Genome analysis of NDM-1 producing Morganella morganii clinical isolate

Genome analysis ofNDM-1 producing Morganellamorganii clinical isolateExpert Rev Anti Infect Ther Early online 1ndash9 (2014)

Abiola OlumuyiwaOlaitan1Seydina M Diene1Sushim Kumar Gupta1Amos Adler2Marc Victor Assous3

and Jean-Marc Rolain1

1Unite de recherche sur les maladies

infectieuses et tropicales emergentes

(URMITE) CNRS-IRD UMR 6236

Mediterranee Infection Faculte de

Medecine et de Pharmacie

Aix-Marseille-Universite Marseille

France2National Centre for Infection Control

Ministry of Health Jerusalem Israel3Microbiology and Immunology

Laboratory Shaare-Zedek Medical

Center Jerusalem Israel

Author for correspondence

Tel +33 491 324 375

Fax +33 491 387 772

jean-marcrolainuniv-amufr

Objective To analyze the resistome and virulence genes of Morganella morganii F675 amultidrug-resistant clinical isolate using whole genome sequencing (WGS) MethodsM morganii F675 was isolated from a patient from Jerusalem Israel WGS was performed usingboth 454 and SOLiD sequencing technologies Analyses of the bacterial resistome and othervirulence genes were performed in addition to comparison with other available M morganiigenomes Results The assembled sequence had a genome size of 4127528 bp with G+Ccontent of 51 The resistome consisted of 13 antibiotic resistance genes including blaNDM-1

located in a plasmid likely acquired from Acinetobacter spp Moreover we characterized for thefirst time the whole lipid A biosynthesis pathway in this species along with the O-antigen genecluster the urease gene cluster and several other virulence genes Conclusion The WGS analysisof this pathogen further provides insight into its pathogenicity and resistance to antibiotics

KEYWORDS intrinsic colistin resistance bull multidrug-resistant bacteria bull NDM-1 bull O-antigen bull resistome bull virulence

genes

Morganella morganii is a member of Enterobac-teriaceae family that shares closest features withProteus and Providencia genera together referredto as the Proteeae tribe It displays only 20relatedness by DNAndashDNA hybridization tomost enteric bacteria including Proteus [1] It isfrequently implicated in opportunistic infec-tions of the urinary tract the respiratory tractwounds (especially in patients with diabeticfoot ulcer) and septicemia among other infec-tions [2] M morganii is becoming an importantnosocomial pathogen and has been attributedto possess a high mortality rate in some infec-tions [3] Apart from its intrinsic resistance tocolistin it is also naturally resistant to someother antibiotics such as macrolides fosfomycinand certain b-lactams [4]

Bacterial whole-genome sequencing (WGS)is an emerging technology that has proved to beinvaluable in the study of bacterial pathogenssuch as the recent investigations of the Shiga-toxin-producing Escherichia coli O104H4 out-breaks and the Haitian cholera outbreak [56]The latest advances in WGS technology haveled to improved quality and cost reduction andit is believed that this technology can beimplemented in clinical microbiology as a real-time routine diagnostic and surveillance tool

including prediction of antimicrobial suscepti-bilities based on the bacterial resistome [7ndash9]

Although New Delhi metallo-b-lactamase1 (NDM-1)-harboring M morganii have beenreported previously [10ndash15] there is no genomicreport describing its resistome NDM-1 positiveM morganii are serious health threat as they areintrinsically resistant to polymyxins (one of thelast few options for treating carbapenem-resistant bacteria) thereby limiting therapeuticoptions available for treating such pathogenHere we sequenced the genome of M morganiiF675 an NDM-1 positive clinical isolate thatwas isolated in 2011 from the biopsy of a78-year-old female with diabetic foot ulcer inJerusalem Israel [16] The objectives of this studywere to utilize WGS to analyze the bacterialpathogenicity by determining the bacterial resis-tome and also analyzing virulence genes presentin this bacterium as compared with other closelyrelated bacteria

Materials amp methodsGenome sequencing amp assembly

Genomic DNA of M morganii F675 wasextracted by phenol chloroform method andsequenced using both paired-end pyrosequenc-ing strategy on the 454-Titanium instrument

informahealthcarecom 101586147872102014944504 2014 Informa UK Ltd ISSN 1478-7210 1

