Study on Survival of Chlamydia trachomatis in the Presence of Antichlamydial Drugs

Problem statement: Recurrent genital Chlamydia trachomatis infections due to treatment failures may results in complex sequelae leading to reproductive complexity and morbidity. It can be resulted by the heterotypic resistance with decreas ed drug susceptibility characteristic of the isolat e. Studies are needed to understand the treatment fail ures and resistance characteristic of C. trachomatis. Hence, in vitro study was conducted on C. trachomatis isolate in the presence of antichlamydial drugs. Approach: Our aim was to study geD gene in C. trachomatis clinical isolate having decreased drug susceptibility profile and to analyze HeLa cells ph enotypically upon infection in presence of antichlamydial drugs. Sequencing was done to check any mutational change (s) in ygeD gene of C. trachomatis isolate (CT-244), mRNA expression was analyzed in presence of antichlamydial drugs by Real Time RT-PCR. Transduction study was carried ou t in infected HeLa cells to detect changes at cellular level in presence of antichlamydial drugs by transducing with GFP/RFP-tagged proteins and analyzed by FACS. Results: A point mutation was detected in ygeD gene of C. trachomatis isolate. Further, mRNA expression level of ygeD gene was observed to be increased at 8 hpi in pres enc of doxycycline while in presence of azithromycin it wa s increased at 24 hpi. GFP-tagged plasma membrane protein expression in infected HeLa cells found to be reduced as compare to the uninfected cells. Upon infection, the RFP-tagged actin protein expression was up-regulated in comparison to the uninfected HeLa cells. No difference in expression of plasma membrane and actin protein was observed in susceptible serovar D and CT-244 isolat e. Conclusion: The present study suggest that C. trachomatis isolate with decreased drug susceptibility profile may have an active efflux strategy for its survival in the presence of antichlamydial drugs an d it may not affect its host cell plasma membrane o r actin organization for its survival in order to res ist the antichlamydial drugs.


