Isolation and Antimicrobial Susceptibilities of Chlamydial Isolates from Western Barred Bandicoots

A range of species of Chlamydiales have previously been detectedin a variety of Australian marsupials, including koalas andwestern barred bandicoots. Thirty-seven ocular, urogenital,or nasal swabs were obtained from 21 wild western barred bandicoots.Chlamydia culture and antibiotic susceptibility testing wereperformed for cycloheximide-treated HEp-2 cells in 96-well microtiterplates. Chlamydia spp. were isolated from 11 specimens from9 (42.8%) bandicoots. All isolates were identified as Chlamydialesby conventional PCR with 16S and 23S rRNA gene primers specificto Chlamydiales and were confirmed to be Chlamydia pneumoniaeby a C. pneumoniae-specific ompA-based real-time PCR assay and16S rRNA and 23S rRNA gene signature sequence analyses. TheMICs of azithromycin, doxycycline, ciprofloxacin, and enrofloxacinfor 10 C. pneumoniae isolates from these bandicoots ranged from0.015 to 1 µg/ml, 0.25 to 1 µg/ml, 0.25 to 2 µg/ml,and 0.25 to 0.5 µg/ml, respectively. The MICs at which90% of isolates were inhibited and the minimal bactericidalconcentrations were within the ranges reported previously forhuman isolates of C. pneumoniae.

INTRODUCTION

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Introduction

The range of hosts known to be infected by Chlamydiales hasbeen expanding. Recent studies using a combination of molecularmethods and cell culture have identified several existing andsome novel species of Chlamydiales infecting a wide range ofAustralian marsupials, including koalas and western barred bandicoots(3, 4). Formerly found over much of Western Australia, westernbarred bandicoots (Perameles bougainville) are now an endangeredspecies found in the wild only on Bernier and Dorre Islandsoff the coast of Western Australia (8). Detection of chlamydialorganisms, including Chlamydia pecorum and unidentified Chlamydiales,in these animals has previously been associated with clinicaldiseases in the form of conjunctivitis, eyelid proliferation,and corneal opacity, as well as an asymptomatic state (16).The in vitro susceptibilities of Chlamydia spp. isolated fromAustralian marsupials, including bandicoots, have not been reported.The purpose of this study was to isolate Chlamydia species intissue culture from specimens obtained from these unique hostsand to determine their growth characteristics, species, andin vitro antibiotic susceptibilities.

MATERIALS AND METHODS

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Materials and Methods

Specimen collection. Ocular, urogenital, or nasal swabs were obtained from westernbarred bandicoots from Dryandra, Western Australia, and BernierIslands, Western Australia. All animals sampled were from wildpopulations. Thirteen of these animals showed clinical signsof disease in the form of conjunctivitis, proliferation of theeyelid, and possible pneumonia. Five were noted to have cataractsor corneal scarring suggestive of previous ocular disease.

Cell culture. Chlamydia spp. were isolated in cycloheximide-treated HEp-2cells grown in 96-well microtiter plates as previously described(14). Culture confirmation was done by staining with a Chlamydiagenus-specific fluorescein-conjugated monoclonal antibody (PathfinderChlamydia confirmation system; Kallestad Diagnostics, Chaska,MN). Each specimen was passaged up to 6 times. Isolates foundpositive by culture were propagated for further characterization,including susceptibility testing and molecular diagnostic studies.

In vitro susceptibility testing. Susceptibility testing was performed in cell culture using HEp-2cells as previously described (10). Azithromycin (Pfizer, CT),ciprofloxacin (Miles Pharmaceuticals, West Haven, CT), enrofloxacin(Bayer Corp., Shawnee Mission, KS), and doxycycline were suppliedas powders and solubilized according to the manufacturers' instructions.Each well was inoculated with 0.1 ml of the test isolate dilutedto yield 10³ to 10⁴ inclusion-forming units per ml, centrifugedat 900 x g for 1 h, and then incubated at 35°C for 1 h.Wells were then aspirated and overlaid with 0.2 ml of overlaymedium containing 1 µg/ml of cycloheximide and serialtwofold dilutions of the test drug. After incubation at 35°Cfor 72 h, cultures were fixed and stained for inclusions witha fluorescein-conjugated antibody to the lipopolysaccharidegenus antigen. The MIC was the lowest antibiotic concentrationat which no inclusions were seen. The minimal bactericidal concentration(MBC) was determined by aspirating the antibiotic-containingmedium, washing wells twice with phosphate-buffered saline,and adding antibiotic-free medium. Cultures were frozen at –70°C,thawed, passed onto new cells, incubated for 72 h, and thenfixed and stained as described above. The MBC was defined asthe lowest antibiotic concentration that resulted in no inclusionsafter passage. The MIC₉₀ and MBC₉₀ were defined as the concentrationsof the antibiotic that inhibited or killed 90% of the isolatestested, respectively. All tests were run in triplicate.