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(454 Life Sciences Branford CT) [17] and SOLiD version4 paired-end sequencing technology (Applied Biosystems FosterCity CA) [18] The assembly of the paired-end and shotgun readswas done using Newbler with 90 identity and 40-bp as overlap

Genome annotation analysis amp comparative genomics

Functional annotation of the assembled genome sequence ofM morganii F675 was done using Rapid Annotation usingSubsystems Technology Server [19] tRNA sequences were pre-dicted using findtRNA tool and rRNA genes predicted byRNAmmer 12 [20] M morganii F675 genome was comparedwith other two available genomes of M morganii strains KTand SC01 from National Center for Biotechnology Informa-tion (NCBI) with the accession numbers of CP004345 andAMWL00000000 respectively using lsquoin silicorsquo DNAndashDNAhybridization with blastn analysis Prophage sequences werechecked for using PHAgeSearch Tool [21]

Analysis of antibiotic resistance genes amp other virulence

genes

All antimicrobial resistance (AR) genes including mutated genesinvolved in antibiotic resistance were retrieved by blasting thesequences obtained from the genome (both chromosomal andplasmidic sequences) against the newly created ARG-ANNOTdatabase using tblastx on Bioedit [22] Phenotypic antibiotictesting was done for most of the antibiotics whose resistancegenes were retrieved Subsequently the genetic environment ofthe blaNDM-1 gene in M morganii F675 was compared withother NDM-1 platforms from different bacteria in NCBI

In order to understand the putative reason(s) for intrinsiccolistin resistance in M morganii ditto in Proteeae all the genesknown to be involved in polymyxin resistance were retrievedfrom the genomes of intrinsically colistin-resistant bacteria (Pro-teeae and non-Proteeae Enterobacteriaceae bacteria) and naturally

colistin-susceptible bacteria (within Enter-obacteriaceae) Comparisons of these geneswere done as well as phylogenetic analysis

The putative O-antigen and capsulargene clusters in M morganii F675 weresearched for by genome comparisonagainst Providencia stuartii (CP003488)and Proteus mirabilis (AM942759)genomes in NCBI database being thecloset bacteria to M morganii Other viru-lence genes such as the flagellar geneoperon and urease gene cluster along withtheir organizations as compared with otherProteeae members were also investigated

Further search for capsule

Transmission electron microscopy wasfurther used to examine the presence ofcapsule in M morganii F675 using ruthe-nium red fixation method as described byLuft [23]

ResultsGenome features

The genome of M morganii F675 was assembled into four scaf-folds corresponding to a genome size of 4127528 bp with GCcontent of 51 and two plasmidic scaffolds ndash pNDM-F675 witha size of 26605 bp and pF675_2 of 8441 bp (TABLE 1)M morganiiKT was found to be the closest genome to M morganiiF675 with 995 identity as compared with 906 identity forM morganii SC01 The total number of predicted coding sequen-ces (CDSs) in strain F675 genome were 4075 of these CDSsRapid Annotation using Subsystems Technology function-basedcomparison with the other two M morganii genomes showedthat 271 of these proteins (SUPPLEMENTARY TABLE S1 [supplementarymaterial can be found online at wwwinformahealthcarecomsuppl147872102014944504]) were absent in the genomes ofboth strain KT and SC01 Most of these proteins were eitherhypothetical proteins or phage relatedM morganii F675 genomecontained a total of nine prophage regions (six complete and threeincomplete prophage regions SUPPLEMENTARY TABLE S2) which repre-sents 73 of the total bacterial genome content Of these nineprophage regions only prophage regions 4 and 7 (both similar)were present in the other twoM morganii genomes

Resistome of M morganii F675

Thirteen AR genes were detected in the genome mutationsknown to confer resistance to quinolones were detected in thequinolones-resistance determining region of DNA gyrase (gyrAand gyrB) as well as in topoisomerase IV (parC and parE)within the genome The resistance genes covered a total ofseven different antibiotic families (TABLE 2) Eight of the ARgenes including blaNDM-1 were located on plasmid Some anti-biotic resistance genes (tet(A) ampH and catA) were found onthe chromosome as previously reported for M morganii