INTRODUCTION
Chlamydia trachomatis an obligatory intracellular pathogen causes a spectrum of clinically important chronic inflammatory diseases of human. C. trachomatis infection is one of the most prevalent sexually transmitted diseases in the world (Gerbase et al., 1998;Beagley and Timms, 2000). In females, C. trachomatis causes cervicitis, urethritis, ectopic pregnancy, pelvic inflammatory disease, tubal factor infertility and chronic pelvic pain (Morre et al., 2000). Studies have also implicated association of C. trachomatis infection with cervical and ovarian cancer and increase in HIV infectivity (Luostarinen et al., 2004). Antibiotics have the major role in treating chlamydial infections; azithromycin and doxycycline are considered as first line drugs by the Centers for Disease control and prevention (CDC) (Workowski and Berman, 2010). Efficacy of these drugs for treatment of chlamydial infections are high, however many researchers report the problem of recurrent infections and treatment failures Wang et al. (2005). It has also been reported that in women with persistent or recurrent infections, the infection can spread upwards from the endocervix to the fallopian tubes and may result in infertility or ectopic pregnancy (Hillis et al., 1997). Recurrent C. trachomatis infections often results from failure of antibiotic therapy or from reinfection due to unprotected sexual contact with either an untreated existing partner or a new infected Partner (Hillis et al., 1994). However, C. trachomatis atypical intracellular characteristics as persistent bodies are suggested to have a role resulting in refractory to antichlamydial drugs and recurrent infections (Beatty et al., 1994). Further, the emerging antibiotic resistance in chlamydia may create severe problems in the treatment of disease. There are few documented in vitro reports of antibiotic resistance in chlamydia but no examples of natural and stable antibiotic resistant strains collected from humans. Samra et al. (2001) few studies with clinical isolates of C. trachomatis from treatment failure patients demonstrated in vitro heterotypic resistance. Recently, 4 clinical isolates demonstrated in vitro resistance to macrolides were shown to carry mutations in the 23S rRNA gene (Misyurina et al., 2004). In vitro studies suggest that antibiotic-resistant genotypes of C. trachomatis can be generated and transferred to C. trachomatis, C. suis or C. muridarum isolates with capability of expressing significant resistant phenotypes (Sandoz and Rockey, 2011). Hence, emerging heterotypic bacterial resistance against antichlamydial drugs resulting in treatment failures in clinical settings cannot be neglected. Studies are needed for characterization of C. trachomatis clinical isolates showing decreased susceptibility towards the antichlamydial drugs which may results in resistant characteristics of the bacteria and can be concluded with respect to the patient's treatment failure (s) or reinfection (s).
Further, it has also been suggested that genotypic changes may not be only responsible for the resistant characteristics of clinical C. trachomatis isolate (s) obtained from multiple treatment failure patients. Different drugs have different targets for their action in bacteria hence; mutation in a single gene may not be suggested to result in multiple treatment failures. It has been reported that in gram-negative pathogens, efflux is the predominant mechanism of tetracycline resistance including Chlamydia suis Dugan et al. (2007). Hence, studies are needed to explore the role of efflux gene(s) in emerging resistance in C. trachomatis. In addition, in the presence of stress conditions host cell might play a role in altered drug sensitivity profile of bacteria. Resistant bacteria may act on various system of a cell directly or indirectly for its survival in the presence of drugs. According to many studies C. trachomatis changes host cell plasma membrane and actin organization by modifying its arrangements to complete its life cycle (Kumar and Valdivia, 2008a;2008b).
In India, a high prevalence (>30%) of C. trachomatis infections in symptomatic female patients have been reported. (Singh et al., 2003) In our previous study the antibiotic susceptibility profile was studied towards the first line antichlamydial drugs and decreased in vitro susceptibility was observed in isolates (Bhengraj et al., 2010). Few of them appeared as of heterotypic resistant isolates in cell culture in the presence of antichlamydial drugs. Further we characterized them for presence of possible mutational changes at the reported resistant marker genes (L4, L22, 23SrRNA) (Bhengraj et al., 2011). However, genotypic characterization did not revealed any mutational changes at the drug target site(s), hence further characterization is needed.
Thus the aim was to study the efflux (ygeD) gene in C. trachomatis heterotypic resistant isolate for presence of any mutational change(s) and mRNA expression in cell culture condition in the presence of azithromycin and doxycycline. In addition to that host HeLa cell plasma membrane and actin was also studied to know if C. trachomatis indirectly affects on it in the presence of antichlamydial drugs to complete its life cycle, which may result in vitro altered drug susceptibility characteristics.
Antimicrobial agents: Azithromycin and doxycycline (Sigma-Aldrich) were dissolved according to the manufacturer's instructions and dilutions were prepared in DMEM cell culture medium without antibiotics.

DNA Isolation and Polymerase Chain Reaction (PCR):
HeLa cells infected with C. trachomatis isolate were subjected to DNA extraction using QIAamp Viral RNA mini Kit (Qiagen, CA, USA) according to manufacturer's instructions. Briefly infected cells were harvested at 48 h. post infection (hpi) and the cell suspension was centrifuged at 3000 rpm for 10 min at 4ºC. Supernatants were centrifuged at 16000 rpm for 1hr at 4ºC; pellets were collected and processed for DNA isolation. Concentration of DNA was quantified spectrophotometrically at 260 nm (Biometra, USA). The amplification of efflux gene was carried out by POLYMERASE CHAIN REACTION (PCR) in a DNA Eppendorf Mastercycler personal Thermal Cycler (Eppendorf GmbH, Germany). The primer sequences are 5' ACGATCTTTCCGTGCATTGGTCGT3' for forward primer and 5'GCCATGTAAGAGCCGACACCCA3' for reverse primer (MWG-Biotech, Germany). The thermal conditions for amplification were initial denaturation at 95°C for 10 min followed by 35 cycles of denaturation at 94°C for 30s, primer annealing at 60°C for 1min and extension at 72°C for 2 min, then a final extension at 72°C for 10 min. The PCR product was visualized by electrophoresis on a 1.5% agarose gel stained with ethidium bromide on Alpha Imager gel documentation system (AlphaInnotech, San Leandro, USA).

