Amoebae feed on bacteria and few bacteria can resist t - PDF document

pathogens from clinical specimens. Amoebae-resisting BacteriaIsolated from Human Nasal Swabsby Amoebal Coculture G. Greub’s current affiliation is Institute of Microbiology, University Patients and Study Designipants. All participants took part freely in the study andgave their written informed consent. Amoebal coculturemethod of T.J. Rowbotham (19). Peptone yeast-extract glu-in 5 Lof distilled water. Page’smodified Neff’s amoebae saline (PAS): 120 mg NaCl, 4 mgin 1 Lof distilled water. Nonnutritive agarplates: 1.5 g agar (Research Organics, Cleveland, OH) wasdiluted in 100 mLof PAS. All media were autoclaved 15m pore membrane (Corning, New York, NY). with 30 mLPYG. After 5 days, the amoebae were harvest-resuspended in 50 mLof PAS. Centrifugation and resus-pension steps were repeated twice. After the last centrifu-gation, the amoebae were resuspended in PAS, and 1 mL/mLwas distributed in each well ofNasal swabs were vortexed for 30 seconds in 1 mLofPAS in individual sterile tubes. The suspension was cen-(1,000 rpm) for 10 minutes. Two hun-amoebal microplates. The pellet was spread on nonnutri-Enterobacter aerogenesin PAS, drained, andatmosphere. The amoebal cocultures were subcul-persons). Amoebal cocultures were examined regularly foramoebal lysis. When amoebal lysis was observed and atamoebal bacteria. This screening was achieved by gentlyshaking the microplates to suspend the amoebae. Then 150Lof the suspension was cytocentrifuged at 150 x the malachite green-stained amoebae. When Gimenez-tured on BCYE agar (BioMérieux, Marcy L’Etoile,phere and examined daily for amoebae-like cells. Whennonnutritive agar plates seeded with viable or UV-inacti-Enterobacter aerogenes. After several subculturesE. aerogenesstudied by Gram and Gimenez staining. To confirm that/mLsuspensions were injected with theisolated strain and incubated at 32°C. After 4 days’incuba-DNAwas extracted according to the manufacturer’sCA) or FastDNAkit (Bio101, Carlsbad, CA) andstrain recovery. Polymerase chain reaction (PCR) amplifi-cation of the 16S rDNAgene was performed by using thefD1 and rP2 primers (21) and Taq DNApolymeraseto manufacturer instructions. The success of the amplifica- Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 3, March 2004471 Statistical Analysistest (STATAsoftware version 7.0., Stata Corporation, College Station,ResultsWe observed trophozoites growing on nonnutrient agarg/mL). Thus, no free-living amoebaeand 41% of cultures from control samples. These fungistrains of ARB from nasal swabs of 4 of 444 participants.volunteers; p = 0.017). All ARB were gram-negative bacil-of 16S rDNAsequence analysis, the isolated ARB were(n = 2). The Table shows the taxonomy and the 16S rDNAARB. When we used a 16S rDNAsequence similarity ofand genus level, respectively (33), four (57%) ARBrecovered from a 44-year-old healthy homeless man. The16S rDNAsequence of isolate 1 shared on BLASTanaly-sp.implicated ina catheter-related bacteremia (X.Y. Han, unpub. data) andR. gilardiistrain ATCC 49956(Table).,as it shares 93.9%–98.9% 16S rDNAtively. Isolate 1 clustered with spp.by phylo-genetic analysis of the 16S rDNAgene (Figure 1). Thus,bootstrap values of 98%, 99%, and 90% in neighbor-join-tively, supported the fork separating (including isolate 1)from their closest relatives,Craurococcus roseusParacraurococcus ruberimportantly, the isolate was clearly different from the typeR. gilardii100% in neighbor-joining, minimum evolution, and parsi-de l’Institut Pasteur, Paris, France, as strain CIP107751Isolate 2 was recovered from a 27-year-old homelessphadenopathy. The 16S rDNAsequence of isolate 2 sharedon BLASTanalysis 99.3% homology withMethylobacterium extorquens(Table).M. extorquensM. extorquensMoreover, isolate 2 clustered with by phylogenetic analysis of the 16S rDNAgene (Figure 1). Isolate 3 was recovered from a 48-year-old HIV-posi- Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 3, March 2004473 Figure 1. 16S rDNAtree showing relationship of isolates 1 to 5 withrelated alpha- and beta-proteobacteria. The tree was constructedplete sequence (1,289 nt) of the 16S rDNAgene. Bootstrap valuesresulting from 100 replications are at branch points.Staphylococcus aureus Streptococcus pneumoniae. The 16SrDNAsequence of isolate 3 shared on BLASTanalysis(Table).,as it shares 98%–99% 16S rDNAsequencespp.by phylogenetic analysis of the 16S rDNAgene(Figure 1). However, it clustered with ,with100% in neighbor-joining, minimum evolution, and parsi-mony analyses, respectively. The morphology of isolate 3, as seen by electron microscopy, isered from an 80-year-old woman with pacemaker-associat-ed endocarditis. The 16S rDNAsequence of isolate 4shared on BLASTanalysis 98.7% homology withRhizobium