Table 1 Genome features of Morganella morganii F675 and otheravailable genomes

Feature M morganii F675 M morganii KT M morganii SC01

Genome size 4127528 bp 3826919 bp 4150412 bp

GC content 510 512 508

No of scaffold or

contigs

4dagger 58Dagger 90Dagger

No of predicted genes 4075 3565 4099

No of predicted tRNAs 119 72 74

No of predicted rRNA 21 21 4 sect

Number of suspected

plasmid

2 0 ndash

Number of phage

sequences

9 6 11

daggerScaffoldsDaggerContigssectNumber obtained from analyzed WGS data

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Table

2ResistomeofMorganellamorganiiF6

75

Resistance

phenotype

Gene

Genename

Size

(aa)

GC

Coverage

identity

Best

blast

hit

Accessionnumber

Location

b-lactams

AXAMCTIM

CRO

CTX

FO

XIPM

dha-5

b-lactamase

DHA-5

380

532

100

99

Morganella

morganii

AEL22919

Chromosome

ampH

Penicillin-binding

protein

AmpH

408

543

100

99

Morganella

morganii

WP_0

04241422

Chromosome

pse-1

b-lactamase

305

402

100

100

Morganella

morganii

ACA48667

Plasm

id

blaNDM-1

New

Delhimetallo-

b-lactamase-1

271

615

100

100

Providen

ciastuartii

YP_005351834

Plasm

id

Aminoglycosides

CNdaggerTOBdaggerK

aphA6

Aminoglycoside3rsquo-

phosphotran

sferase

260

336

100

100

Providen

ciastuartii

YP_005351836

Plasm

id

aadA2

Aminoglycoside

adenylyltransferase

270

521

100

99

Proteusmirab

ilis

ABV60287

Plasm

id

Phenicols

catA1

Chloramphenicol

acetyltransferase

220

449

100

100

Shigella

flexneri2b

NP_0

52903

Chromosome

catA2

Chloramphenicol

acetyltransferase

2

214

411

100

100

Morganella

morganii

subspmorganiiKT

YP_007506298

Chromosome

Macrolides

ERY

ereA

2Erythromycin

esterase

409

500

100

99

Providen

ciastuartii

AAC78336

Plasm

id

mph(A)

Macrolide2rsquo-

Phosphotransferase

302

656

100

99

Pseudomonas

aeruginosa

VRFPA02

EOQ80852

Plasm

id

ereB

Erythromycin

esterase

typeII

420

36

100

100

Escherichia

coli

BAE54319

Plasm

id

Tetracycline

TETTGC

tet(A)

Tetracyclineefflux

protein

TetA

402

432

100

100

Salmonella

enterica

subspenterica

serovar

Heidelberg

YP_006956556

Chromosome

Trimethoprim

T

dfrA19

Dihydrofolate

reductase

170

465

100

100

Morganella

morganii

AEH59670

Plasm

id

Fluoroquinolones

Dagger

CIPOFX

gyrA(S83R)sect

gyrB(S464Y)

parC

(S80I)

parE

(S458Y)

DNAgyrase

Topoisomerase

IV

ndashndash

ndashndash

ndashndash

Chromosome

daggerInterm

ediate

resistance

DaggerMutational

analysis

sectPositionsofmutations

aa

Amino

acids

AMCAmoxicillin-clavulanic

acidAXAmoxicillinCIPCiprofloxacinCNGentamicinCROCeftriaxoneCTXCefotaximeERYErythromycinFO

XCefoxitinIPMIm

ipen

emKKanam

ycin

OFX

OfloxacinTTrimethoprimTET

TetracyclineTGCTigecycline

TIM

Ticarcillin-clavulanateTO

BTobramycin

Genome analysis of NDM-1 producing M morganii clinical isolate Original Research

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KT [24] M morganii F675 was resistant to imipenem andshowed high-level resistance to fluoroquinolones (ciprofloxacinand ofloxacin) (TABLE 2) The plasmid of M morganiiF675 (pNDM-F675) displayed closest resemblance (99

identity and about 33 coverage) to blaNDM-1-bearing plasmidsfrom Acinetobacter spp its blaNDM-1 and associated genes dis-played similar organization to that of pNDM-BJ02 from Acine-tobacter lwoffii (FIGURE 1) Phenotypic antimicrobial susceptibility

testing showed that M morganiiF675 was only susceptible to aztreonamand amikacin

The genome of M morganii F675 con-tained all the genes involved in lipid Abiosynthesis (lpxA lpxC lpxD lpxHlpxB lpxK kdtA lpxL lpxP and lpxM)(SUPPLEMENTARY TABLE S3) with extra copy oflpxP The genes involved in lipid Amodifications (phosphoethanolamineand 4-amino-4-deoxy-L-arabinose modifi-cations) all known to be involved in colis-tin and other cationic antimicrobialpeptides resistance were identified in thebacterial genome (SUPPLEMENTARY TABLE S3) Twocopies of arnT gene were detected in thegenome of M morganii and other Proteeaemembers whereas non-Proteeae bacteriahave just one copy ArnT is known totransfer the synthesized L-Ara4N to core-lipid A in the final step of lipid A modifica-tion with L-Ara4N [25] The phoPphoQtwo-component regulatory system thatactivates the arnBCADTEF-pmrE operon