DNA sequencing:
The PCR products were purified using Qiagen gel extraction kit as per manufacturer's instructions. The sequencing of purified PCR products was carried out using BigDye terminator v3.1 (Applied Biosystems, CA, USA) as per recommendations. Briefly, 75-150 ng µL −1 of purified PCR product and sequencing primers (1 pmol/µL) were added to 4 µL Big Dye Terminator Reaction mix and final volume was made up to 10 µL with autoclaved MilliQ water. Sequencing PCR was set up with 30 cycles of 30 sec denaturation at 96°C, 30 sec annealing at 55°C and 4 min extension at 60°C. After sequencing PCR, the products were purified and re-suspended in Hi-Di formamide (Applied Biosystems). The samples were denatured at 94°C for 5 min followed by a brief incubation on ice and loaded on the 3130XL Genetic Analyzer (Applied Biosystems). Sequence analysis was carried out using Sequence Analysis software (Applied Biosystems) and SeqMan module of DNASTAR v5.07 software.

RNA isolation and real-time RT-PCR analysis:
HeLa cells monolayers were prepared by seeding (3×10 5 cells/well) in six-well tissue culture plates and infected with C. trachomatis inoculum at MoI of 2 as described earlier Bhengraj et al. (2010). Dilutions of drugs (0.5, 5 and 10 µg mL) −1 were added at 2 hpi and cultures were incubated at 35°C in 5% CO 2 . Total RNA was isolated at 8, 24 and 48 hpi using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions and quantified using a UV-VIS spectrophotometer. RNA was treated with DNase I to prevent DNA carryover. The isolated RNA was further tested by PCR to check any carryover DNA contamination. There was no amplification of product detected and the RNA was considered as DNA free.
Complementary DNA was prepared using SuperScript™ First-Strand Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Real-time PCR was performed with the DyNAmo™ SYBR® Green qPCR Kit (Finnzymes, Espoo, Finland). The primer sequences used for efflux (ygeD) gene were F 5'ACGATCTTTCCGTGCATTGGTCGT3 and R 5' GCCATGTAAGAGCCGACACCCA3' and for endogenous control (16S rRNA) gene were 5'CTGCAGCCTCCGTAGAGTCTGGGCAGTGTC3'a nd 5'TTCAGATTGAACGCTGGCGGCGTGGATG 3' as described earlier. Mpiga and Ravaoarinoro (2006) Primers were of HPLC-purified grade and were commercially synthesized (MWG-Biotech AG, Ebersberg, Germany). The negative control consisted of nuclease free water substituted for cDNA. PCR amplification was performed in an Applied Biosystems 7000 Real-Time PCR System (Applied Biosystems, CA, USA). For data analysis, the 2 -∆∆Ct method was used to calculate fold change (Livak and Schmittgen, 2001).

Transduction of HeLa cel:
For targeting host cell actin and plasma membrane proteins HeLa cells were transduced with Cellular-Lights and Organelle-Lights transduction reagents (Molecular probes, Invitrogen, Carlsbad, CA, USA) respectively. The transduction was based on the BacMam technology of viral delivery for specific expression of a targeted (fluorescent) protein in mammalian cell.
Transduction was carried out according to the manufacturer's instructions. Hela cells ~1×10 6 -4×10 6 were seeded in 50 cm 2 tissue culture Flask (Greiner, Germany), allowed to adhere and grow for approximately 24 h at 37°C, 5% CO 2 . Cellular-Lights transduction solution was prepared in Dulbecco's Phosphate Buffered Saline (D-PBS) without Ca 2 + or Mg 2 +. Upon reaching 70-80% confluency of the adhered cells, culture medium was aspirated and 5.5mL of the diluted transduction solution were added. The cells were incubated at room temperature (20-25°C) in the dark for 4 h with gentle rocking. Transduction solutions from the culture flask were again aspirated and culture medium without serum plus 1X enhancer were added. Cells were incubated for 2 h at 37°C and 5% CO 2 . After incubation enhancer solution from the culture flask were replaced with the appropriate culture medium and incubated at 37°C, 5% CO 2 for >16 h. Same method was followed for transduction of HeLa cells with organelle-lights.
Transduced HeLa cells were plated in 6-well tissue culture plates with cell density of 3×10 5 cells/well in EMEM containing 10% FCS. On reaching the subconfluence, monolayer were washed twice with PBS and infected with chlamydial EBs at MoI of 2. For homogenous infection tissue culture plates were placed on a rocker for 2 h at 35°C after addition of serum free media containing EBs. Media containing unbound EBs were aspirated and supplemented with complete DMEM containing 10% FCS. Infected HeLa cells were incubated at 35°C with 5% CO 2 . Thereafter at 2 hpi media was aspirated and replaced with fresh media containing azithromycin or doxycycline. After 48 hpi cells were analysed for fluorescence using flow cytometer (BD FACS Caliber) in FL-1 and FL-2 channel. For negating auto-fluorescence same pool of untransduced cells were used and appropriate setting was used for further acquisition and analysis. Flow histogram was analysed for geometric mean using FCS V3 express (DeNovo Inc). Every experiment was done in triplicate.
Statistical analysis: Differences between two groups were evaluated using Student t test and p<0.05 was considered significant.

