Detection of a microbial biofilm in intraamniotic infection
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Transcript of Detection of a microbial biofilm in intraamniotic infection
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etection of a microbial biofilm in intraamniotic infectionoberto Romero, MD; Christoph Schaudinn, MSc; Juan Pedro Kusanovic, MD; Amita Gorur, MSc; Francesca Gotsch, MD;aul Webster, PhD; Chia-Ling Nhan-Chang, MD; Offer Erez, MD; Chong Jai Kim, MD; Jimmy Espinoza, MD;uis F. Gonçalves, MD; Edi Vaisbuch, MD; Shali Mazaki-Tovi, MD; Sonia S. Hassan, MD; J. William Costerton, PhD
BJECTIVE: Microbial biofilms are communities of sessile microor-anisms formed by cells that are attached irreversibly to a substratumr interface or to each other and embedded in a hydrated matrix ofxtracellular polymeric substances. Microbial biofilms have been im-licated in �80% of human infections such as periodontitis, urethritis,ndocarditis, and device-associated infections. Thus far, intraamnioticnfection has been attributed to planktonic (free-floating) bacteria. Aase is presented in which “amniotic fluid sludge” was found to con-ain microbial biofilms. This represents the first report of a microbialiofilm in the amniotic cavity.
TUDY DESIGN: “Amniotic fluid sludge” was detected by transvaginalonography and retrieved by transvaginal amniotomy. Bacteria weredentified with scanning electron microscopy and fluorescence in situybridization for conserved regions of the microbial genome; the ex-
002-9378/$34.00 • © 2008 Mosby, Inc. All rights reserved. • doi: 10.1016
gglutinin lectin method. The structure of the biofilm was imaged withonfocal laser scanning microscopy.
ESULTS: “Amniotic fluid sludge” was imaged with scanning electronicroscopy, which allowed the identification of bacteria embedded in
n amorphous material and inflammatory cells. Bacteria were demon-trated with fluorescent in situ hybridization using a eubacteria probe.xtracellular matrix was identified with the wheat germ agglutinin lectintain. Confocal microscopy allowed 3-dimensional visualization of theicrobial biofilm.
ONCLUSION: Microbial biofilms have been identified in a case of in-raamniotic infection with “amniotic fluid sludge.”
ey words: amniocentesis, amniotic fluid sludge, clinicalhorioamnionitis, intraamniotic infection, microbial invasion of the
ite this article as: Romero R, Schaudinn C, Kusanovic JP, et al. Detection of a microbial biofilm in intraamniotic infection. Am J Obstet Gynecol 2008;198:35.e1-135.e5.
icrobial invasion of the amni-otic cavity has been detected in
he mid trimester of pregnancy in ap-arently healthy women1-5 and in themniotic fluid of women with cervicalnsufficiency,6,7 preterm labor with in-act membranes, preterm prelaborupture of membranes, idiopathic vag-nal bleeding,8 premature rupture of
embranes at term,9,10 spontaneous
labor at term with intact membranes,11
and clinical chorioamnionitis. Intra-amniotic infection is a common mech-anism of disease in obstetrics; bacteriacan attack the fetus and cause systemicinflammation (a fetal inflammatory re-sponse syndrome12,13) and multipleorgan damage.14
The microbiologic diagnosis of infec-tion has been based on the use of culti-
vation techniques in which bacteria arerecovered from amniotic fluid. Growthof microorganisms in the laboratory de-pends on the provision of suitable nutri-tive and environmental conditions. Thispractice was developed after the seminalobservations of Robert Koch, who estab-lished the time-honored isolation andpure culture techniques. However, thereare 2 major limitations of cultivation-based techniques. First, approximately99% of bacteria in aquatic and soil eco-systems resist cultivation (a phenome-non referred to as “the great plate countanomaly”15); second, bacteria grow incommunities called “biofilms,” which typ-ically adhere to surfaces.16 Molecular mi-crobiologic techniques have been used re-cently to detect microorganisms in theamniotic cavity.17-21
The biofilm theory states that mostbacteria that grow in matrix-enclosedbiofilms differ from their planktoniccounterparts22 (isolated bacteria seen inGram stain examinations of biologic flu-
rom the Perinatology Research Branch, NICHD/NIH/DHHS, Bethesda, MD, and Detroit,I (Drs Romero, Kusanovic, Gotsch, Nhan-Chang, Erez, Kim, Espinoza, Gonçalves,aisbuch, Mazaki-Tovi, and Hassan); the Departments of Obstetrics and Gynecology (Drsomero, Kusanovic, Nhan-Chang, Erez, Espinoza, Gonçalves, Vaisbuch, Mazaki-Tovi, andassan) and Pathology (Dr Kim), Wayne State University/Hutzel Women’s Hospital, and
he Center for Molecular Medicine and Genetics, Wayne State University (Dr Romero),etroit, MI; and the Center for Biofilm, School of Dentistry, University of Southernalifornia, Los Angeles (Dr Costerton, Mr Schaudinn, and Ms Gorur), and the House Ear
nstitute (Dr Webster), Los Angeles, CA.
