0095-1137/83/080395-05$02.00/0
Copyright01983,American SocietyforMicrobiology
Vol.18, No. 2
Comparison
of
Epidemiological
Markers Used in the
Investigation of
an
Outbreak of
Methicillin-Resistant
Staphylococcus
aureus Infections
GORDON L. ARCHER*ANDC.GLEN MAYHALL
Divisionof Infectious Diseases, Departmentof Medicine, Medical College of Virginia, Virginia CommonwealthUniversity, Richmond, Virginia23298
Received4March1983/Accepted 19 April 1983
An outbreak of nosocomial infections was caused by a single strain of methicillin-resistant (MR) Staphylococcus aureus. This strainwas followed as it
wastransmitted from the indexcase to 17patients, 3hospitalpersonnel, and12
items in thehospital environment. The MRS. aureusstrainwastraced byusing
four specific epidemiological markers: antibiogram, phage type, production of
aminoglycoside-inactivating enzymes,andplasmid pattern.These markerswere
assessed for theirreliability indifferentiating theepidemicS. aureusstrainfrom resident nonepidemic strains and for the ease and rapidity with which they determined differences. Theepidemic strainwasresistanttobeta-lactam antibiot-ics,gentamicin, erythromycin, clindamycin, and rifampin. Resistancetorifampin
wasthe only unique marker in the antibiogram which distinguishedtheepidemic strain from the indigenous strains, and it was the easiest marker to use for
screening isolates from culture surveys. Phage typing was poorly reproducible
and didnot yield results rapidly enough tobe usefulfor ongoingepidemiology.
Theepidemic strainproduced aunique aminoglycoside-inactivating enzyme
(3'-phosphotransferase) which distinguisheditfromindigenous gentamicin-resistant
staphylococci, but this markerwas not easily identified, nor was identification
helpfulduringthecourseof theinvestigation.Plasmidpattern analysiswasrapidly
performed(inless than 24h),allowed manyisolatestobe examinedatatime,was
stable andreproducible,andyieldedaunique fingerprintwhichdistinguishedthe epidemic strain from all indigenous isolates. Plasmid pattern analysis is a
promising epidemiological tool for MR S. aureus outbreaks in which epidemic strains lackuniqueantibiotic resistance markers.
The incidence of nosocomial infections caused by methicillin-resistant (MR)
Staphylo-coccus aureus strains isincreasing dramatically
in hospitals in the United States (5). MR S.
aureus isolates have been shown to be fully
virulent, causing staphylococcal endocarditis
andsepticemia at afrequency similartothat of
methicillin-sensitive S. aureus isolates (16; E.
Lewis and L. D. Saravolatz, Program Abstr. Intersci. Conf. Antimicrob.Agents Chemother.
22nd, MiamiBeach, Fla., abstr. no. 366, 1982). Furthermore,theseisolatesareusuallyresistant to multiple additional antibiotics, including
cephalosporins, aminoglycosides, macrolides,
and lincosamides (2, 8, 10, 16). The antibiotic resistance ofS. aureus strains allows increased colonization ofpatientsinareasofhigh antibiot-ic useand impedes effective antibiotic therapy for infections. Therefore, it is important to be abletodocumentthespread ofanepidemicMR
S. aureus isolate sothat effective control
mea-sures canberapidlyinstituted.
ThefirstMRS.aureusisolate identifiedatthe MedicalCollegeofVirginiaHospitalswas intro-duced by apatientwho wastransferred froma
Florida hospital where several such isolates
wereendemic. Before thispatientwasidentified
as being infected, he had contact withmedical
personnel and otherpatients. Therefore,an epi-demiological investigation was instituted to
trace contacts and document environmental
contamination. In the process of tracing the
epidemic isolate, many surveillance cultures
wereobtainedfrompatients,personnel, and the
environment. The separation of the epidemic isolate from other, resident S. aureus isolates required thatspecific markers forthisisolate be identified. Inthisstudy,wecomparedthe tradi-tional epidemiological markers, antibiograms andphage typing, with newermarkers,
amino-395
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glycoside-inactivating enzyme analysis and plas-mid pattern fingerprinting, for their ability to
identify MRS. aureusduring theinvestigation of
theoutbreak.