P started pMR0211(JN687470)

E coli HK-01 pNDM-HK(HQ451074)

M morganii F675 pNDM-F675

A iwoffii WJ10659 pNDM-BJ02(JQ060896)

E coli N10-2337 pNDM102337(JF714412)

orfA

orfA

insB

insB

aphA

6

ISAba

125

aphA

6

ISAba

125

blaNDM

-1

bleM

BL

sul1

ISCR1

hp

hp

iso

iso tat

cutA

1gr

oES

groE

L

insE

orfA

insB

aphA

6

ISAba

125

blaNDM

-1

bleM

BL

hp iso tat

cutA

1gr

oES

groE

L

insE

tniA

orfB

aphA

6

ISAba

125

blaNDM

-1

bleM

BL

tniB

iso tat

cutA

1gr

oES

groE

L

rcr2

7

aacC

2

ISAba

125

blaNDM

-1

blaNDM

-1

blaDHA-1

ampR

hypA

Figure 1 Comparison of the genetic environment of blaNDM-1 region of pNDM-F675 from Morganella morganii F675 withother reported NDM-1 harboring plasmids similar arrows show genes that are similar or the same GenBank accession num-bers are in bracket

E coli MG1655 (U00096)

Shigella boydii CDC 3083-94 (CP001063)

Salmonella enterica 24249 (CP006876)

K pneumoniae MGH 78578 (CP000647)

Enterobacter cloacae EcWSU1 (CP002886)

Serratia marcescens WW4 (CP003959)dagger

Proteus mirabilis HI4320 (AM942759)

Morganella morganii F675

Providencia stuartii MRSN 2154 (CP003488)96

100

10098

71

100

000005010015020025

Na

tura

l co

listin

resi

sta

nce

Na

tura

l co

listin

susc

eptib

le

Figure 2 Phylogenetic analysis of the concatenated PhoPQ two-componentsystem and its negative feedback regulator MgrB protein sequence amongsome selected Enterobacteriaceae Sequences were aligned using CLUSTALX andphylogenetic inferences obtained using neighbor joining method within Mega 5 softwareBootstrap values are expressed by percentage of 1000 replicates and are shown at thebranch point The accession numbers for the sequences used in the study are shownin bracketsdaggerSerratia marcescens is colistin heteroresistant

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was detected as well as its feedback inhibitor mgrB But surpris-ingly pmrApmrB was not detected in the genome ofM morganiiand other Proteeae members Phylogenetic analysis of theconcatenated PhoP PhoQ and MgrB protein sequences showedthat the intrinsic colistin-resistant bacteria formed a separatecluster from the naturally colistin susceptible ones (FIGURE 2)

Localization of O-antigen gene cluster

The O-antigen gene cluster has been reported to be localizedbetween cpxA and yibK housekeeping genes in Providencia sppand between cpxA and secB genes in Proteus spp [2627] Wecompared this region in M morganii F675 against the sameloci in Proteus mirabilis HI4320 and Providencia stuartiiMRSN 2154 (FIGURE 3) The cpxA-yibK locus in M morganiiF675 contained 10 genes blast results showed these genes werehighly similar to O-antigen genes in other gram-negative bacte-ria including Pseudomonas aeruginosa (TABLE 3)

Search for capsular genes

No capsular gene cluster was found in the genome of M morga-nii F675 Some genes reported to be involved in surface expres-sion of capsular polysaccharides were detected within theO-antigen gene cluster of Proteus mirabilisHI4320 and P stuartiiMRSN 2154 [2628] but none of these genes was present inM morganii F675 Of the five regulatory genes implicated incapsule synthesis (rcsA rcsB rcsC rcsD and rcsF) which were

present in both Proteus mirabilis HI4320 and P stuartiiMRSN 2154 genomes only four were found in the genome ofM morganii F675 (rcsA was absent) In addition the result oftransmission electron microscopy further showed the absenceof capsule in M morganii F675 (FIGURE 4)