eceived Oct. 21, 2007; accepted Nov. 14, 2007.
ddress correspondence to Roberto Romero, MD, Perinatology Research Branch, NICHD/NIH/HHS, Wayne State University/Hutzel Women’s Hospital, 3990 John R-Box #4, Detroit, MI8201. [email protected].
his research was supported in part by the Intramural Research Program of the National Institutef Child Health and Human Development, NIH, DHHS.
polymeric matrix was identified by histochemistry by the wheat germ
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ds or of pure cultures). It is now recog-
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ized that biofilms play a major role inuman disease. Periodontitis, otitis me-ia, endocarditis, prostatitis, biliary tract
nfections, and many other infections inhich there is a device (eg, prostheticalves, catheters) involve bacterialiofilms.23
Microbial biofilms are important be-ause bacteria in these communities areore resistant to antimicrobial agents
nd to the host response to infec-ion.24-27 Moreover, since bacteria iniofilms are more difficult to isolate inulture, these infections are oftenverlooked.The presence of particulate matter in
he amniotic fluid in close proximity tohe cervix was recognized recently asamniotic fluid sludge.”28 This findingas associated with microbial invasionf the amniotic cavity, impending pre-erm delivery, and histologic chorioam-ionitis.28 Subsequently, these resultsere confirmed in asymptomatic pa-
ients with sludge in the mid trimester ofregnancy29 and in those with cervicalerclage.30 The finding that “amnioticuid sludge” represents the presence ofacteria and intraamniotic inflamma-ion has been reported recently.31
Because of the original description ofamniotic fluid sludge,” it has been spec-lated that this material may reflect theresence of biofilms in the amnioticuid,28 which is a hypothesis with bio-
ogic, diagnostic, and therapeutic impli-
FIGURE 1Two-dimensional transvaginalultrasound image shows thepresence of “amniotic fluidsludge”
omero. Microbial biofilm in intraamniotic infection. Am Jbstet Gynecol 2008.
ations. The evidence in support of the i
35.e2 American Journal of Obstetrics & Gynecol
ontention that microorganisms canorm a biofilm in the amniotic cavity ishe subject of this case report.
ATERIALS AND METHODSo determine whether amniotic fluid
ludge (Figure 1) represents a biofilm,he material was aspirated by transvagi-al needle amniotomy under ultrasounduidance in a patient at 28 weeks of ges-ation with spontaneous preterm labornd clinical chorioamnionitis. This wasone in accordance with an Institutionaleview Board–approved protocol; theatient provided written informed con-ent at the time of enrollment, before theollection of the amniotic fluid samples.mniotic fluid studies indicated a glu-ose concentration of �10 mg/dL, ahite blood cell count of 19,650 cells/m3, and the presence of Gram-positive
occi. The patient was treated with am-icillin and gentamycin for the clinicaliagnosis of chorioamnionitis, and laborrogressed quickly to a spontaneous vag-
nal delivery of a female infant whoeighed 1135 g, with Apgar scores at 1
nd 5 minutes of 8 and 8. The amnioticuid culture obtained from the sludgeaterial was positive for Mycoplasma
ominis, Streptococcus mutans, and As-ergillus flavus. Histologic examinationf the placenta revealed marked acuteecrotizing chorioamnionitis and acute
unisitis. The details of this case haveeen reported previously.31 The new-orn infant was admitted to the neonatal
ntensive care unit and experienced met-bolic acidosis and respiratory distressyndrome that resolved in the first weekf life. There was no evidence of pneu-onia; a neurosonogram was normal,
nd microbial cultures of the cerebrospi-al fluid and blood were negative. How-ver, the newborn was treated with am-icillin and gentamycin because ofuspected sepsis. After 45 days, the infantas discharged home in good condition.
