxwang@hqu.edu.cnmds6430@126.com †E-mail:mxwang@hqu.edu.cnmds6430@126.com mxwang@hqu.edu.cn Yun Leung Laboratory for Molecular Diagnostics, School of Biomedical Sciences and Institute of Molecular Medicine, Huaqiao University Engineering Research Center of Molecular Medicine, Ministry of Education, Xiamen 361021, China Yun Leung Laboratory for Molecular Diagnostics, School of Biomedical Sciences and Institute of Molecular Medicine, Huaqiao University Engineering Research Center of Molecular Medicine, Ministry of Education, Xiamen 361021, China Department of Neurosurgery, Linyi People’s Hospital, Linyi 276000, China Department of Neurosurgery, Linyi People’s Hospital, Linyi 276000, China Linyi Health School of Shandong Province, Linyi 276000, China Linyi Health School of Shandong Province, Linyi 276000, China Institute of Nanomedicine, Department of Medical Laboratory, Weifang Medical College, Weifang 261053, China Institute of Nanomedicine, Department of Medical Laboratory, Weifang Medical College, Weifang 261053, China Department of Clinical Laboratory, Quanzhou First Hospital Affiliated to Fujian Medical University, Quanzhou 362000, China Department of Clinical Laboratory, Quanzhou First Hospital Affiliated to Fujian Medical University, Quanzhou 362000, China Bacteria of the genus Myroides ( Myroides spp.) are rare opportunistic pathogens. Myroides sp. infections have been reported mainly in China. Myroides sp. is highly resistant to most available antibiotics, but the resistance mechanisms are not fully elucidated. Current strain identification methods based on biochemical traits are unable to identify strains accurately at the species level. While 16S ribosomal RNA (rRNA) gene sequencing can accurately achieve this, it fails to give information on the status and mechanisms of antibiotic resistance, because the 16S rRNA sequence contains no information on resistance genes, resistance islands or enzymes. We hypothesized that obtaining the whole genome sequence of Myroides sp., using next generation sequencing methods, would help to clarify the mechanisms of pathogenesis and antibiotic resistance, and guide antibiotic selection to treat Myroides sp. infections. As Myroides sp. can survive in hospitals and the environment, there is a risk of nosocomial infections and pandemics. For better management of Myroides sp. infections, it is imperative to apply next generation sequencing technologies to clarify the antibiotic resistance mechanisms in these bacteria. 芳香黄杆菌广泛耐药，一旦感染很难治愈。其耐药机制尚不清楚，需要进一步研究。本文对国内外的芳香黄杆菌病例进行全面总结，分析该细菌的抗生素耐药情况、耐药机制、病人预后、医院内爆发感染和流行的风险，以及目前实验室应用的诊断方法，指出全基因组测序应用于阐明其耐药机制的可行性和迫切性。 Myroides sp. Antibiotic resistance Identification methods 16S ribosomal RNA gene sequencing Next generation sequencing 芳香黄杆菌 抗生素耐药 鉴定方法 16S rRNA 基因测序 下一代测序 Introduction 1 The genus Myroides ( Myroides spp.) comprises yellow-pigmented, non-motile, Gram-negative, rod-like bacteria (Holmes et al., 1977 1977 ; Cho et al., 2011 2011 ) that release a fruity odor during growth (Holmes et al., 1977 1977 ). The first strain, Stutzer, of the genus Myroides was isolated from the stools of patients with intestinal infections (Holmes et al., 1977 1977 ) and was assigned the species name Flavobacterium odoratum (Stutzer and Kwaschnina, 1929 1929 ). For easier clinical recognition, the bacteriological features, pigmentation, biochemical characteristics, and antimicrobial profiles of 10 isolates were examined (Holmes et al., 1977 1977 ). Myroides spp. were found to be non-fermentative organisms resistant to many antibiotics (Holmes et al., 1977 1977 ). In 1996, after extensive polyphasic taxonomic analysis of 19 strains of F. odoratum , the genus Myroides was established and included two species, M. odoratus and M. odoratimimus (Vancanneyt et al., 1996 1996 ). Later, more strains were isolated from forest soil (strain TH-19(T), named M. xuanwuensis sp. nov. (Zhang et al., 2014 2014 )), seawater (strain JS-08(T), named M. marinus sp. nov. (Cho et al., 2011 2011 ), strain SM1(T), named M. pelagicus sp. nov. (Yoon et al., 2006 2006 )), deep-sea sediment (strain D25T (Zhang et al., 2008 2008 )), human saliva (strain MY15T, named