C. trachomatis ygeD gene: C. trachomatis efflux (ygeD)
gene was checked for any changes in the genetic level. A fragment of 822bp was amplified and single band was observed in 1.5% agarose gel. Product was sequenced in both the directions and reviewed by assembling into alignments using reference sequence C. trachomatis serotype D (GenBank accession numbers NC000117). The sequence showed variation with a point mutation T to G in 734318 position of the studied C. trachomatis clinical isolate (CT-244).
Real-time RT-PCR analysis: C. trachomatis isolate (CT-244) efflux ygeD gene was studied for any changes in gene expression in the presence of doxycycline and azithromycin in host HeLa cells.
Increased expression of ygeD gene was detected at 8, 24 and 48 hpi in the absence of doxycycline. However, in the presence of doxycycline significantly (p<0.05) increased expression was observed only at 8 hpi while at 24 and 48 hpi it was found to be decreased in presence of all three concentrations of doxycycline (Fig. 1). On addition of azithromycin, no significant changes were detected at 8 and 48 hpi with all the three concentrations of drug however, at 24 hpi expression was observed to be significantly (p<0.05) increased (Fig. 2).

Host cell analysis: Upon infection with serovar D and
CT-244 isolate expression of Green Fluorescent Protein (GFP)-tagged plasma membrane protein in HeLa cells were found to be significantly reduced as compare to the uninfected transduced cells. On addition of drugs, expression was found to be upregulated in comparison to the absence of drugs and comparable to the uninfected cells. Further no difference in expression was observed in serovar D and CT-244 isolate in the absence of antichlamydial drugs. However at higher concentration of azithromycin and lower concentration of doxycycline non-significant difference was observed in the protein expression (Fig. 3). Host cells were further studied for any changes in actin protein expression in the presence of antichlamydial drugs. The red fluorescent protein (RFP)-tagged actin protein expression was found to be up-regulated on infection with isolates in comparison to the uninfected HeLa cells. No difference in expression of actin was observed in serovar D and CT-244 isolate, however, addition of drugs increased the expression of RFP-tagged proteins in infected transduced cells (Fig. 4).