canning electron microscopymniotic fluid was dehydrated in araded ethanol line, critical point driedEMS 850), mounted on a stub, sputter-oated with 8-nm platinum, and exam-
ned with a scanning electron micro-ogy JANUARY 2008
cope with 5 kV in the secondarylectron mode (XL30 SFEG; FEI Inc,illsboro, OR).
luorescent in situ hybridizationFISH) and confocal lasercanning microscopymniotic fluid was transferred to 100%thanol for 24 hours and washed withhosphate-buffered saline solution. Vol-mes of 100 �L of the amniotic fluidere hybridized with the probe EUB338-y3 (final concentration, 5 ng �L�1; In-
egrated DNA Technologies, Coralville,A) for 90 minutes at 46°C in the dark.he samples were washed with buffer for0 minutes at 46°C in the dark.
heat germ agglutinin lectintainistochemistry was used to detect the
resence of extracellular matrix that isharacteristic of a biofilm. This was per-ormed by the wheat germ agglutinin
ethod (final concentration, 20 ngL�1), as described in the followingebsite: Vector Laboratories, Burlin-ame, CA; www.vectorlabs.com. Stain-ng was performed at room temperature
FIGURE 2Scanning electron micrographof a floc of “amniotic fluidsludge” shows the bacterialcells and the exopolymericmatrix material that constitutea biofilm
n the center of the image, cocci are resolvedmong a fibrous mass of matrix material (the barepresents 5 microns). The lectin-based evi-ence for a matrix and molecular evidence foracterial presence are demonstrated in Figure 4.omero. Microbial biofilm in intraamniotic infection. Am Jbstet Gynecol 2008.
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or 20 minutes, which was followed by 3ashing steps with double-distilled wa-
er. The amniotic fluid was mounted onslide and examined with confocal laser
canning microscopy (LSM 5 PASCAL;arl Zeiss, Thornwood, NY). These
echniques have been used extensively inhe study of biofilms and published else-here in detail.32,33
ESULTScanning electron microscopy showedocs of “amniotic fluid sludge” that con-isted of bacterial cells and the exopoly-
eric matrix material that are typical of aiofilm (Figure 2). Cocci are resolvedmong a fibrous mass of matrix material.igure 3, A shows a microbial biofilmith neutrophils. Figure 3, B shows bac-
erial cells (coccoid) in the form of indi-idual cells or as a chain of cocci on theurface of a fetal epithelial cell and par-ially covered in amorphous slime. Fig-re 3, C shows large aggregates of biofilmnd most of the bacterial cells that werenclosed in amorphous matrix material.igure 3, D shows the presence of well-efined bacterial cells on the surfacef an epithelial cell; bacteria in this sur-ace have formed amorphous matrix
aterial.The evidence that “amniotic fluid
ludge” is a biofilm is presented in Figure. Figure 4, A is a confocal scanning lasericrograph of a floc. The presence of
acteria or bacterial fragments is dem-nstrated by Figure 4, B by FISH usinghe Eubac 338 probe. The presence of anxtracellular matrix is demonstrated inigure 4, C with the use of wheat germgglutinin lectin, which reacts with the-acetylglucosamine of the matrix ex-polymer. Figure 4, D is the composite
mage of the previous 3 images and illus-rates bacteria within the biofilm. Figure
is a 3-dimensional reconstruction ofhe biofilm with the use of confocal lasercanning microscopy. (A video clip ofhe bacterial biofilm is available online).
OMMENThe observations reported herein repre-
ent the first evidence that bacteria canorm a microbial biofilm within the am-
iotic cavity and that such biofilm was gound in a case with “amniotic fluidludge.”
The following evidence determineshat the material retrieved from the am-iotic cavity represents a biofilm: (1)acteria detected with FISH, with the usef a probe against the conserved se-uence of prokaryotes; (2) bacterial ag-regates were separated by material thatesembled a matrix, and (3) lectin-baseddentification of exopolymeric matrixhat stained with wheat germ agglutinin.
