Co-transmission of Rahnella aquatilis between hospitalized patients

braz j infect dis.2015;19(6):648–650

www .e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Brief

communication

Co-transmission

of

Rahnella

aquatilis

between

hospitalized

patients

Willames

Martins

_,

_Cecília

_Godoy

_Carvalhaes

_,

_Rodrigo

_Cayô

_,

_Ana

_Cristina

_Gales

_,

Antonio

Carlos

Pignatari

a_{LaboratórioEspecialdeMicrobiologiaClínica(LEMC),DivisionofInfectiousDiseases,DepartmentofInternalMedicine,EscolaPaulista} deMedicina/UniversidadeFederaldeSãoPaulo,SãoPaulo,SP,Brazil

b_{LaboratórioDASA,SãoPaulo,SP,Brazil} c_{Hospital9deJulho,SãoPaulo,SP,Brazil}

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Articlehistory:

Received10April2015 Accepted14July2015

Availableonline26September2015

Keywords: Rahnellaspp. Bacteriaidentification MALDI-TOFMS Environmentalbacteria

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Rahnella aquatilis is an environmental Gram-negative bacillus that is rarely reported ashuman pathogen,being mainly associatedwith infections inimmunocompromised patients.HereinwedescribetwocasesofR.aquatilisisolatesrecoveredfromendotracheal aspirateculturesofdifferentpatientsinatertiaryhospitallocatedinthecityofSãoPaulo, Brazil.Matrix-assistedlaserdesorption/ionizationtime-of-flightmassspectrometryand16S rDNAgenesequencingwereperformedtoconfirmbacterialidentificationaftertheisolates beingerroneouslyidentifiedasPantoeaspp.byautomatedsystem.Bothisolatesshowedthe samePFGEpatternandpresentedthe␤-lactamaseencodinggeneblaRAHN-1,responsiblefor

resistancetocephalothin.Theisolatesweresusceptibletobroad-spectrumcephalosporins, carbapenems,fluoroquinolones,aminoglycosides,andpolymyxinB.Thisreportshowsthe presenceandtransmissionofuncommonbacteriain thenosocomialenvironmentand alertsusabouttheneedfornewtoolsofcorrectmicrobiologicdiagnosis.

©2015ElsevierEditoraLtda.Allrightsreserved.

Rahnella spp. is a facultative anaerobic and nitrogen fixer member ofthe Enterobacteriaceaefamily.1 _Thisgenuswas

describedin1976,andwasinitiallydenominatedasaGroup HofEnterobacteriaceae,basedonnumericaltaxonomy.With the advent of molecular techniques, this new genus was renamed Rahnella. Ithas been mainly isolated from water and soil, and therefore it can be abundantly found in freshproducts.TheRahnellagenuscomprisesR.aquatilis,R.

∗ _{Correspondingauthor.Currentaddress:LaboratórioEspecialdeMicrobiologiaClínica–LEMC,UniversidadeFederaldeSãoPaulo–UNIFESP,}

RuaLeandroDupret,188,VilaClementino,04025-010SãoPaulo,SP,Brazil. E-mailaddress:willamesbrasileiro@hotmail.com(W.Martins).

genomospecies2andR.genomospecies3,thatcanbe differ-entiatedonlybymoleculartechniques.2_{Todate,fewcasesof}

infection caused byR.aquatilis havebeenreported in liter-ature,affectingmainlyimmunocompromisedpatients.1,3 _In

2001,Bellais andcolleagues reportedanovelnon-inducible and chromosomally encoded Ambler class A ␤-lactamase, namedRAHN-1,whichisintrinsicofR.aquatilisstrains.4_Two

variants of blaRAHN have been described to date (blaRAHN-1

http://dx.doi.org/10.1016/j.bjid.2015.07.009

brazj infect dis.2015;19(6):648–650

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andblaRAHN-2),and contributeto oneofthefew resistance

mechanismsdescribedinthispathogen.Althoughclinicaland environmentalRAHN-producingR.aquatilisstrainsreported thus far were usually resistant to penicillins and narrow-spectrumcephalosporins,thesestrainsremainedsusceptible toceftazidime,imipenem,andwereinhibitedbyclavulanic acid.5

TheidentificationofR.aquatilisbyclassicalmicrobiologic features is difficult, since there is not a single biochem-ical test that could differentiate R. aquatilis isolates from otherEnterobacteriaceae.6 _{R.aquatilisisanon-motilebacillus}

at35◦C(motileonlyat25◦C),usecitrateasthecarbonsource, andisabletoferment maincarbohydrates,after48h incu-bation at 35◦C. However, in MacConkey agar, a few weak lactose colonies are usually observed.2 _{R. aquatilis maybe}

misidentifiedasEnterobacter agglomerans,sincethese bacte-riaspecies share similar biochemical profile. Thus, correct identification by molecular techniques maybe required. In the last years, matrix-assisted laser desorption/ionization time-of-flightmassspectrometry(MALDI-TOFMS)hasbeen establishedasapreferablealternative methodforaccurate bacterialidentification.Herein,wereportthemicrobiological featuresofRAHN-1-producingR.aquatilisisolatesrecovered fromtwopatientsadmittedtoatertiaryhospitalinthecityof SãoPaulo,Brazil.

Two different Gram-negative rods were recovered from endotracheal aspirate cultures of distinct hospitalized patients,inthesameperiod,ataneurologicalintensivecare unit(ICU).Inthefirstcase,a64y-omalehospitalizedfora long-term due to autoimmune encephalopathy associated to Lyme disease was submitted to tracheostomy due to respiratorydisorders.Duringhisstay,hedevelopedvarious infections (respiratory and urinary) and received several coursesofantimicrobialtherapy. However, thepatient had notbeen on antimicrobialtherapy in the month before R. aquatilisisolation.Afterbeing hospitalizedforfivemonths, thepatientwas diagnosedwithapulmonaryinfectiondue tothepresenceofpurulentendotrachealaspirateand radio-logical image compatible with parenchymal lung infiltrate.

Pseudomonas aeruginosa and Pantoea spp. were identified by Vitek®_{2 automated system (bioMérieux, Marcy l’Etoile,}

France) in the endotracheal aspirate specimen, both at >105_{UFC/mL.Meropenemtherapywasintroducedand}

clini-calimprovementwasobservedsevendaysthereafter.Inthe secondcase,a37y-omalewithadegenerativeneurological syndrome(Hallervorden-Spatzsyndrome)admittedtoa long-term institution developed septicshock due to pulmonary infection in the left lower lobe,requiring mechanical ven-tilationand vasoactivedrugsupport. Antimicrobialtherapy withteicoplaninandmeropenemwasinitiatedwithclinical improvement afterone week. No pathogen was recovered fromanyclinicalsamplescollectedafterimprovement. How-ever,aftertwo-monthseveralbacterialisolateswerecultured frombloodsamplesinaperiodofonemonth,includinga KPC-producingKlebsiellapneumoniae,followingbyKPC-producing

Enterobacter aerogenes and Staphylococcus haemolyticus. The patient wastreated with multiplecoursesof antimicrobial agents including tigecycline, amikacin, teicoplanin, and meropenem.Oneweekafterfinishingantimicrobialtherapy thepatientpresentedwithfeverandacarbapenem-resistant

Fig.1–PFGEofR.aquatilisisolates.Lines1and4–Lambda

LadderPFGMarker50–1000kb;Lines2and3–R.aquatilis

isolates.

P. aeruginosa and Pantoeaspp. isolateswere recoveredfrom endotrachealaspirate.Thepatientreceivedanewcourseof tigecyclineandamikacintherapywithclinicalimprovement. Both Pantoea spp. isolates were referred to a reference laboratory for confirmation of bacterial identification and antimicrobialsusceptibility.MassspectrometrybyVitek®_MS

(bioMérieuxS/A,MarcyI’Etoile,France)identifiedbothisolates asR.aquatilis(scoreof99%)and16SrDNAgenesequencing confirmed thoseresultswith100%similarity andcoverage.

Rahnella species were recentlyincluded inthe commercial massspectrometrydatabases,thus,allowingtheir identifica-tion fromclinicalandenvironmentsamples.7_Todetermine

theclonalrelationship,pulsed-fieldgelelectrophoresis(PFGE) usingSpeIastherestrictionenzymewasperformedas pre-viouslydescribed.8_{Theanalysiswasperformedaccordingto}

theTenovercriteria.9_{TheRahnellaisolatesbelongedtoa}

sin-glePFGEpatterns(Fig.1),suggestingthatapatient-to-patient transmissionhadoccurred,sincetheywereadmittedtothe same ICU and shared the same hospital health care staff inthesameperiodoftime.Caroffandcolleaguesdescribed one of the first transmission of R. aquatilis in nosocomial environment.10_{Inthatstudy,becausethepatientswere}

unre-lateditwassuspectedthatprolongedparentalnutritiongiven tobybothpatientswasthesourceofspread.Other studies identifiedthewaterusedinthebronchialwashingprocessand citratesolutionusedinparenteralnutritionsolutionasthe

650

mainsourcesofcontaminationbyR.aquatilis.3,6,10_Fortunately,

nofurthertransmissionofR.aquatilishasbeenobservedand investigationofthesourcewasnotevaluated.