DISCUSSION
To avoid the severe sequelae of C. trachomatis infection, antibiotic strategies are important to eradicate the pathogen. First-line antichlamydial drugs have proven successful for the treatment of C. trachomatis infection however, treatment failures have been observed in notable number of cases Horner (2006). Few studies suggest resistance as a cause for clinical treatment failures (Jones et al., 1990;Somani et al., 2000). The obligate intracellular nature of Chlamydia may limit the emergence of antibiotic resistance in vivo (Abdelrahman and Belland, 2005). However, the extensive use of drugs has been known to favor the selection of resistance in pathogens, including Chlamydia suis in pig (Lefevre and Lepargneur, 1998). The role of genetic resistance in the recurrence of chlamydial infections is still not clear and needs further attention.
Besides well known mechanisms, a further resistance mechanism, active drug efflux, has become increasingly important in the current threat of multidrug resistance. It involves certain bacterial transport proteins which pump out antimicrobial compounds from the cell as a result of over expression of these pumps due to mutations hence decreasing intracellular antibiotic concentration. Efflux pumps possessed by various pathogens are likely to contribute their pathogenic mechanisms by escaping a number of antimicrobial compounds (Poole, 2005). Hence, we studied the efflux ygeD gene of heterotypic resistant C. trachomatis isolate in order to explore the resistant characteristics. The studied sequence showed variation with a point mutation T to G with reference sequence of serotype D. There was no difference in the products of the mutated nucleotide as reference sequence has CTT-Leucine amino acid and mutated nucleotide has CTG which also code for the leucine. This may be a nonsignificant mutation in developing in vitro resistance. In another study of efflux (ygeD) gene in clinical isolates of C. trachomatis-resistant to high and intermediate level of FQ concentrations several silent mutations and mutations resulting in amino acid substitutions were observed (Misiurina et al., 2004). Hence, this can be concluded that the mutation may not be directly related to the resistant characteristic of the bacteria but it might be possible that it has some indirect role, which may make bacteria more refractory to the drugs. Further expression of the efflux gene was also analyzed in the isolate and it was observed that efflux gene was actively expressed at 8hpi in presence of doxycycline, suggesting its expression may have helped in reducing doxycycline pressure at the initial time point. On addition of azithromycin, expression of ygeD gene was observed to be significantly increased at 24 hpi suggesting that in the presence of azithromycin efflux gene was capable in reducing the drug pressure at 24 hpi but not at the initial time point. Hence, we may conclude that C. trachomatis isolate with altered drug susceptibility profile may have an active efflux strategy for its survival in the presence of antichlamydial drugs.
Antimicrobial susceptibility profile of C. trachomatis may be dependent on the host cell environmental conditions and host cell-specific factors. It is reported that oxygen concentrations in female urogenital tract affects the removal of chlamydia upon antibiotic treatment (Shima et al., 2010). In addition it has been observed that pathogenic microbes exploit the host cytoskeleton for entry, colonization and intracellular survival in eukaryotic cells (Rottner et al., 2005). C. trachomatis also co-opts host actin and intermediate filaments to form a dynamic scaffold for providing structural integrity to the chlamydial vacuole and minimizing immune detection for its survival. (Kumar and Valdivia, 2008a;2008b) Hence, host cell factors should be studied to know if this affects the antibiotic susceptibility profile Therefore, host HeLa cells harbouring the heterotypic resistant C. trachomatis isolate were studied for any phenotypic changes at the cellular level. The significantly reduced expression of GFP-tagged plasma membrane protein in HeLa cells detected may be due to the use of proteins for the invagination of infectious elementary bodies of C. trachomatis. However, on addition of drugs expression was found to be comparable to the uninfected cells. Further for detecting any changes in actin protein expression in the presence of antichlamydial drugs host cells were studied for RFP-tagged actin protein expression and it was found to be up-regulated upon infection. However, addition of drugs increased the expression in infected cells. There is no difference observed in expression of plasma membrane and actin protein in between the serovar D and CT-244 isolate. Hence, this may be suggested that C. trachomatis isolate with altered drug susceptibility profile do not affect its host cell plasma membrane or actin organization for its survival in order to resist the antichlamydial drugs.

CONCLUSION
In conclusion, our study supports the emergence of clinical antibiotic resistance, not an impossible scenario for C. trachomatis despite their isolated niche which limits the opportunity for acquisition of antibiotic resistance genes from other organisms (McOrist, 2000). Successful treatment is necessary for preventing sequelae of chlamydial infections hence, treatment failures and in vitro antibiotic resistance characteristics of C. trachomatis is of great concern. The results of present study in characterizing resistance in clinical isolate may enhance the understanding of chlamydial therapy and the nature or transmission of resistant C. trachomatis. Further, studies are needed in more number of C. trachomatis clinical isolates to know its biological relevance to in vivo conditions.

ACKNOWLEDGMENT
Science publication is acknowledged for sponsoring the article for publication. University Grants Commission, New Delhi is acknowledged for providing research fellowship to Apurb Rashmi Bhengraj. Indian Council of Medical Research (ICMR) India is also acknowledged for providing financial assistance in the form of fellowship to Harsh Vardhan, Pragya Srivastava and Suraj Singh Yadav. The study was funded by Indian Council of Medical Research.