Chlamydia trachomatis antimicrobial susceptibility in colorectal and endocervical cells

Chlamydia trachomatis antimicrobial susceptibility in colorectal and endocervical cells Abstract Background Rectal Chlamydia trachomatis infections represent one of the most common sexually transmitted infections in the MSM population. Although current treatment guidelines suggest the use of either azithromycin or doxycycline, several clinical studies reported on azithromycin treatment failures in the case of rectal C. trachomatis localizations. In this context, the biological reasons behind the lack of azithromycin efficacy for C. trachomatis infections at the rectal level are still poorly understood. Objectives To evaluate the in vitro antimicrobial susceptibility of several C. trachomatis strains in two different cell lines, mimicking the urogenital localization and the rectal site of infection. Methods The susceptibility to macrolides (i.e. azithromycin and erythromycin), doxycycline and levofloxacin was assessed for 20 C. trachomatis strains, belonging to the most frequently reported genovars (D, E, F and G), both in human endocervical cells (HeLa cells) and in colorectal cells (Caco-2 cells). Moreover, a correlation between MIC values and C. trachomatis bacterial load was investigated in both cell lines. Results For all the C. trachomatis strains, regardless of the genovar, macrolides showed higher MIC and MBC values (2-fold dilutions) in Caco-2 cells compared with HeLa cells, whereas for doxycycline and levofloxacin, no significant differences were found between the two cell lines. Moreover, azithromycin MICs were significantly higher with increasing levels of C. trachomatis elementary bodies on Caco-2 cells. Conclusions The higher azithromycin MICs observed in colorectal cells, together with the positive correlation between MICs and C. trachomatis loads found, could explain azithromycin treatment failure for C. trachomatis infections at the rectal site. Introduction Chlamydia trachomatis genovars D through to K represent the agents of the most common bacterial sexually transmitted infection worldwide.1 Urogenital C. trachomatis infections (i.e. urethritis and cervicitis) are often asymptomatic and, if left untreated, can lead to several complications, including pelvic inflammatory disease, tubal infertility and epididymo-orchitis.2 Besides urogenital localizations, C. trachomatis can be found at extra-genital sites, such as pharyngeal and rectal mucosa, particularly in the MSM population.3 In considering the frequent asymptomatic nature of extra-genital C. trachomatis infections, they can act as an important reservoir for further transmission.4 In the absence of a chlamydial vaccine, public health control of C. trachomatis infections relies primarily on effective, accessible and affordable antimicrobial treatment, combined with appropriate prevention, diagnostics and epidemiological surveillance. In this context, international guidelines suggest, as recommended regimens for urogenital C. trachomatis infections, the use of either 1 g of azithromycin orally in a single dose or 100 mg of doxycycline orally twice a day for 7 days. Erythromycin and quinolones (i.e. levofloxacin or ofloxacin) represent alternative regimens.5,6 For rectal infections, several non-randomized clinical studies showed higher efficacy rates for doxycycline (98.8%–100%) than for azithromycin (74%–87%) at this anatomical site.6–8 Considering the important limitations of these studies and the low quality of data supporting the superiority of doxycycline over azithromycin, both regimens continue to be recommended as first-line for treating rectal infections.6 Randomized controlled trials will probably allow to better compare the efficacy of azithromycin versus doxycycline for the treatment of rectal infections.9 Although recently it has been suggested that azithromycin treatment failure can be associated with high bacterial loads,10 the reasons behind its failure in rectal C. trachomatis localizations are still poorly understood. To understand better the biological basis of these findings, the aim of this study was to evaluate the in vitro antimicrobial susceptibility of several C. trachomatis strains in two different cell lines, providing a simplified model of both the urogenital localization and the rectal site of infection. In particular, the