DNA extraction. DNA from culture-positive isolates was extracted by using theDNeasy tissue kit (QIAGEN, Valencia, CA) according to the manufacturer'sinstructions and was stored at –20°C prior to PCRanalysis.

C. pneumoniae species identification. (i) 16S and 23S rRNA gene signature sequencing. The 16S and 23S rRNA gene-based signature sequences were determinedin order to identify chlamydial isolates to the species level,as recommended by Everett et al. (7). The amplification/sequencingprimers used for 16S rRNA and 23S rRNA gene products were 16SIGF(5'-CGGCGTGGATGAGGCAT-3'), 16SIGR (5'-TCAGTCCCAGTGTTGGC-3'),U23F (5'-GATGCCTTGGCATTGATAGGCGATGAAGGA-3'), and 23SIGR (5'-TGGCTCATCATGCAAAAGGCA-3'),as described by Everett et al. (7). PCR was performed, usinga QIAGEN ProofStart DNA polymerase kit, at 95°C for 5 min,followed by 45 cycles of 94°C for 30 s, 60°C for 30s, and 72°C for 60 s, and a final extension for 5 min at72°C. PCR products were purified with a QIAGEN QIAquickPCR purification kit and were sequenced in both directions (GeneWiz,North Brunswick, NJ).

(ii) C. pneumoniae-specific real-time PCR TaqMan assay. Chlamydial isolates were further tested using a C. pneumoniae-specificompA-based real time PCR as a second and concurrent test todetect the presence of C. pneumoniae. The C. pneumoniae-specificprimers and probe targeting an 85-bp highly conserved and specificregion of the ompA gene were as follows: primers QMOMP1 (5'-GATCCGCTGCTGCAAACTATACT-3')and QMOMP2 (5'-GTGAACCACTCTGCATCGTGTAA-3') and probe QMOMPS(5'-6-carboxyfluorescein-TAGGCCGGGTTAGGTCTATCTACGGCAGT-6-carboxytetramethylrhodamine-3')(1). Real-time PCR was performed using a Roche LightCycler (version2.0) at 95°C for 10 min, followed by 45 cycles of 95°Cfor 5 s, 65°C or 60°C for 10 s, and 72°C for 10s. A low-titer TW-183 isolate of C. pneumoniae (ATCC VR-2282)was used as a positive control, and double-distilled water wasused as a negative control, in all the in vitro experiments,PCRs, and sequencing reactions.

Sequence analysis. The sequences were analyzed using BLAST 2 (http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/)and were compared to the sequences of 16S and 23S rRNA genesof Chlamydiales available in GenBank, including the three previouslydescribed biovars of C. pneumoniae.

Nucleotide sequence accession numbers. The signature sequences of the 16S rRNA and 23S rRNA genes frombandicoot C. pneumoniae are currently available in GenBank underaccession numbers DQ444323 and DQ465990, respectively.

RESULTS

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Results

Culture. Thirty-seven ocular, throat, and cloacal specimens were obtainedfrom 21 bandicoots. A total of 11 specimens from 9 (42.8%) bandicootswere positive for Chlamydia spp. after 3 to 6 passages, confirmingthe presence of viable chlamydiae in the specimens. Inclusionsvaried in size and granularity but were very similar in appearanceto those of human isolates (Fig. 1). Of the 11 positive swabs,4 were obtained from the left eye, 3 from the right eye, 1 fromthe nose, and 3 from the throat.

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FIG. 1. Photomicrograph of a bandicoot C. pneumoniae isolate showing characteristic chlamydial inclusions in HEp-2 cell culture. Magnification, x200.