M. phaeus sp. nov. (Yan et al., 2012 2012 )), as well as strains from urine, sputum, surgical exudate (Andreoni, 1986 1986 ), and patients’ matter (Table 1 1 ). Thus, Myroides spp. are widely distributed in nature (Mammeri et al., 2002 2002 ; Ktari et al., 2012 2012 ; Suganthi et al., 2013 2013 ; Ravindran et al., 2015 2015 ). M: male; F: female; N/A: not applicable; ND: not described; CSF: cerebrospinal fluid; iv: intravenous injection Patient No. Age (year)/gender Underlying diseases or reasons Site of isolation Antibiotic resistance status Treatment strategy Outcome Reference 1 87/M Trauma, old age Wound Resistant to all antibiotics except ciprofloxacin. Sensitive to trimethoprim-sulfamethoxazole iv ciprofloxad Favorable Sun and Zhang, 2006 2006 2 34/F Hydatid cyst of liver Drainage Resistant to neomycin, streptomycin, gentamicin, ampicillin, tobramycin. Sensitive to norfloxacin Norfloxacin Favorable An, 1992 1992 3 2/M Young age CSF Resistant to cefazolin, penicillin, chloramphenicol. Sensitive to ampicillin, polymyxin, kanamycin, erythromycin, neomycin ND ND Shi and Zhou, 1993 1993 4 71/M Chronic bronchitis, old age Sputum Resistant to meropenem, imipenem, ampicillin, cefradine, tobramycin, cephalothin, amoxicillin-clavulanic acid, ampicillin-shubatan, ceftriaxone, gentamicin, cefoxitin, ceftazidime, piperacillin. Sensitive to cefepime, levofloxacin ND ND Guo and Liu, 2011 2011 5 30/F Burn Blood, central venous catheter, urine Sensitive to amikcin, norfloxacin Amikacin Favorable Wu, 1998 1998 6 28/F Injury and surgery Wound Resistant to gentamicin, sulfamethoxazole, ciprofloxacin, cefoperazone-sulbactam, tetracycline, tobramycin, cefoperazone, cefepime, imipenem, piperacillin-tazobactam, cefoselis, amikacin, piperacillin, levofloxacin, netilmicin, ceftazidime, cefotaxime, aztreonam, ampicillin-sulbactam. Sensitive to minocycline. Moderately sensitive to meropenem Debridement, skin transplantation, iv cefperazone-sulbactam and oral minocycline for 3 d, then oral minocycline for another 3 d Cured Hu et al., 2013 2013 7 76/M Chronic obstructive pulmonary disease and heart failure, old age Blood, wound Resistant to piperacillin, ceftazidime, ceftriaxone, cefepime, aztreonam, imipenem, meropenem, amikacin, gentamicin, ciprofloxacin, levofloxacin, tetracycline, trimethoprim-sulfamethoxazole, ampicillin-sulbactam, cefoperazone-sulbactam, piperacillin-tazobactam Oral minocycline for 9 d Cured Huang et al., 2014 8 4/M None Blood Resistant to ampicillin, ampicillin-sulbactam, piperacillin, piperacillin tazobactam, aztreonam, cefazolin, cefoxitin, ceftazidime, cefotaxime, azole cefepime, ceftazidime, ceftriaxone, cefepime Piperacillin and tobramycin for 14 d Cured Huang and Lin, 2003 2003 9 58/ND Diabetes mellitus complicated by heel bursitis Drainage Sensitive to cefoperazone and amikacin Incision and drainage, cefoperazone and amikacin for several days (more than 3 d) Cured Yang and Wang, 2001 2001 10 28/F None Pus Resistant to kanamycin, penicillin, erythromycin. Sensitive to ceftriaxone, norfloxacin, trimethoprim-sulfamethoxazole Abscess incision drainage and norfloxacin and trimethoprim-sulfamethoxazole Cured Song et al., 1995 11 24 d/M Neonate Blood Resistant to penicillin, chloramphenicol, carbenicillin, streptomycin, cefazolin. Sensitive to amikacin, erythromycin, ampicillin, benzylpencilline Ampicillin, and oxacillin for 19 d Cured Wang and Su, 1992 1992 12 11 d/M Preterm birth Blood, CSF Resistant to ampicillin, cefazolin, gentamicin, cefoperazone, cefotaxime, cefatrizine, ceftazidime. Sensitive to amikacin, piperacillin, ampicillin, sulbactam-cefoperazone Antimicrobial treatment for 5 d (the antibiotic was not described) Failed Zhang and Zhang, 1996 1996 13 69/F Lung cancer and surgery, old age Pleural effusion and sputum Resistant to tobramycin, gentamicin, ampicillin, erythromycin, clindamycin, tetracycline. Sensitive to amikacin, tobramycin, ceftriaxone Antimicrobial treatment, but not described in detail Died Song, 2005 2005 14 10 months/F Child Blood Sensitive gentamicin, tobramycin, cephalexin, sulbactam-cefoperazone, ceftriaxone Cefoperazone, tobramycin for 10 d Cured Zhao, 2000 2000 