Antimicrobialsusceptibilitywasevaluatedbydetermining the minimal inhibitory concentrations (MICs) using broth microdilutionaccordingtotheCLSIguideline.11_The

antimi-crobialagentstestedwereampicillin,amikacin,gentamicin, cefepime,cephalothin,ceftriaxone,ceftazidime,aztreonam, meropenem, imipenem, ciprofloxacin, levofloxacin, and polymyxin B. According to CLSI breakpoints,12 _both

iso-lates were susceptible to levofloxacin (MIC, ≤0.25␮g/mL), ciprofloxacin (MIC, ≤0.125␮g/mL), gentamicin (MIC, ≤0.125␮g/mL), amikacin (MIC, ≤0.5␮g/mL), ampicillin (MIC, 8␮g/mL), cefepime (MIC, 0.25␮g/mL), ceftriaxone (MIC, 1␮g/mL), aztreonam (MIC, 1␮g/mL), imipenem (MIC, 0.25␮g/mL),meropenem(MIC,≤0.06␮g/mL),andpolymyxin B(MIC, 4␮g/mL). Since the isolates were resistant only to cephalothin(MIC,128␮g/mL),thepresenceof␤-lactamases encoding genes was investigated byPCR followed byDNA sequencing using the ABI3500 Genetic Analyzers (Applied Biosystems, Foster City, CA). The presence of blaRAHN-1

gene was detected in both isolates, justifying resistance to cephalothin observed in these isolates. Although the twoR.aquatilisisolatesweresusceptibletobroad-spectrum cephalosporins, previous studies have demonstrated that RAHN-1 shows hydrolytic activity against cefotaxime and ceftriaxone.4 _{So far,all isolatesofRahnellaspp.reported in}

theliterature,weresusceptibletomostantimicrobialsagents tested. Thus, broad-spectrum cephalosporins are usually employed forthe treatment of infections caused by these pathogens. In contrast, insome cases when R. aquatilis is isolatedwithanotherpathogen,theantimicrobialtreatment musttakeintoaccountthecoverageagainstbothpathogens. HumaninfectionscausedbyR.aquatilisareinfrequentand hadnotbeenpreviouslyreportedinBrazil,whichcouldbe jus-tified,atleastinpart,bytheinabilityofautomatedsystems toidentifyenvironmentalmicroorganisms.SinceR.aquatilis

biochemicalprofileisverysimilartothatofother Enterobac-teriaceae,likePantoeaspp.andEnterobacterspp.,morereliable techniquesarecrucialtoidentifyenvironmentalisolatesfrom clinicalspecimensinordertobetterdefinethe epidemiolog-icalroleplayedbythesespeciesinthenosocomial environ-ment.Inthiscontext,MALDI-TOFMSseemstobeareliable toolintheidentificationofsuchisolates.Duetotheisolation ofmulti-drugresistantP.aeruginosastrainsinthesameclinical specimenswhereR.aquatiliswerealsoisolated,itisdifficultto definetheexactroleofR.aquatilisasthepathogenresponsible forventilator-associatedpneumonia.However,the isolation of environmentalmultisusceptible bacteria in the hospital setting could represent a real threat, since, generally, this microorganismscarried virulentdeterminantsand areable toeasilyacquireresistantgenescarriedbymotilegenetic ele-mentsfromothercommonpathogens.Thus,theemergence ofsuchmicroorganismsas clinicallysignificant pathogens, especiallyinimmunocompromisedpatients,isworrisome.

Conflicts

of

interest

A.C.G.recentlyreceivedresearchfundingand/orconsultation feesfromAstraZeneca,MSD,Novartis,ThermoFisher Scien-tific,andBioMérieux.Otherauthorshavenothingtodeclare.

Acknowledgements

Weare gratefultoFernandaRodrigues-Costaand Ioná Tor-resMagalhãesfortheirexcellenttechnicalcontributiontothe manuscript.

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