Urease gene cluster

The noninducible urease gene cluster consisting of ureABCEFGDwas detected in M morganii F675 having an overall average GC of 534 It lacked the ureR regulatory gene that was detectedin Proteus mirabilis HI4320 and Providencia rettgeri DSM1131 (urea-inducible urease gene cluster) as shown in FIGURE 5APhylogenetic tree analysis of the concatenated urease geneoperons among some selected Enterobacteriaceae revealed thatthe urease operon in M morganii is more related to that of Yersi-nia enterocolitica Edwardsiella ictaluri and Photorhabdus lumines-cens than that of Proteus mirabilis and P rettgeri (FIGURE 5A amp 5B)

Flagellar gene operon amp chemotaxis

A total of 49 flagellum-related genes including genes involvedin chemotaxis and two hypothetical proteins were identified inthe genome of M morganii F675 the overall GC of theflagellar-encoding genes is 529 These flagellum-related geneslike in Providencia spp were located in separate loci within thegenome unlike in P mirabilis [28] and at the same time showedclosest match to that of Providencia spp (SUPPLEMENTARY TABLE S4)

Morganella morganii F675

Providencia stuartii MRSN 2154(CP003488)

Proteus mirabilis strain HI4320(AM942759)

yibK

galE

wzc wzb wza ugp

GT wcag

udg

hp hp hpCpx

A

GT GT pbp

yibK

wbgZ

wbgY

wbgX

GT GT wlbK hp wbpE Cpx

A

wbpB

ugp

secB

CpxA

gpsa

cysE

wemP

yibK

wbnF

ugp

cpsF

wemM

wemL

wemK

wemJ

weml

wzxwzy

Figure 3 Comparative structural organization of the putative O-antigen gene cluster locus in Morganella morganii F675 tothat of Providencia stuartii and Proteus mirabilis GenBank accession numbers are in brackets the small rectangles show thehousekeeping genes bounding the putative O-antigen gene clusters and the big square shows the boundary reported in Proteus sppProvidencia stuartii MRSN 2154 (CP003488)- cpsF Glycosyl transferase cpxA Copper sensory histidine kinase CpxA galE UDP-glucose4-epimerase GT Glycosyl transferase GT Glycosyl transferase group 1 hp Hypothetical protein pbp Polysaccharide biosynthesis proteinUdg Uridine diphosphate galacturonate 4-epimerase ugp UDP-glucose dehydrogenase wcag Capsular polysaccharide biosynthesisprotein wza Polysaccharide export lipoprotein wzb Low-molecular-weight protein-tyrosine-phosphatase wzc Tyrosine-protein kinaseyibK MethyltransferaseProteus mirabilis strain HI4320 (AM942759)- secB protein-export protein cpxA Copper sensory histidine kinase CpxA cysE Serineacetyltransferase gpsa Glycerol-3-phosphate dehydrogenase ugd UDP-glucose 6-dehydrogenase wbnF nucleotide sugar epimerasewemI Putative O antigen biosynthesis protein wemJ Glycosyl transferase wemK Glycosyl transferase wemL Putative O antigenbiosynthesis protein wemM Glycosyl transferase wemP Putative Serine acetyltransferase wzx O unit flippase wzy O antigenpolysaccharide unit polymerase yibK MethyltransferaseThe names of the genes found within this locus in M morganii F675 are explained in TABLE 3

Genome analysis of NDM-1 producing M morganii clinical isolate Original Research

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A duplicated fliI gene (flagellum-specific ATP synthase) wasobserved elsewhere in the genome Some nonflagellum-relatedgenes observed in M morganii KT genome such as the lysRfamily transcriptional regulator and insecticidal toxin geneswere likewise observed in M morganii F675 Interestingly a329 kb complete phage sequence (phage region 1) having49 CDSs (15 CDSs among these showed best blast hit to bacte-rial proteins SUPPLEMENTARY TABLE S2) was found between flgF and flgFgenes inM morganii F675 genome

Hemolysin genes

Genes coding for hemolysin (hmpA) and hemolysin activatorprotein (hmpB) were identified in the genome these two genesare important for hemolytic activity of M morganii