15 60/M Common bile duct stones Blood, bile, peritoneal effusion Resistant to tobramycin. Sensitive to piperacillin, cefoperazone, amikacin, gentamicin, ceftriaxone, cefotaxime Amikacin and cefoperazone Cured Meng et al., 1999 16 44/F None Blood, bone marrow Sensitive to norfloxacin, ciprofloxacin, cefazolin, amikacin, ceftazidime ND ND Geng et al., 2000 2000 17 67/M Old age Sputum (this strain was isolated with Serratia marcescens , Acinetobacter lwoffi ) Resistant to ampicillin, piperacillin cefazolin, cefuroxime, cefotaxime, ceftazidime, cefotaxime, aztreonam, gentamicin, norfloxacin, trimethoprim-sulfamethoxazole Antimicrobial treatment, but not described in detail Cured Liu and He, 2001 2001 18 45/M None Urine Resistant to ampicillin, amikacin, azithromycin Application of cefoperazone, cefotaxime, nitrofurantoin, and tobramycin for 3 weeks Cured Wuer et al., 2000 19 N/A ND Blood, sputum, bile, cerebrospinal fluid, urine, all these three isolates were from patients (no further details were available) All 12 isolates were resistant to erythromycin, penicillin, streptomycin, ampicillin, oxacillin, piperacillin, carbenicillin N/A N/A Li and Zhao, 1995 1995 20 ND Chronic nephritis 3 Urine 3 Two isolates were resistant to meropenem. All three isolates were resistant to ampicillin-sulbactam, piperacillin-tazobactam, cefuroxime, cefotetan, ceftriaxone, aztreonam, gentamicin, ciprofloxacin, levofloxacin, ampicillin, piperacillin, cefazolin, cefuroxime axetil, ceftazidime, cefepime, imipenem, amikacin, tobramycin, levofloxacin, trimethoprim-sulfamethoxazole 3 One isolate was sensitive to meropenem 3 ND 3 Li et al., 2010 2010 21 ND Diabetes mellitus 22 ND/F Cervical cancer 23 61/F Coma, cerebral hemorrhage Sputum N/A Ceftazidine, chloramphenicol, penicillin G, gentamicin by atomization inhalation, ketoconazole by nasal feeding Died Jin and Xiao, 1995 1995 24 49/M Chronic alcohol misuse Blood Intermediately sensitive to imipenem Treatment with amoxicillin-clavulanic acid was changed to ciprofloxacin, imipenem-cilastatin used for 10 d, then oral ciprofloxacin for 21 d Cured Bachmeyer et al., 2007 2007 25 55/F Liver cirrhosis bilateral lower extremity cellulitis and open wounds Blood, wound Resistant to amikacin, gentamicin, tobramycin, aztreonam, ceftriaxone, ciprofloxacin, tetracycline, trimethoprim-sulfamethoxazole, vancomycin. Intermediately sensitive to piperacillin-tazobactam, cefepime, imipenem, and cilastatin iv vancomycin, piperacillin-tazobactam, and levofloxacin for 18 h, then iv imipenem-cilastatin, daptomycin, clindamycin, then imipenem-cilastatin and doxycycline Died Crum-Cianflone et al., 2014 26 13/M Soft tissue infection Pus Resistant to piperacillin-tazobactam, aztreonamaminoglycosides. Intermediately susceptible to imipenem. Sensitive to all quinolones tested, cotrimoxazole, chloramphenicol, and amoxicillin-clavulanic acid Drainage of osteolytic lesions combined with iv ciprofloxacin for 10 d and continued with oral ciprofloxacin for an additional 10 d Cured Maraki et al., 2012 27 48/F Cystitis (contaminated) Urine 9 Fully resistant to streptomycin, gentamicin, kanamycin, ampicillin, carbenicillin, tetracycline, polymyxin B. Fully resistant or moderately resistant to sulfamethoxazole, trimethoprim-sulfamethoxazole, cephaloridine, erythromycin, chloramphenicol. Moderately sensitive to nalidixic acid 9 N/A 9 N/A 9 Holmes et al., 1977 28 34/M Infected cut finger Wound 29 59/F ND Urine 30 ND/ND Urinary retention Urine 31 ND/ND Further details are not available Urine 32 ND/ND Varicose ulcer Wound 33 76/F Leg ulcer Ulcer 34 67/F Breast lump Urine 35 48/M Chronic renal insufficiency Urine 36 66/M Urinary tract infection Urine 7 Resistant to all β-lactam and non-β-lactam antibiotics tested, including imipenem, vancomycin, ciprofloxacin, chloramphenicol, tigecycline, rifampicin Imipenem, colistin Failure 7 Ktari et al., 2012 2012 37 44/M 3 Bladder colonization Urine No treatment Favorable 38 44/M Urine No treatment Favorable 39 47/M Urine No treatment Favorable 40 77/M 3 Urinary tract infection Urine Ifampicinþ ciprofloxacin Cured 41 65/M Urine Ifampicinþ ciprofloxacin Cured 42 80/M Urine Ifampicinþ ciprofloxacin Cured 43 59/M Urinary tract infection Urine Resistant to amikacin, gentamicin, imipenem, meropenem, cefazolin, ceftazidime, cefotaxime, cefepime, aztreonam, ampicilllin, piperacillin, amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam, colistin, trimethoprim-sulfamethoxazole, chloramphenicol, ciprofloxacin, levofloxacin, moxifloxacin, tetracycline Levofloxacin was used only temporarily and orally Failure Our case Myroides sp. is a rare opportunistic pathogen (Schröttner et al., 2014 2014 ). Nevertheless, management of Myroides sp. infection is troublesome due to its high resistance to most antibiotics (as summarized in Table 1 1 ). For accurate strain identification of Myroides sp., current diagnostic methods, such as the Vitek Jr. system (Vitek Systems, bioMerieux) (Spanik et al., 1998 1998 ), are based on bacteriological and biochemical characteristics, and can determine Myroides sp. at the species level in most cases. However, they and 16S ribosomal RNA gene sequencing (16S rRNA sequencing), a standardized bacterial strain identification method (Yoon et al., 2006 2006 ; Zhang X.Y. et al., 2008 2008 ; Zhang Z.D. et al., 2014 2014 ) still not widely applied in Chinese hospitals, fail to provide any information on the status and mechanisms of antibiotic resistance in Myroides sp. Whole genome sequencing technologies could address these questions, and should be applied to Myroides sp. promptly. Antibiotic resistance status of clinical Myroides sp. infections 1 Myroides sp. infections are rare. By searching the PubMed database of English literature using “ Myroides ” or “ Flavobacterium odoratum ” as key words, only a few reports could be found. In immunocompetent people, primary infections by Myroides sp. have been rarely reported, such as a case of M. odoratimimus cellulitis resulting from a pig bite in an immunocompetent child (Maraki et al., 2012 2012 ). However, secondary infections can frequently arise when human immunity is impaired, such as post catheterization (Holmes et al., 1977 1977 ; Spanik et al., 1998 1998 ), in patients with cancer (Holmes et al., 1977 1977 ; Spanik et al., 1998 1998 ; Song, 2005 2005 ) or diabetes mellitus (Yang and Wang, 2001 2001 ), and in neonates (Wang and Su, 1992 1992 ; Zhang and Zhang, 1996 1996 ; Zhao, 2000 2000 ). Myroides sp. can cause soft tissue infection (Benedetti et al., 2011 2011 ), cellulitis (Bachmeyer et al., 2007 2007 ), necrotizing fasciitis (Crum-Cianflone et al., 2014 2014 ), ventriculitis (Macfarlane et al., 1985 1985 ), and urinary tract infections (Yağci et al., 2000 2000 ). M. odoratimimus even caused an outbreak of urinary tract infection in a hospital (Ktari et al., 2012 2012 ). By using the same key words to search the China National Knowledge Infrastructure (CNKI) database, we found that most reports of Myroides or F. odoratum infections contained a single case (Table 1 1 ). Two papers reported 23 (Table 2 2 ) and 11 strains (Table 3 3 ), respectively. Translated from Lan and Bao ( 2009 2009 ) with permission of the authors. K-B method: Kirby-Bauer disk diffusion method; CSF: cerebrospinal fluid; R : resistant; I : immediately sensitive; S : sensitive Site of isolation (total samples of positive isolation) Antibiotic R I S Sputum (8); Amikacin 5 10 8 Urine (6) Cefazolin 11 9 3 Blood (4); Cefoperazone 9 9 5 CSF (3); Sulfamethoxazole 4 10 9 Bile (2) Sulfadiazine 5 7 11 Ceftazidime 10 6 7 Erythrocin 3 10 10 Azithromycin 4 9 10 Translated from Chen et al. ( 2009 2009 ) with permission of Chin. J. Pract. Med. Tech . R : resistant; I : immediately sensitive; S : sensitive; WBC: white blood cell Patient information Antibiotic R I S 13 2–76 years (average 53 years), 9 males, 2 females. All patients suffered from urinary retention or urinary tract stones, but none of them had symptoms of urinary tract infection or other discomfort. In nine urinarily catheterized patients, the urinary culture when the catheter was in situ was Myroides sp. positive, but the urinary testing showed no WBC in these urinary samples, and pus cells were found in only three of them. The urinary culture of Myroides sp. became negative after