MATERIALS ANDMETHODS
Collection of cultures.Isolates analyzed for specific markers wereobtained fromthewounds, blood,nares, and secretions of infected patients; the nares and wounds ofasymptomatic contacts; and the hospital environment. Culture swabswererolledonthesurface of S110 agar(BBL Microbiology Systems, Cockeys-ville, Md.) containing 20,ugof methicillin perml and incubatedat 37°C for 72 h. ColoniesgrowingonS110 methicillin agar werepicked and tested forcoagulase
production. As confirmation of their resistance to methicillin, somecoloniesgrowing onthisagarwere inoculatedinto drug-free broth andgrownovernightat 37°C; 0.1 ml of the brothwas then replatedon agar containing20 ,ugof methicillinperml. Thepresenceof subpopulationsonthisagarafter 72 hof incubationat 37°C confirmed the methicillin resistance of the colo-nies.
Antibioticsensitivity.Thesensitivity ofallisolatesto antibiotics other than methicillin was determined by standard agar dilutiontechniques on Mueller-Hinton agar(9).
Phage typing.Phagetypingwasperformed by Gary Hancockatthe Centers for DiseaseControl, Atlanta, Ga., and by Harry Dalton in the Medical College of Virginia Clinical Microbiology Laboratory. Phages weretestedatthe MedicalCollege of Virginia onlyat thestandard(1/100) dilution. The Centers for Disease Control used both the standardand a1/10 dilution.
Aminoglycoside-inactivatingenzyme analysis. Select-edgentamicin-resistant S. aureus isolates werelysed andtestedfortheproduction of aminoglycoside-inacti-vating enzymes by David Bobey at Bristol Labora-tories, Syracuse, N.Y. Lysis was done with lysosta-phin in Tris bufferby the method of Scottetal.(15). Theassay was avariationof thenitrocellulose-binding
assaydescribed by Haas andDowding (4).
Plasmid pattern determination. Staphylococcal iso-lates werelysed and theirDNA wasvisualizedbythe
followingscheme:S.aureusisolatesweregrown
over-night onagar, andthegrowth from one-fourth ofan agar platewastransferred with a swabto 10ml ofa
solution containing0.1 MNaCland 0.05M EDTAat pH6.9.Bacteriawerewashedtwice andresuspended in1mlof this salt solutiontowhichlysostaphin (Sigma Chemical Co., St. Louis, Mo.) was added to a final concentration of70 p.g/ml.After 30 minof incubation at37°C,thesolutionoflysedbacteriawasspunathigh
speed(40,000xg)for 45min,and the cleared superna-tant wasremoved andtreatedwith 10 ,ugofpancreatic RNase A(Sigma) per ml for1 hat 37°C,followedby treatmentwith 10 ,ug ofproteinaseK(Sigma) per ml for1 hat 37°C.A 40-,ul portionoftheclearedlysate wasthenelectrophoresedat80 V(35mA) for3hon an 11.5-cmverticalslabgel through 0.7%agarosein Tris-borate bufferaspreviouslydescribed(1). Thegelwas washed, stained with ethidium bromide, transillumi-nated with UV light, and photographed. This lysis procedure produced a lysate with abundant plasmid DNAandvery littlechromosomalDNA. Plasmid size was determined by comparing thegel migration
dis-tance ofunknown plasmids with that ofplasmids of knownmolecular size.
RESULTS
Antibiotic sensitivity. MR S. aureusorganisms
were recovered from 17 patients (8 infected, 9
colonized), 3 nurses, and 12 items in the
envi-ronment, including charts, telephones, and
stethoscopes. No MR S. aureus organisms were
recovered from air samples. Detailed results of
the epidemiological investigation will be
pre-sented in a separate report.
Theepidemic MRS. aureusisolatewasfound
to be resistant to thefollowing antibiotics:
naf-cillin, cefazolin, gentamicin, amikacin,
clinda-mycin, chloramphenicol, tetracycline,
erythro-mycin, and rifampin. Resistance to methicillin,
gentamicin, rifampin,andclindamycinwasused
asthe characteristic antibiotic resistance
mark-ersfor the epidemic isolate.
During the investigation of the spread of the
epidemic MR S. aureus isolate, surveillance
culturesuncovered avariety of antibiotic
resist-ance patterns. Some S. aureus isolates were
sensitive to allantibiotics; some were resistant
togentamicin andclindamycin, but sensitive to
methicillin andrifampin; and one, found at the
affiliated McGuire Veterans Administration
Medical Center, was resistant to methicillin,
gentamicin, and clindamycin but sensitive to
rifampin. All of these isolates were used as
controls forevaluating theotherepidemiological
markers considered below.
Phagetyping. Acomparisonof all markers for
both theepidemic MRS. aureusisolateand the
controls is shown in Table 1. Although the
epidemic isolate was typed with phage 85, a
reaction was only obtained at a 1/10 phage
dilution aftertwo previous typingattempts had
classified the isolates as nontypable. Three of
thenonepidemicS.aureusisolates were
nontyp-able even at the 1/10 dilution, including one of the MR S. aureus isolates recovered at the
Veterans Administration Medical Center.