Genomic characterization of M morganii into subspecies

amp biogroup

Phenotypically M morganii are divided into subspecies siboniiand morganii based on their ability or inability to ferment treha-lose respectively and further subdivided into biogroups basedon the production of ornithine decarboxylase (ODC) andorlysine decarboxylase (LDC) [29] The genome of M morganiiF675 lacked the trehalose operon (treR treB and treP) needed fortrehalose fermentation this was found to be present inP mirabilis HI4320 and P stuartii MRSN 2154 (trehalosefermenters) This thereby placed M morganii F675 into subspe-cies morganii Furthermore it contained ODC gene (speF) andthe putrescine-ornithine antiporter (potE) but lacked the LDCgene operon (cadBA operon) Thus these results allow placingM morganii F675 into biogroup A (ODC+ LDC-)

Table 3 Characteristics of the putative O-antigen gene cluster found in Morganella morganii F675 genome

Gene Gene functions Similar protein similarity GC

wbgZ UDP-N-acetylglucosamine 46-dehydratase Providencia alcalifaciens (AFV53200) 65 345

wbgY Lipid carrier UDP-N-acetylgalactosaminyltransferase

Providencia alcalifaciens (AFV53199) 64 277

wbgX 4-keto-6-deoxy-N-Acetyl-D-hexosaminyl-(Lipid

carrier) aminotransferase

Providencia alcalifaciens (AFV53198) 83 385

GT Putative glycosyltransferase Xenorhabdus nematophila ATCC 19061

(YP_003710479)

56 303

GT Glycosyltransferase Acinetobacter lwoffii (WP_005108328) 68 318

hp Putative membrane protein wlbK Bordetella pertussis (CAA62254) 33 277

hp Hypothetical protein Photorhabdus temperata subsp temperataM1021 (EQB98509)

36 263

wbpE Glutamate ndash UDP-2-acetamido-2-deoxy-D-ribo-

hex-3-uluronic acid aminotransferase (PLP

cofactor)

Pseudomonas aeruginosa NCMG1179

(GAA18855)

74 320

WbpB UDP-2-acetamido-2-deoxy-D-glucuronic acid

dehydrogenase (NAD + cofactor)

Pseudomonas denitrificans ATCC 13867

(YP_007660052)

77 323

ugp UDP-glucose dehydrogenase Pseudomonas aeruginosa (AAM27657) 72 333

100 nm

500 nm

A

B

Figure 4 Transmission electron micrograph of thin sectionof Morganella morganii F675 (A) and the magnified sectionof the cell membrane showing the absence of capsulearound the outer membrane (B)

Original Research Olaitan Diene Gupta Adler Assous amp Rolain

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DiscussionM morganii is an opportunistic pathogen that is often found col-onizing patients as observed for Proteus mirabilis [28] and is ableto cause a large variety of diseases [2]

We observed that prophages and hypothetical genes substan-tially accounted for the interstrain genetic variability seenbetween our strain F675 and the other two M morganiistrains (KT and SC01) [30] The acquisition of NDM-1 genetogether with other plasmid- and chromosomally encoded ARgenes in strain F675 further emphasizes the clinical importanceof this bacterium We believed that the blaNDM-1 gene orpNDM-F675 may have been acquired horizontally from Acine-tobacter spp or similar bacteria harboring such plasmid due tothe similarity of this plasmid and the genetic arrangement ofthe blaNDM-1 gene to that of Acinetobacter spp This furtheremphasizes the propensity for inter-genus transmission of ARgenes and plasmids harboring blaNDM-1 [31]

The upregulation of capsular polysaccharide had beenimplicated in polymyxin resistance in K pneumoniae [32]such mechanism is unlikely to be involved in intrinsic resis-tance to colistin and other cationic antimicrobial peptidesamong the Proteeae tribe since M morganii is noncapsulatedAll the known genes mediating lipid A biosynthesis andmodifications with respect to colistin resistance were foundin M morganii genome [25] in addition to this clustering ofthe concatenated PhoPQ-MgrB proteins among the intrinsiccolistin-resistant Enterobacteriaceae was observed Wehypothesized that the nature of lipid A (structure composi-tion and modifications) among intrinsic colistin-resistantEnterobacteriaceae (especially the Proteeae) is likely to be dis-tinctly different from that of the naturally susceptible ones asobserved in Burkholderia spp (intrinsically colistin-resistantbacteria) [33] Multiple presence of some genes such as arnTas well as the absence of pmrApmrB among the Proteeae tribecould as well be a significant factor contributing to theirintrinsic colistin resistance nature