Biofilms are defined as communities ofessile organisms characterized by cellshat are attached to a substratum or in-erface or to each other, that are embed-ed in a hydrated matrix of extracellularolymeric substances they have pro-uced, and that exhibit an altered phe-otype with respect to growth rate and
FIGURE 3Scanning electron micrograph of “
, The biofilm, in the center of the image, and soccoid bacteria are on the surface of a fetal epitf individual cells (arrow 1), a chain of cocci (aartially “buried” in amorphous slime (arrow 3)hair shed from the fetus; most of the bacteria, The presence of 4 very well-defined bacteripithelial cell, although other bacteria that hamorphous matrix material.omero. Microbial biofilm in intraamniotic infection. Am J O
ene transcription in comparison to c
JANUARY 2008 America
lanktonic cells.22 It has been postulatedhat most bacteria grow in matrix-en-losed biofilms adherent to surfaces in allutrient-sufficient aquatic ecosystems,nd that these sessile bacterial cells differrofoundly from their planktonic coun-erparts, which accounts for most physi-logic processes in these ecosystems.16,22
ndeed, based on direct microscopic ob-ervations and quantitative recoveryechniques, it has been demonstratedhat �99% of the bacteria grow in bio-lms on a wide variety of surfaces.22
Bacterial biofilms have been shown tolay a major role in many chronic infec-ions. Indeed, a public announcement ofhe National Institutes of Health hastated that biofilms account for �80% of
icrobial infections of the body. Bio-lms have been implicated in vaginitis,
niotic fluid sludge”
ral neutrophils are identified by arrows. B, Theal cell; these bacterial cells are seen in the form
2), and a biofilm aggregate in which they areSeveral large aggregates of biofilm are next to
lls are enclosed in amorphous matrix material.ells (arrows) on the rugose surface of a fetalcolonized this surface have begun to accrete
t Gynecol 2008.
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is. Moreover, they are also important inolonizing medical devices such as uri-ary, venous, and arterial catheters. Bio-lms have been found in pacemakers,eart valves, vascular grafts and stents,rtificial joints and pins, and even breastmplants.
This communication is based on thebservations of a single case. The preva-
ence of microbial biofilms in intraamni-tic infection is unknown. However, theecognition that bacteria can form bio-
FIGURE 4Demonstration that “amniotic fluid
hese images were generated with confocal lasef “amniotic fluid sludge” without staining of antained with the EUB338-Cy3 probe for eubacomponent of bacteria. Bacteria and bacterial fraeen stained with wheat germ agglutinin, whomponent of the matrix material that formsuperposition of the 3 images (A, B, and C) shoome unstained material that is likely to represenepresents 100 microns.omero. Microbial biofilm in intraamniotic infection. Am J O
lms within the amniotic cavity is im- t
35.e4 American Journal of Obstetrics & Gynecol
ortant for both diagnostic and thera-eutic reasons. First, the diagnosis oficrobial invasion in the presence of
iofilms is extremely challenging, andurrent cultivation techniques are inad-quate to detect such infections. Theonsequence is that the frequency of in-ection of the amniotic cavity may be un-erestimated and that molecular micro-iologic techniques will be required to
mprove diagnosis.17-21 Second, the op-imal treatment of biofilm-related infec-
ludge” is a biofilm
canning microscopy. A, The structure of a flocmponent. B, The same structure that has been
a, which reacts with the 16S ribosomal RNAents are seen in red. C, The same floc that has
reacts with the N-acetylglucosamine of thee structural framework of the biofilm. D, Abacteria (red dots), matrix material (green), andost components trapped by the biofilm. The bar
t Gynecol 2008.
ions represents a challenge in clinical
ogy JANUARY 2008
edicine. Antimicrobial agents appearo be inactivated or fail to reach bacteriaithin a biofilm. Interestingly, bacteriaithin biofilms have increased resistance
o antimicrobial compounds, evenhough the bacteria can be sensitive tohe same agent if grown under standardonditions.24-27 Thus, the difficulties inhe treatment of intraamniotic infection
ay be due to the refractoriness of bio-lms to conventional antibiotic treat-ent. Third, biofilms in the amniotic
uid may represent a unique form ofhese structures that can be dislodged byetal movement and result in the seedingf planktonic bacteria and the eliciting ofn inflammatory response. This studylso demonstrates that biofilms in themniotic fluid can be formed by multiplerganisms. Further research is requiredo characterize, with specific probes, the
icrobial constituents of amniotic fluidiofilms. The mechanisms responsibleor the formation of a microbial biofilmn amniotic fluid are unknown and maynvolve a combination of microbial andost factors. f
FIGURE 5Three-dimensionalreconstruction of sequential Zstack images of a bacterialbiofilm in amniotic fluid byconfocal laser scanningmicroscopy
acteria are stained in red because of the hy-ridization with the probe EUB338-Cy3 (a videolip is available online).omero. Microbial biofilm in intraamniotic infection. Am Jbstet Gynecol 2008.
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