Aminoglycoside-inactivating enzymes. The
epi-demic MR S. aureus isolate contained
amino-glycoside-inactivating enzymes with substrate
profiles different from those of the resident
gentamicin-resistant,methicillin-sensitive S.
au-reusisolates (Table 1). Furthermore,virtually all
of the resident gentamicin-resistant S. aureus
isolates at ourhospital have this resistance
en-codedon aself-transmissibleplasmid (G.Archer
and J. L. Johnston; Program Abstr. Intersci.
Conf. Antimicrob. Agents Intersci. Chemother.
22nd, Miami Beach, Fla. abstr. no. 366, 1982),
but gentamicin resistance could not be
trans-ferred fromtheepidemicMRS.aureusisolateto
a suitable recipient by the filter mating
tech-niquesdescribed by Schabergetal. (14).
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397 TABLE 1. Markers forepidemic MR andnonepidemic S.aureusisolates
Isolate Phagetype Antibiogram' Aminoglycoside-inactivatingenzymeb (megadaltons)
Plasmids
Epidemic(W-1) 85 Mr Gr RrCLr 3'-APH(IandII),6'-AAC(I) 34, 1.75,1.5
Nonepidemic
S-i 77, 84 Mr Gr Rs CLr NDc 34
W-2 Nontypable MrGs RsCLr ND None
985 96 MSGs Rs CLr ND 20
962 Nontypable MSGS Rs CLs ND 25
605 Nontypable MSGS RS CLs ND 1.65
866 52,52A, 79,80,6,42E, MsGr R5 CLr 2"-APH, 6'-AAC(I) 37, 1.65
47,53,54, 75, 85
265 Nontypable Ms Gr Rs CLr 2"-APH,6'-AAC(I),4'-ANT(II) 34,31
aM,
Methicillin;
G,gentamicin; R,rifampin;CL,cindamycin.
bAPH, Phosphotransferase; AAC, acetyltransferase; ANT, adenyltransferase. Roman numerals indicate
differentisozymicforms of the enzyme.
cND, Not done.
Plasmid pattern analysis. All of the epidemic
MR S. aureus cells contained three plasmids,
approximately 34, 1.8, and 1.5 megadaltons in molecular size. None of the otherisolates
exam-ined had thisprofile, which readily identified the
epidemic isolate (Table 1 andFig. 1). Attempts
totransfergentamicin (seeabove),clindamycin,
erythromycin, orchloramphenicol resistance to a suitable recipient from the epidemic MR S.
aureusisolate by filtermatingormixed culture
(6) were unsuccessful. With the lysis method outlined above, cells obtained in the morning
could be completely processed (lysis,
electro-phoresis,andphotography) by late afternoon.
DISCUSSION
Theepidemic MR S. aureusisolate
responsi-ble for the outbreak in the Medical College of
Virginia Hospital had a unique antibiogram
which made its identification relatively easy; it
was resistant to rifampin. Rifampin resistance
was a stable chromosomal markernot seen in
any other staphylococcal isolate cultured from
patients,contacts, orthehospital environment.
However, auniqueantibiotic resistance marker
such asrifampin resistance has notbeenfound
in themajorityofMR S. aureusisolates associ-ated with outbreaks in otherhospitals (2, 8, 10, 16). Thereliabilityof the antibiogram in identify-ingaparticularnosocomial staphylococcal iso-late associated with an outbreak can be
ques-tioned for several reasons. First, methicillin resistance is a phenotypic trait which may be
difficulttodetectby standard antibiotic
suscep-tibility testing methods.Itsexpressionmay vary
withinoculum size,incubationtemperature,
me-diumcomposition, andexposure tobeta-lactam antibiotics (12).Second, staphylococciresistant tomultipleantibiotics, including methicillinand
gentamicin, may be present in the hospital
be-fore the introduction of an epidemic strain.
