myroides species , previously named as flavobacterium odoratum , are aerobic gram - negative bacteria that can be found in environmental sources such as soil and water , but are not residents of the normal human microflora .
the genus myroides comprises two species , myroides odoratus and myroides odoratimimus . taking into consideration their role as causative agents in human disease , both m.  odoratimimus and m.  odoratus
are seen rarely as opportunistic pathogens , causing infections in severely immunocompromised patients and rarely , in immunocompetent hosts .
myroides odoratimimus strains have been found to be responsible for urinary tract infections , cellulitis   in immunocompromised patients , and also for septic shock , pneumonia and soft - tissue infections   in immunocompetent hosts .
in addition , three outbreaks of m.  odoratimimus , two of urinary tract infections and the third of central venous catheter - associated bloodstream infections due to contaminated water , have been reported .
antibiotic resistance patterns of myroides strains show variable susceptibility to -lactams , with a conserved decreased susceptibility to cephalosporins and carbapenems .
this decreased susceptibility is the result of the production of a chromosome - encoded -lactamase mus-1 .
this enzyme is a member of the subclass b1 of metallo--lactamases and is distantly related to other metalloenzymes , being most closely related to ind-1 from chryseobacterium indologenes ( 42% amino acid identity ) .
however , phylogenic analysis showed that mus-1 belongs to the same phylogenic lineage of subclass b1 enzymes that groups the subclass b1 -lactamases of flavobacterium species . here
we report a novel variant , mus-2 , of the chromosome - encoded -lactamase mus-1 from m.  odoratimimus isolated from livestock animals in lebanon .
three imipenem - resistant m.  odoratimimus strains ( 32 , 35a and 104b ) were isolated from two livestock farms of cows and pigeons by rectal swabs ( 32 : farm 1 , cow ; 35a : farm 1 , pigeon ; 104b : farm 2 , cow ) in january 2014 .
these strains were collected during a study that aimed to detect carbapenemase - encoding genes in animals in lebanon .
they were isolated on macconkey agar supplemented by ertapenem ( 1  mg / l ) , from alive and non - sick animals without contact with any other animals .
strains were sub - cultured on trypticase soy agar plates at 37c for 1824  h and identification was confirmed by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof / ms ) ( microflex ; bruker daltonics , bremen , germany ) with flexcontrol software ( bruker daltonics ) .
hinton agar by standard disc diffusion method as recommended by the european committee on antimicrobial susceptibility testing ( eucast ; www.eucast.org ) .
seventeen antibiotics were tested , including ticarcillin , ticarcillin - clavulanic acid , piperacillin , piperacillin - tazobactam , ceftazidime , cefotaxime , cefepime , aztreonam , amikacin , tobramycin , gentamicin , ciprofloxacin , rifampicin , ertapenem , meropenem , imipenem and colistin ( bio - rad , marnes - la - coquette , france ) .
the mic for imipenem was determined using the etest method ( biomrieux , la balmes les grottes , france ) and the result was interpreted according to the eucast breakpoint for enterobacteriaceae ( susceptible if mic    2  mg
/ l ) isolates were screened for carbapenemase production using the modified hodge test , the edta , the modified carbanp test as previously described [ 1113 ] and also by maldi - tof ms ultraflex assay .
screening for class a , b and d carbapenemases was carried out using pcr according to previous protocols and previously described primers including for blavim , blaimp , blandm-1 , blakpc and all the blaoxa variants [ 1520 ] .
in addition , amplification of the chromosomal mus gene was performed using primers designed in this study ( mus - f / r 5-cgttaggcacaccagaagaa-3'/3-tccacaccattctgagcgta-5 ) .
these primers were designed outside the region containing the blamus-1 located on the chromosome of a random m.  odoratimimus genome deposited in genbank .
pcr products were purified and sequenced using the big dye terminator chemistry on an abi 3730 automated sequencer ( applied biosystems , foster city , ca , usa ) .
the sequences obtained were analysed using blastn and blastp against the ncbi database ( www.ncbi.nlm.nih.gov ) .
the full length of the blamus-2 from m.  odoratimimus was amplified using herculase ii fusion enzyme with dntps combo ( agilent technologies , santa clara , ca , usa ) with primer mus_f / mus_r .
the gene was then cloned into the high - copy - number plasmid pcr - bluntii - topo using the zero blunt topo pcr cloning kit ( invitrogen , calrsbad , ca , usa ) .
the antibiotic resistance profile of the top10 e.  coli containing ptopo - mus vector was then determined .
the three isolates were typed by the pulsed - field gel electrophoresis ( pfge ) method as previously described .
the dna was digested with smai restriction enzyme and the fragments of dna were separated using a genepath system ( bio - rad ) .
during a previous study that was aiming to detect carbapenemases encoding genes in animals in livestock farms in lebanon , three isolates of m.  odoratimimus were isolated and were studied separately .
these isolates were obtained after culture of animal faeces in macconkey agar supplemented with ertapenem ( 1  mg / l ) and were identified by maldi - tof ms as m.  odoratimimus ( score : 2.439 ) .
the results of antibiotic susceptibility testing revealed that the isolates were resistant to almost all antibiotics , including -lactams , aminoglycosides and colistin but remain susceptible to sulfamethoxazole - trimethroprim , rifampicin and fluoroquinolones ( table  1 ) .
in addition , all the isolates showed resistance to carbapenems with mic for imipenem at 8  mg / l .
the isolates were then analysed via the modified hodge test , the maldi - tof ms ultraflex assay , the edta - disc test and the modified carbanp test and they were all positive on the modified hodge test , edta - disc and modified carbanp test positive .