larrymooreistrain 3–10(Table). Although theAgrobacterium [34]),it sharedgenetic analysis of the 16S rDNAgene with a node sepa-rating them from the closest type strain neighbor, larrymoorei and 100% in neighbor-joining, minimum evolution andparsimony trees, respectively. Isolate 4 has been depositedin the Collection de l’Institut Pasteur, Paris, France, asstrain CIP10774980-year-old woman with a pacemaker-associated endo-carditis. The 16S rDNAsequence of isolate 5 shared onBLASTanalysis 99.5% homology with Achromobacterstrain ATCC 9220(Table). Genetic analysisAchromobacterAchromobacter Bordetella Achromobacter spp.by phylogenetic analysis of the 16SrDNAgene (Figure 1). Thus, bootstrap values of 100% inneighbor-joining, minimum evolution, and parsimonytrees supported the fork’s separating the Achromobacterspp. (including isolate 5)from Bordetella Candidatus Amoebinatus massiliae,(like iso-late 3) was recovered from a 48-year-old HIV-positive man. The 16S rDNAsequence of isolate6 shared on BLASTanalysis 99.2% homology with that of(Table). On genetic analysis, isolate 6 shared93.1%–96.0% 16S rDNAsequence homology with, respectively. Phylogenetic analysis ofthe 16S rDNAgene also suggested that isolate 6 belonged(Figure 3). Thus, bootstrap values of 100%, 100%, and34% in neighbor-joining, minimum evolution, and parsi-mony analyses, respectively, supported the fork’s separat-6)from their closest relatives. Isolate 6 has been depositedin the Collection de l’Institut Pasteur, Paris, France, asCandidatus Amoebinatus massiliaestrain CIP107750isolates 4 and 5) was recovered from an 80-year-old woman 474Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 3, March 2004 RESEARCH sp. (isolate 3)in the process of being phagocy-as seen on electron microscopy. 8,900x magnification. Bar repre-sents 200 nm. B. -like rod (isolate 7)within (arrows), as seen on electron microscopy. Arrow showmagnification. Bar represents 200 nm. AB with pacemaker-associated endocarditis. The 16S rDNAsequence of isolate 7 shared on BLASTanalysis 98.0%(Table). On genetic analysis, isolate 7 shared 92.5%–97.1%16S rDNAsequence homology with respectively. Phylogenetic analysis of the 16S rDNAgene(Figure 3). Thus, bootstrapvalues of 100%, 100%, and 97% in neighbor-joining, min-imum evolution, and parsimony analyses, respectively, sup-ported the fork’s separating isolate 6 from its closestneighbor, an unpublished species of as seen by electron microscopy, is shown in Figure 2.Pasteur, Paris, France, as strain CIP107752,and ing number of ARB. Our study also shows that humans areexposed to unknown ARB and that the amoebal cocultureis an effective tool for the recovery of new species fromUntil this study, the ARB included Bradyrhizobiaceae Procabacter acanthamoeba,a betaproteobacteria) (42). In this study, we recovered additionalextorquans, . We alsoAchromobacter xylosoxidans(betaproteobacteria)and two new C. Amoebinatus massiliaeof humans to these bacteria. From the nasal mucosa, ARBto intracellular life. The fact that ARB were more likely towhether patients were colonized with ARB during theirhospitalization, whether colonization with ARB may be athe isolated ARB may itself be the etiologic agent of theEnterobacteriaceae, StaphylococciStreptococciin a nasal sample). Thus, using thatfrom human feces (43). Moreover, the amoebal coculturecrobial susceptibility. Relative to a broad amplificationstrategy for microbial rDNAfound in nasal washings,ceptibility, and virulence may then be further character-heavily contaminated sample. As amoebal resistance may Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 3, March 2004475 Figure 3. 16S rDNAtree showing relationship of isolates 6 and 7. The tree was constructed by usingsequence (1,283 nt) of the 16S rDNAgene. Bootstrap valuesresulting from 100 replications are at branch points.Staphylococcus aureus be associated with virulence (11–16), the amoebal cocul-growing potential human pathogens. The main limitations 37°C. Another limitation is that, contrary tothe amoebal host, if any, present in the sample. In addition,We did not isolate where the survey was performed. This finding may alsoAcknowledgmentsWe thank the Swiss National Science Foundation for fund-reviewing the manuscript; P. Brouqui for allowing us to swab theDiseases, Hôpital Nord, Marseille; and T.J. Rowbotham for kind-Dr. Greub is a specialist in medical microbiology and infec-and other emerging intracel-1.Rodriguez-Zaragoza S. Ecology of free-living amoebae. Crit Rev2.Michel R, Hauröder-Philippczyk B, Muller KD, Weishaar I.3.Amann R, Springer N, Schönhuber W, Ludwig W, Schmid EN,spp.Appl Environ Microbiol1997;63:115–21.4.Rowbotham TJ. 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Appl 476Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 3, March 2004 RESEARCH