susceptibility to macrolides (azithromycin and erythromycin), doxycycline and levofloxacin was assessed for 20 C. trachomatis strains, belonging to the most frequently reported genovars (D, E, F and G),3,11 both in human endocervical cells (HeLa cells) and in colorectal cells (Caco-2 cells). Moreover, a correlation between MIC values and C. trachomatis bacterial load was investigated in both cell lines. Materials and methods C. trachomatis strains and genotyping A total of 20 C. trachomatis strains were tested. Of these, four were reference strains: serovar E strain Bour (ATCC®-VR-348B), serovar D strain UW-3/Cx (ATCC®-VR-885), serovar F strain IC-Cal-3 (ATCC®-VR-346) and serovar G strain UW-57/Cx (ATCC®-VR-878). The remaining 16 were randomly selected from a broad collection of strains isolated during the years 2005 and 2008 in the Microbiology Unit, S. Orsola Hospital, Bologna (Italy). In particular, these strains were recovered from urethral swabs (n = 9) of male patients with non-gonococcal urethritis and from cervical swabs (n = 7) of women with cervicitis.12 At the time of C. trachomatis isolation, all the patients were not under antibiotic treatment and most of them (70%) were symptomatic, complaining about various urogenital disorders (dysuria, genital discharge and dyspareunia). When C. trachomatis infection was diagnosed the subjects were treated with doxycycline twice a day for 7 days. After the isolation, C. trachomatis strains were propagated for ∼2/3 weeks in LLC-MK2 cells (ATCC® CCL-7TM). Afterwards, C. trachomatis elementary bodies (EBs) were purified from cell debris and reticulate bodies by Renografin density gradient centrifugation,13 harvested in sucrose–phosphate–glutamate (SPG) buffer and stored at −80 °C until use. The infectivity titre of each C. trachomatis strain was determined by a serial dilution method, inoculating suitable dilutions into susceptible cell cultures and calculating the number of inclusions inside the host cells. The infectivity titre of EBs was expressed as the number of inclusion-forming units (IFUs)/mL. C. trachomatis molecular genotyping was performed for the clinically isolated strains by omp1 gene sequencing, as previously described.12 Globally, six genovar E strains, five genovar D strains, five genovar G strains and four genovar F strains were included in the study. Cell lines Experiments were conducted using both HeLa cells (ATCC® CCL-2TM), an epithelial cell line originating from a human cervix adenocarcinoma, and Caco-2 cells (ATCC® HTB-37TM), epithelial cells derived from a colorectal adenocarcinoma (kindly provided by Dr Elisa Michelini, FaBiT, University of Bologna, Italy). Cells were grown to confluent monolayers in individual tubes containing sterile coverslips in 5% CO2 at 37 °C. The cell lines were cultivated in DMEM (EuroClone, Pero, Italy), supplemented with 10% fetal bovine serum and 1% l-glutamine, without antibiotics. In the case of antimicrobial susceptibility testing, 1 mg/mL of cycloheximide was added to the medium. Antimicrobial susceptibility The antimicrobial drugs levofloxacin (GlaxoSmithKline, Verona, Italy), doxycycline, erythromycin and azithromycin (Sigma–Aldrich, Milan, Italy) were provided as powders and solubilized according to the manufacturers’ instructions. Antimicrobial susceptibility testing was performed in both HeLa cells and Caco-2 cells, as described elsewhere with slight modifications.14,15 The confluent cell monolayers were inoculated with a total of 5 × 103 IFUs of each C. trachomatis strain. In particular, a single SPG aliquot of purified EBs stored at −80 °C was thawed slowly in ice and vigorously vortexed. Afterwards, the stock solution was diluted with SPG to a concentration of 5 × 104 IFUs/mL and 100 μL (corresponding to 5 × 103 IFUs) was added to each of the individual tubes, containing 900 μL of antibiotic-free medium. After centrifugation at 1700 g for 1 h, the medium was removed and replaced with medium containing scalar concentrations of the different antimicrobial drugs. The concentrations tested for each antimicrobial ranged as follows: levofloxacin from 0.06 to 2 mg/L, doxycycline from 0.006 to 2 mg/L, erythromycin from 0.006 to 4 mg/L and azithromycin from 0.003 to 2 mg/L. After incubation at 37 °C for 48 h, infected monolayers were washed with PBS, fixed with methanol and stained with a monoclonal antibody