16S and 23S rRNA gene-based PCR and sequence analysis. PCR was performed on 10 of the 11 culture-positive specimens.All 10 specimens were shown to belong to the Chlamydiales by16S and 23S rRNA gene primers specific to Chlamydiales. Sequenceanalysis of 16S and 23S rRNA gene signature regions revealedthat all 10 bandicoot isolates were identical to each other.BLAST analysis of the 551-bp segment of the 23S rRNA gene signaturesequence demonstrated that bandicoot isolates were 99.6% and99.1% identical to human (2-bp difference) and equine (5-bpdifference) biovars of C. pneumoniae, respectively. BLAST analysisof the 294-bp 16S rRNA gene signature segment showed that bandicootisolates were 99.3% identical to the human biovar (2-bp difference),99.5% identical to the koala biovar (1-bp difference), and 98.9%identical to the equine biovar (3-bp difference) (GenBank accessionnumbers AE 017160, AF 100957, and U68426, respectively).

ompA-based real-time PCR. All 10 isolates tested positive by a C. pneumoniae-specificompA-based PCR assay, confirming the identity of the isolatesas C. pneumoniae.

In vitro susceptibility testing. The MIC₅₀, MIC₉₀, and MBC₉₀ values for bandicoot isolates ofC. pneumoniae are shown in Table 1. The ranges of MICs and MBCswere 0.015 to 1 µg/ml for azithromycin, 0.25 to 1 µg/mlfor doxycycline, 0.25 to 2 µg/ml for ciprofloxacin, and0.25 to 0.5 µg/ml for enrofloxacin.

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TABLE 1. In vitro antibiotic susceptibilities of 11 bandicoot isolates of C. pneumoniae

DISCUSSION

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Discussion

We were able to isolate Chlamydia spp. in tissue culture frommultiple anatomic sites from 42.8% of bandicoots in this sample.Tissue culture using cycloheximide-treated HEp-2 cells appearsto be an effective system for isolation and propagation of theseorganisms. The isolates were propagated for further analysisby PCR. All isolates were confirmed to be Chlamydiales by stainingin cell culture with monoclonal antibodies specific to the Chlamydiaceaeand by conventional PCR with 16S and 23S rRNA gene primers specificto Chlamydiales, and they were subsequently identified as C.pneumoniae by a highly species specific ompA-based real-timePCR assay using specific primers and a specific probe. In addition,16S rRNA and 23S rRNA signature sequence analysis revealed similaritiesof 99.3% and 99.6%, respectively, to human isolates of C. pneumoniae;similarity of more than 95% is enough to confirm the species(7). C. pneumoniae is considered to be a primarily human respiratorypathogen, although recent reports have identified koalas, frogs(2, 11, 12), and horses (15) as additional hosts and have detectedthe koala biovar in human carotid plaque specimens obtainedfrom patients undergoing elective endarterectomy in Australia(6). The identification of C. pneumoniae infections in bandicootsprovides further evidence for the expanding host range of thisspecies. The identification of novel hosts and biovars of C.pneumoniae raises interesting questions about the evolutionand epidemiology of this pathogen, specifically the presenceof animal reservoirs and additional modes and or directionsof transmission across species. Additional studies are neededto gain further understanding of these issues.

Although molecular techniques have been increasingly used inthe past few years for the detection of sequences of Chlamydialesin a wide range of animal and environmental sources, and althoughthese techniques, in combination with sequencing, have facilitatedthe discovery of novel Chlamydiales (3) and an increasing rangeof chlamydial hosts, isolation and propagation of the organismin culture allows further antigenic and molecular characterizationof isolates and remains essential for determination of in vitrosusceptibilities. Several of the animals in this study wereinitially treated with beta-lactam antibiotics without responsebut subsequently responded to intramuscular tetracycline. Invitro testing of the susceptibilities of these C. pneumoniaeisolates to azithromycin, ciprofloxacin, and doxycycline demonstratedantibiotic susceptibilities similar to those reported for humanisolates (9). Enrofloxacin, a veterinary quinolone, has beendemonstrated to be very active against Chlamydia psittaci invitro (5) and has been used for the treatment and preventionof psittacosis in birds (13). Enrofloxacin has not been testedagainst C. pneumoniae. Although in vitro susceptibilities maynot necessarily predict in vivo efficacy, these data indicatea possible role for enrofloxacin in the treatment of C. pneumoniaeinfections in Australian marsupials.

ACKNOWLEDGMENTS

We thank the Australian veterinarians who did the field workand collected the specimens.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pediatrics, Division of Infectious Diseases, State University of New York, Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203. Phone: (718) 270-7588. Fax: (718) 270-3210. E-mail: swati.kumar{at}downstate.edu.

Published ahead of print on 22 November 2006.

S.K. and A.K. contributed equally to this work.

REFERENCES

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