removal of urinary catheter in these nine urinarily catheterized patients even though they were not treated. Ampicillin 11 0 0 Piperacillin 11 0 0 Cefuroxime 11 0 0 Cefoperazone-sulbactam 11 0 0 Ceftazidine 10 1 0 Cefepime 10 1 0 Aztreonam 11 0 0 Imipenem 11 0 0 Meropenem 11 0 0 Levofloxacin 8 3 0 Ciprofloxacin 9 2 0 Trimethoprim-sulphamethoxazole 0 0 11 Amikacin 11 0 0 From Tables 1 1 , 2 2 , and 3 3 , we conclude that Myroides spp. are resistant to broad antibiotics, and that their extensive antibiotic resistance has resulted in treatment failure and fatalities. We observed a case in July 2009 in which a patient presented with a post-injury urinary tract infection caused by M. odoratimimus strain PR63039 (Table 1 1 , our case). Using antibiotic sensitivity testing (AST), the strain was found to be resistant to ampicillin, amoxicillin, clavulanate, amikacin, aztreonam, chloramphenicol, cephalosporin, imipenem, gentamycin, levofloxacin, meropenem, shubatan, sulfamethoxazole, tetracycline, ciprofloxacin, and tazobactam. Even though many antibiotics, such as cefazolin oxime, amikacin, tetracycline, moxifloxacin, ciprofloxacin, and nitrofurantoin, were administered to the patient for 47 d, the infection was not cured. The reports analyzed in Table 1 1 reveal that the antibiotic resistance of Myroides sp. varies among strains isolated from different sources. For example, a strain from a patient suffering from a hydatid cyst of the liver was sensitive to norfloxacin (An, 1992 1992 ), but another strain isolated from pulmonary infection patient was reported to be resistant to norfloxacin (Liu and He, 2001 2001 ). Two strains isolated from patients with cellulitis and a leg amputation, respectively (Hu et al., 2013 2013 ; Crum-Cianflone et al., 2014 2014 ) were resistant to ciprofloxacin, while another two isolated from trauma and septicemia patients, respectively, were sensitive to ciprofloxacin (Geng et al., 2000 2000 ; Sun and Zhang, 2006 2006 ). Why is there so much variation in the antibiotic resistance profiles of Myroides sp. strains isolated from different sources In our opinion, the subtypes and genotypes of Myroides sp. might have a great influence on their sensitivity to certain antibiotics. Therefore, it is imperative to obtain accurate information on strain subtype and genotype. Preliminary opinions on the antibiotic resistance mechanisms of Myroides sp. 1 In China, there have been no reports on the antibiotic resistance mechanisms of Myroides sp. Although several foreign researchers have investigated this topic, very little information is available. Hummel et al. ( 2007 2007 ) showed that the β-lactamase gene was responsible for the variable patterns of resistance to β-lactam antibiotics and the decreased susceptibility to carbapenems of different Myroides sp. strains. In a study investigating a number of clinical cases involving systemic infections, Mammeri et al. ( 2002 2002 ) claimed that resistance to β-lactams was due to the production of the chromosome-encoded β-lactamases TUS-1 and MUS-1 in M. odoratus and M. odoratimimus . The β-lactamases produced by Gram-negative and Gram-positive bacteria play a vital role in resistance against β-lactam antibiotics. However, their study showed that the β-lactamases TUS-1 and MUS-1 could only partly explain the intrinsic resistance of Flavobacteriaceae species to β-lactams. Also, a common observation was that these Escherichia coli expressed metalloenzymes were much less resistant to β-lactam than those of primitive origin (Mammeri et al., 2002 2002 ). Even Flavobacteriaceae and Myroides sp. belong to the same family, the mechanism of resistance conferred by TUS-1 and MUS-1 in Flavobacteriaceae species cannot be assumed to operate in Myroides sp. Then, why does Flavobacteriaceae serve as a source for a variety of metalloenzymes As observed for other environmental species, this might be due to the combined biosynthesis of carbapenem derivatives and hydrolyzing β-lactamases (Mammeri et al., 2002 2002 ). Suganthi et al. ( 2013 2013 ) investigated whether the antibiotic sensitivity of plasmid-containing M. odoratimimus SKS05-GRD was correlated with the