in addition , the maldi - tof ms ultraflex assay was positive for all of them  suggesting a carbapenemase production , more specifically a metallo--lactamase as the edta test was positive .
screening for carbapenemase - encoding genes by pcr using carbapenemase gene primers showed a negative result for blavim , blaimp , blandm-1 , blakpc and all the blaoxa variants .
sequencing of the chromosomal mus gene of the three isolates and nucleotide alignment between the blamus-1 and the new blamus sequence showed some differences .
the blamus-2 differed from blamus-1 due to substitution in nucleotides at positions 69 ( ag ) , 525 ( ag ) , 171 ( ct ) , 177 ( ct ) , 397 ( ct ) , 671 ( ct ) , 179 ( tc ) , 514 ( tc ) and 675 ( cg ) , resulting in a different protein mus-2 that differ from mus-1 protein due to substitution of amino acid at position 60 ( valinealanine ) , 133 ( histidinetyrosine ) and 224 ( threonineisoleucine ) ( fig .  1 ) .
the new enzyme variant was designated mus-2 by the curators of the lahey database of -lactamases ( http://www.lahey.org/studies/webt.asp ) and deposited in genbank under accession number kp658209 .
cloning of the blamus-2 successfully gave plasmids harbouring this gene ; these plasmids were transformed into top10 electrocomp e.  coli .
mic values of -lactams for the e.  coli top10 strain , which harbours recombinant plasmid ptopo - mus-2 , showed that the bacteria was resistant to amoxicillin and ticarcillin and had a reduced susceptibility to piperacillin , in addition it showed an increased resistance to extended - spectrum cephalosporins and carbapenems by at least four - fold of mic ( table  2 ) .
finally , pfge analysis showed that the three strains of m.  odoratimimus were genetically clonal since their pfge profiles were similar ( fig .  2 ) .
to date , infections caused by m.  odoratimimus in immunocompromised and immunocompetent hosts had been rarely reported [ 1,2,48 ] .
the fact that this bacterium might cause infection in immunocompetent hosts is a real health problem because it is resistant to -lactams including carbapenems , aminoglycoside but also to colistin .
( formerly f.  odoratum ) was performed in 1985 by sato et  al . , but the molecular characteristics of -lactamase were not reported .
in fact , m.  odoratimimus species produce chromosomal mus-1 -lactamase , which belongs to the subclass b1 of ambler classification , and their antibiotic resistance patterns show a constant decreased susceptibility to imipenem .the -lactamase produced by the three m.  odoratimimus isolates in our study shared 98.78% amino acid identity with the known mus-1 protein . at the present time , each bacterial species of the flavobacteriaceae family that has been investigated for -lactamase characterization produces a subclass b1 metalloenzyme .
our study confirms a previous study performed on m.  odoratimimus species ; however , here we report a new variant of this enzyme in lebanon .
pfge analysis showed that these isolates belong to the same clone because they had the same pfge profile .
this confirms that contamination between farms has occurred and precautions should be established to limit the emergence of this clone in livestock farms because that could be a source of human infection .
taking into consideration the biochemical criteria established by rasmussen and bush to classify metallo--lactamases , mus-1 and most probably mus-2 belong to functional subgroup 3a , as their catalytic efficiencies for penicillins are at least 60% of that for imipenem .
however , these data are discrepant with those from rasmussen and bush , who mentioned that a metallo--lactamase produced by f.  odoratum strain belongs to subgroup 3b , which groups true carbapenem - hydrolysing -lactamases .
regarding this conflict , it is very difficult to estimate the exact role of either mus-1 or mus-2 in the intrinsic -lactam resistance of m.  odoratimimus because the metalloenzymes expressed in e.  coli give much lower levels of resistance to -lactams than those seen in the original producers as seen in the top10 e.  coli after cloning .
finally , it remains unknown if these bacteria could be a source of such a variety of metalloenzymes .
the most serious finding is that animals could be a reservoir for such genes and are a source of new and/or emerging multidrug - resistant bacteria and due to possible links with animals , humans may have high risk factors for colonization / infection with such bacteria . in conclusion ,
we report a novel variant of the chromosome - encoded -lactamase mus-1 that has been designated mus-2 from m.  odoratimimus isolates from livestock animals in lebanon .
the full sequence of the blamus-2 described in this study is deposited in the genbank database with accession no . kp658209 .
this work was supported by azm research centre for biotechnology and its application , lebanese university , the national council for scientific research , lebanon , the ihu mditerrane infection and the french cnrs .
 the aim of the present study was to investigate the molecular mechanism of carbapenem resistance of three imipenem - resistant isolates of myroides odoratimimus recovered from two livestock farms of cows and pigeons by rectal swab in lebanon in january 2014 . 
 investigation of imipenem resistance of these isolates using the modified hodge test , the edta test , the modified carbanp test and the matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ultraflex assay showed a carbapenemase activity due to the presence of a chromosome - encoded -lactamase mus , verified by pcr . 
 however amplification and sequencing of this chromosomal gene showed a novel variant of it designated mus-2 by the curators of the lahey database of -lactamases ( http://www.lahey.org/studies/webt.asp ) . 
 cloning of the blamus-2 was performed , followed by protein expression in escherichia coli top 10 . 
 pulsed - field gel electrophoresis clearly showed that the three isolates belonged to the same clone . 
 this study reports a novel variant of the chromosome - encoded blamus-1 associated with carbapenem resistance in myroides odoratimimus and shows that animals may represent a reservoir of bacteria harbouring several variants of resistance genes . 
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