We localized the putative O-antigen gene cluster inM morganii for the first time to the best of our knowledge astructure that has played an important role in bacterial typingand also serves as host immunologic response [34] There is nodoubt that virulence genes found in M morganii genome suchas urease flagellar and hemolysin gene clusters have contributedto the pathogenicity attributed to this pathogen and other Pro-teeae members such as bacteremia and urinary tract infec-tions [2428] Some of these genes could play a role that hasenabled M morganii in colonizing foot ulcer in diabetic patientsbecause it has repeatedly been isolated in these patients [163536]

Using WGS we were able to characterize the isolate up toits biogroup type by analyzing the genes responsible for its bio-grouping As such WGS technique therefore offers a goodplatform for bacterial genomic typing [37]

ConclusionIn conclusion M morganii is a well-known causative agent ofa large variety of human infections such as bacteremia andwound infections Hence it is well adapted for causing suchinfections due to the presence of several virulence genes in itsgenome and is also able to harbor numerous AR-encodinggenes likely acquired from plasmids Bacterial WGS provides away to comprehensively characterize AR and other virulencegenes this could be utilized for routine investigations of patho-gens This study further shows the importance of real-timeWGS in clinical microbiology that will enhance the study ofbacterial pathogens

Nucleotide sequence accession numberThe chromosome and plasmid sequences of Morganella morga-nii F675 isolate are currently under submission in EMBLdatabase under Project ID PRJEB6425

Acknowledgement

We thank Linda Hadjadj for technical assistance

A B

Regulatorygene

Accessorygene

Structural genes Accessory genes

ureR ureD ureA ureB ureC ureE ureF ureG

ureD ureA ureB ureC ureE ureF ureG

ureA ureB ureC ureE ureF ureG ureD

Morganella morganii F675

Yersinia enterocolitica 8081 (AM286415)

Edwardsiella ictaluri 93-146 (CP001600)

Photorhabdus luminescens TTO1 (BX571866)

Klebsiella pneumoniae MGH78578 (CP000647)

Escherichia coli O26H11 (AP010953)

Proteus mirabilis HI4320 (AM942759)

Providencia rettgeri DSM 1131 (ACCI00000000) 100

100

100

100

100

57

01

Figure 5 Comparison of the urease gene clusters (A) Genetic organization of urease gene operon in Morganella morganii F675 ascompared with other urease operons in Enterobacteriaceae (B) Concatenated phylogenetic tree of urease gene cluster The arrangementof the concatenated urease gene sequences followed that ofM morganii Sequences were aligned using CLUSTALX and phylogeneticinferences obtained using neighbor joining method within Mega 5 software Bootstrap values are expressed by percentage of 1000replicates and are shown at the branch point The accession numbers for the sequences used in the study are shown in brackets

Genome analysis of NDM-1 producing M morganii clinical isolate Original Research

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Financial amp competing interests disclosure

This work was funded by the French Centre National de la Recherche Sci-

entifique (CNRS) and Infectiopole Sud Foundation The authors have no

other relevant affiliations or financial involvement with any organization

or entity with a financial interest in or financial conflict with the subject

matter or materials discussed in the manuscript apart from those disclosed

No writing assistance was utilized in the production of this

manuscript

Key issues

bull Whole-genome sequencing technology is a promising technology that could be implemented in clinical microbiology for studying

pathogens and for antimicrobial resistance surveillance in real time

bull Morganella morganii F675 is a multidrug-resistant clinical isolate harboring a total of 13 antibiotic resistance genes and showing

susceptibility to only two antibiotics

bull The blaNDM-1 of M morganii F675 is likely to have been acquired from Acinetobacter spp harboring such gene

bull M morganii F675 though noncapsulated possesses numerous virulence genes including urease- hemolysin- and flagellar-encoding

genes that have contributed to its distinctive infections

bull The putative O-antigen gene cluster of M morganii is likely located within yibK-cpxA locus similar to that of Providencia spp and

could be the same among the Proteeae bacteria

bull The presence distinct clustering and gene duplications of genes known to be involved in lipid A modifications observed herein among

intrinsically colistin-resistant bacteria (including M morganii) could indicate that these bacteria have a unique lipid A structure different

from that of naturally colistin-susceptible bacteria

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