plasmid or was chromosomally-mediated. They revealed that resistance to kanamycin, amikacin, and gentamicin was plasmid-mediated, and that resistance to ampicillin, cefadroxil, cefoperazone, ceftazidine, ceftriaxone, and netillin was chromosomally-mediated. The Klebsiella pneumoniae carbapenemase (KPC) family is closely related to resistance to carbapenem in a variety of pathogens and the KPC gene is located in a plasmid. However, Myroides sp. WX2856, obtained from an abdominal abscess (Kuai et al., 2011 2011 ), harbored a KPC-2 carbapenemase, but the KPC-2 gene might not be located on a plasmid as in K. pneumoniae . Do these results suggest the possibility of interspecies transmission of the KPC-2 gene This aspect needs further investigation. On the other hand, there are several known features of antibiotic resistance mechanisms in bacteria from the same family of Myroides , such as in F . indologenes , now named Chryserobacterium indologenes (Tian and Wang, 2010 2010 ). First, resistance transfer factors (R-factors) in the cytoplasm determine the bacteria’s resistance to antibiotics. R-factor plasmids can carry and transfer a variety of resistance genes among bacteria. In addition, the thick outer membrane and its low permeability, resulting from multidirectional mutations, and the active discharge system of the bacterial cell membrane of C. indologenes confer inherent multi-drug resistance. The bacteria also produce a β-lactamase with a broad spectrum of β-lactam hydrolytic activity (Tian and Wang, 2010 2010 ). Thus, it is apparent that the antibiotic resistance mechanisms of Myroides sp. are unclear and deserve further investigation. Present diagnostic methods do not clarify the antibiotic resistance mechanisms of Myroides sp. 1 In clinical diagnostic laboratories, traditional bacterial identification methods primarily rely on testing biological traits and biochemical characteristics, and include microscopic inspection and metabolic testing of isolated and cultured bacteria. These methods have shown that Myroides spp. do not have flagella, release a fruity fragrance, are yellow, oxidase-positive, urea- and indole-negative, and are unable to oxidize sugar (Li and Zhao, 1995 1995 ; Chen et al., 2009 2009 ). However, the bacterial strain can be preliminarily identified only as Myroides sp., as further strain type designations cannot be determined using these traditional methods. Nearly all cases of Myroides sp. infections reported in China have used these traditional identification approaches, and did not describe any Myroides sp. subtypes. Using these traditional identification methods, the M. odoratimimus strain PR63039 isolated in our case was first identified as Pseudomonas putida , then as M. odoratimimus , and as the Acinetobacter calcoaceticus - baumannii complex, at different time points throughout the 47 d of the patient’s hospitalization. It was finally confirmed as M. odoratimimus by 16S rRNA sequencing. This case also indicates that these traditional identification methods may not correctly diagnose the strain type. Recently, other microorganism strain identification technologies have been developed, including VITEK 2 (bioMerieux VITEK-2, France), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), 16S rDNA sequencing, and another more frequently used nomenclature, 16S rRNA sequencing (Table 4 4 ). VITEK 2, a routine laboratory method, can help to discriminate among genera but not among species. MALDI-TOF MS and 16S rDNA sequencing/16S rRNA sequencing can be used to identify species and are more frequently used for research purposes (Lee et al., 2014 2014 ; Schröttner et al., 2014 2014 ). According to Schröttner et al. ( 2014 2014 ), the genus Myroides was reliably identified in tests of 22 isolates using VITEK 2. 