These resident resistant staphylococci maythen
be discoveredduringculture surveysperformed aspartofanoutbreakinvestigation.A different
methicillin- and gentamicin-resistant S. aureus
strain whichwascolonizingpatientsatthe
affili-FIG. 1. Plasmidpatterns oftheepidemicMR and controlS. aureus strains. Thebandsrepresent cova-lently closed circular and chromosomalDNA. Lanes Aand B, Epidemic MRS. aureus strain; anisolate from the indexcase(lane A) (strainW-1 inTable1), andapositive culture fromacolonizedhospital staff contact(laneB). Lanes C throughIshow the nonepi-demic isolateslisted in Table1:lane C,S-1; lane D, W-2; lane E, 985; lane F, 96W-2; laneG, 605;laneH, 866; and laneI,265.Thenumbers tothe leftarethesizes
(in megadaltons)of the plasmidsin theepidemic MR
S. aureus strain;CindicateschromosomalDNA.
I. &.F ..
18,
A
B
C
D
E
F
G
H
I
C
10-1.8
iw-
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ated Veterans Administration Hospital was
foundduringouroutbreak investigation. Itwas notdetected earlier because it causedno infec-tions and was associated with no outbreaks at
thathospital. However, had resistanceto rifam-pinnotbeenaspecific marker for theepidemic
strain, the similar antibiogram of this resistant
staphylococcalstrain and of theepidemic strain
mayhaveconfoundedthe outbreakinvestigation
by raising the possibility that the outbreak had spreadtoasecondhospital.
Phage typing was not particularly helpful in marking the epidemic strain in this outbreak.
Theepidemic strain was susceptibleto lysis by only one phage and then only at 10 times the routine test dilution. This result was only ob-tained when theisolateswere sent to theCenters for Disease Control after variable typing results had beenreported by locallaboratories for
sev-eral weeks. The inability ofMR S. aureus iso-latestobe typed by phage has been reported in otheroutbreaks (13).
Thedeterminationoftheproductionof specif-ic proteins, suchas catalases, has been usedto
identify and mark specific staphylococcal
iso-lates (11). Since the epidemic MR S. aureus
isolatewasresistanttogentamicin,wesoughtto
identifyspecificaminoglycoside-inactivating
en-zymeswhich might distinguish it from the
resi-dent gentamicin-resistant staphylococci. The
epidemic strain produced a unique
phospho-transferasethat was not seen in other gentami-cin-resistant staphylococciand wastherefore a
useful marker. However, many gentamicin-re-sistant staphylococci of the same or different species mayproduce the same
aminoglycoside-inactivating enzymes (3, 7). Furthermore, the
detection of theseenzymesisaspecialized
pro-cedure not readily available to clinical labora-tories.
Because of the limitations of the markers discussedabove, wefound thatplasmidpattern
analysis was a useful epidemiological tool for
fingerprinting MR S. aureus strains. It has the
followingadvantages.
First,
theplasmid
patternis stable. We found that all 32 oftheepidemic
(rifampin-resistant) MR S. aureus isolates
ob-tained over7 months had an identical pattern.
Furthermore,thepatternremainedstable forup
to 1 year of storage at -70°C (G. Archer,
unpublished data). Second, it can be used to
differentiate isolates with similar antibiograms.
Thetworifampin-sensitiveMRS.aureusstrains (Table1, strains S-1 andW-2) discoveredduring
culture surveys at the VeteransAdministration
Hospitalweredifferentiated from each otherand
the epidemic strain by their dissimilar plasmid
patterns(Fig. 1).Finally, thelysis,
electrophore-sis, and photography procedures involved in
plasmidpattern analysiscanbeperformed
rapid-ly and inexpensively by unspecialized
person-nel. Multiple isolates can be screened in less
time than is required todetermine a minimum
inhibitory concentrationor aphagetype.
How-ever, there are limitations to the procedure.
Someone with some understanding of DNA
electrophoresis and microbial genetics mustbe availabletointerpret the gels. For instance,two
patterns may bevery similar but notidentical,
differing by only a single plasmid. The loss or
gainofa single plasmid maybe associated with the lossorgain of antibioticresistance; this can
only bedetermined by curingorplasmid transfer studies. Theacquisition by anepidemic MR S.
aureus strain of a chloramphenicol resistance
plasmid was shown by Locksley, et al. (8).
Otherproblems, suchasthe existence ofasingle plasmid in different molecular forms, mayhave to be interpreted aswell. Furthermore,isolates
with no or only one plasmid will not have
distinctpatterns on gels and cannotbe reliably
distinguishedfromsimilar isolates.
In our study of a discrete MR S. aureus
outbreak involving a single strain newly intro-ducedinto thehospital, plasmid pattern analysis
quickly and reliably fingerprinted the epidemic
isolate. Itwassuperiortobothphagetyping and the antibiogram of routine antistaphylococcal antibiotics as amarker. Itshould beeven more
useful forepidemiological investigations of
com-plexMRS. aureusoutbreaks involving multiple
strains in different hospitals.
ACKNOWLEDGMENTS
We thank J. L. Johnston and G.0. Hall for technical assistance.
This study was supported in part by Public Health Service grant HL-19818 from the National Heart, Lung and Blood Institute.
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VOL.18, 1983
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