16S rDNA sequencing further revealed that they shared ≥97% homology, enough for a reliable identification at the species level. Yoon et al. ( 2006 2006 ) applied 16S rRNA sequencing successfully to clarify the phylogenetic position of Myroides sp. strain SM1T, isolated from seawater in Thailand. Yoon et al., 2006 2006 ; Lee et al., 2014 2014 ; Schröttner et al., 2014 2014 Method Trait VITEK2 Only suitable to identify bacteria at the genus level, not at the species level MALDI-TOF MS Able to distinguish between M. odoratus and M. odoratimimus 16S rDNA sequencing/16S rRNA sequencing Able to distinguish microorganisms at the species level Whole genome sequencing Able to identify the microorganism and provide the bioinformatics of microorganism Whole genome sequencing is a feasible way to investigate the antibiotic resistance mechanisms of Myroides sp. 1 In the past decade, many next generation sequencing platforms have been developed, such as 454 invented in 2004 (Margulies et al., 2005 2005 ), Illumina Solexa in 2006 (Bentley, 2006 2006 ), SOLiD in 2007 (Chi, 2008 2008 ), Ion Torrent in 2011 (Rothberg et al., 2011 2011 ), and PacBio in 2012 (Koren et al., 2012 2012 ). This has led to a rapid increase in the sequencing of whole genomes of microorganisms, including eukarya, bacteria, archaea, and viruses. These sequences have been deposited in the National Center for Biotechnology Information (NCBI) RefSeq genome collection database ( http://www.ncbi.nlm.nih.gov/genome http://www.ncbi.nlm.nih.gov/genome ) (Tatusova et al., 2015 2015 ). In 2014, over 10 000 microbial genomes were released (Tatusova et al., 2015 2015 ). Along with the next generation platforms, whole-genome analysis of multi-drug resistance mechanisms has emerged. For example, A. baumannii is a common cause of fatal nosocomial infections because of its extensive antibiotic resistance. Genomic sequencing revealed comprehensive drug-resistance mechanisms, such as a 41.6-kb closely related antibiotic resistance island in the chromosome (Huang et al., 2012 2012 ), the horizontally transmittable carbapenem resistance gene ( bla OXA-23 ) containing a plasmid (in isolate MDR-TJ, with 454 Titanium) among different A. baumannii strains (Huang et al., 2012 2012 ; Lee et al., 2013 2013 ), a list of antimicrobial resistance-associated genes (with 454 and SOLiD) (Rolain et al., 2013 2013 ), a diversified resistance gene list (with Illumina Hiseq2000) (Tan et al., 2013 2013 ), and longitudinally evolved antibiotic resistance gene mutations and mutational pathways under pressure from the antibiotic colistin (with 454 Titanium) (Snitkin et al., 2013 2013 ). Since its invention by Rothberg et al. ( 2011 2011 ), Ion Torrent sequencing technology has been successfully applied to complete genomic sequencing, such as in Clostridium sp. BL8 (with Ion Torrent PGM™) (Marathe et al., 2014 2014 ), and clear characterization of drug-resistant genes, such as in Mycobacterium tuberculosis (Daum et al., 2014 2014 ). Results can be obtained within five days, comparable to the turnaround time required by current drug sensitivity testing (DST) (Daum et al., 2014 2014 ). PacBio single-molecule real-time technology has frequently been used to perform whole-genome sequencing of many microorganisms, such as Neisseria gonorrhea (with the PacBio RSII platform), a Gram-negative β proteobacterium responsible for the sexually transmitted infection gonorrhea (Abrams et al., 2015 2015 ). Yet, little genomic information about Myroides sp. is available. A brief description of the genomes of M. odoratus DSM 2801 and CIP 103059 was found in genome database of NCBI (Table 5 5 ), but this was not suitable for studying its drug-resistance mechanisms. Recently, the genome sequencing of the urethral catheter isolate Myroides sp. A21 was completed (Burghartz et al., 2015 2015 ). The sequence contained 3650 protein-coding sequences (CDSs), 136 RNA-coding genes, and eight copies of the rRNA gene cluster, of which three were resolved as a direct repeat of two rRNA gene clusters. The presence of 106 transfer RNAs (tRNAs) and six noncoding RNAs (ncRNAs) was also predicted. By comparing the genome sequence of Myroides sp. A21 with those of M. odoratimimus CCUG 10230 and M. odoratus DSM 2801, 293 unique CDSs were found in the A21 genome (Burghartz et al., 2015 2015 ). In addition, five genomic islands were predicted by Island Viewer analysis (Burghartz et al., 2015 2015 ). However, as the antibiotic treatment history was not described and the antibiotic resistance status of this strain was not given, the antibiotic resistance mechanisms could not be analyzed from the data. Cited from GenBank assembly accession: GCA_000243275.1 and GCA_000297875.1. INSDC: International Nucleotide Sequence Database Collaboration Strain Name RefSeq INSDC Size (Mb) Total number of genes Total number of proteins rRNA tRNA Other genes GC content (%) Pseudogenes CIP 103059 Master WGS NZ_AGZJ00000000.1 AGZJ00000000.1 4.23 3773 3631 10 67 1 35.8 64 DSM 2801 NZ_CM001437.1 CM001437.1 4.3 3838 3695 9 74 1 35.8 59 To study these aspects in our M. odoratimimus strain PR63039, we extracted its genomic DNA. Agarose gel electrophoresis results from several experiments revealed that it might harbor at least six different types of plasmids which could be related to antibiotic resistance (Fig. 1 1 ). However, the exact number of plasmid type needs further confirmation. Many bacterial drug-resistance genes are plasmid- or chromosome-mediated, but we did not know which mechanism was operating in Myroides sp. The genome of strain PR63039 was sequenced using an Ion Torrent Personal Genome Machine. We generated 610 contigs and 4221 open reading frames. The total sequence numbers with Gene Ontology (GO) and Clusters of Orthologous Groups (COG) of protein were 2741 and 1026, respectively. However, we could not completely assemble the genome and plasmids, so the multi-drug resistance mechanisms of strain PR63039 still could not be clarified. We are now using PacBio single-molecule real-time technology in the hope of generating a complete genome sequence of both the chromosome and plasmids, to elucidate the mechanisms of resistance and pathogenesis of this strain. The genomic DNA of PR63039 was separated by gel electrophoresis using 0.75% (7.5 g/ml) agarose. The genomic DNA was about 23 kb in size. M1: 1 kb DNA marker; M2: DNA marker-G; A: 0.173 μg DNA; B: 0.273 μg DNA; C: 0.328 μg DNA. The genomic DNA was about 23 kb in size. Six different types of plasmids (1–6) were visible All the infection reports from China indicated that Myroides sp. is a serious source of nosocomial infection and has the potential to cause a pandemic. The completion of the Myroides sp. genome sequence and detailed bioinformatics analysis are imperative for the understanding of its mechanisms of antibiotic resistance and pathogenesis. Discussion and outlook 1 Myroides sp. is an opportunistic and extensively antibiotic-resistant pathogen. Infections have not been widely reported, though there have been many cases in China. As the antibiotic resistance mechanisms of Myroides sp. are still unclear, and in view of the risk of nosocomial infection and pandemics, novel technologies, such as whole genome sequencing and further bioinformatic analyses, should be applied urgently to Myroides sp. An outline of a strategy for whole genome sequencing and bioinformatic analyses is presented in Fig. 2 2 . These analyses will also be helpful in developing appropriate management strategies. Moreover, whole genome sequencing might become a routine diagnosis method for all microbial infections in the near future. 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One case of children with Flavobacterium odoratum septicemia Compliance with ethics guidelines Shao-hua HU, Shu-xing YUAN, Hai QU, Tao JIANG, Ya-jun ZHOU, Ming-xi WANG, and De-song MING declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500068 Project supported by the Huaqiao University Graduate Student Scientific Research Innovation Ability Cultivation Plan Projects, the Major Program of Department of Science and Technology of Fujian Province (No. 2012Y4009), the Science and Technology Planning Project of Xiamen (No. 3502Z20123036), the Xiamen Southern Oceanographic Center (No. 14GYY008NF08), the Construction Project for Yun Leung Laboratory for Molecular Diagnostics (No. 14X30127), the Technology Planning Projects of Quanzhou Social Development Fields (No. 2014Z24), and the Major Support Research Project of National Key Colleges Construction of Quanzhou Medical College (No. 2013A13), China 2015-03-19 2016-02-15 2015-11-30 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 17