against the chlamydial lipopolysaccharide antigen conjugated with fluorescein (Meridian, Cincinnati, OH, USA). The MIC was defined as the lowest concentration able to reduce the number of chlamydial inclusions >90%, compared with the level of drug-free controls. When evaluating the number of inclusions, both ‘aberrant’ inclusions (small but intensely bright inclusions lacking granularity) and ‘normal’ inclusions (large chlamydial vacuoles displaying a high degree of granularity) were taken into account. The MBC values were measured by aspirating the antibiotic-containing medium, washing the monolayer twice with PBS and re-incubating in antibiotic-free medium for 48 h at 37 °C. Afterwards, cell monolayers were fixed and stained as described before. The MBC was the lowest concentration of the drug reducing >90% of chlamydial inclusions after the re-incubation of monolayers in antimicrobial-free medium. Each experiment was run in triplicate. Correlation between MIC and C. trachomatis IFUs Azithromycin and doxycycline MICs were determined as previously described, using different amounts of C. trachomatis IFUs (5 × 102, 5 × 103 and 5 × 104 IFUs), both in HeLa cells and Caco-2 cells. Statistical analysis The mean number of IFUs/microscopic field at ×200 magnification ± SD was compared between Caco-2 cells and HeLa cells in drug-free controls, by means of a paired t-test. The antimicrobial MIC values, expressed as mean ± SEM, were compared between the three different amounts of C. trachomatis IFUs (5 × 102, 5 × 103 and 5 × 104 IFUs) in both cell lines, by means of a one-way analysis of variance (ANOVA) test. Statistical analysis was performed by using GraphPad Prism software (GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com). P < 0.05 was considered as statistically significant. Results Antimicrobial susceptibility In drug-free controls, a significantly higher number of chlamydial IFUs was noticed in Caco-2 cells compared with HeLa cells (mean number of IFUs/microscopic field at ×200 magnification ± SD: 195 ± 23.3 versus 117 ± 22.0; P < 0.0001). Table 1 shows the MIC and MBC values of the antimicrobial agents tested for the 20 C. trachomatis strains, subdivided on the basis of the different genovars. In both cell lines, except for levofloxacin, showing MICs comparable to MBCs, the other antimicrobial drugs were characterized by MBC values two to four times the MICs. No significant differences were noticed between different C. trachomatis genovars. Table 1 MIC and MBC values (mg/L) of different antimicrobial drugs for 20 C. trachomatis strains, belonging to different serovars C. trachomatis serovar (no. of strains)  Azithromycin   Erythromycin   Doxycycline   Levofloxacin   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  D (5)  0.03  0.06  0.125  0.25  0.06–0.125  0.125  0.25–0.5  0.5–1  0.06  0.125  0.06  0.125  0.25  0.25  0.25–0.5  0.25–0.5  E (6)  0.03  0.06–0.125  0.125  0.25–0.5  0.06–0.125  0.125  0.25  0.5  0.06–0.125  0.125–0.25  0.125  0.125–0.25  0.25  0.25  0.5  0.5  F (4)  0.03  0.06  0.125  0.25  0.06–0.125  0.125  0.25–0.5  0.5  0.125  0.25  0.125  0.25  0.25  0.25  0.25–0.5  0.25–0.5  G (5)  0.03–0.06  0.06–0.125  0.25  0.5  0.125  0.25  0.5  1  0.06–0.125  0.125–0.25  0.125  0.25  0.25  0.25  0.5  0.5  C. trachomatis serovar (no. of strains)  Azithromycin   Erythromycin   Doxycycline   Levofloxacin   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  D (5)  0.03  0.06  0.125  0.25  0.06–0.125  0.125  0.25–0.5  0.5–1  0.06  0.125  0.06  0.125  0.25  0.25  0.25–0.5  0.25–0.5  E (6)  0.03  0.06–0.125  0.125  0.25–0.5  0.06–0.125  0.125  0.25  0.5  0.06–0.125  0.125–0.25  0.125  0.125–0.25  0.25  0.25  0.5  0.5  F (4)  0.03  0.06  0.125  0.25  0.06–0.125  0.125  0.25–0.5  0.5  0.125  0.25  0.125  0.25  0.25  0.25  0.25–0.5  0.25–0.5  G (5)  0.03–0.06  0.06–0.125  0.25  0.5  0.125  0.25  0.5  1  0.06–0.125  0.125–0.25  0.125  0.25  0.25  0.25  0.5  0.5  Antimicrobial susceptibility testing was performed both in endocervical cell line monolayers (HeLa cells) and in epithelial colorectal cells (Caco-2 cells). Globally, for all the strains, macrolides show higher MIC and MBC values (2-fold dilutions) in Caco-2 cells compared with HeLa cells, whereas for doxycycline and levofloxacin, no significant differences were found between the two cell lines. Correlation between MIC and C. trachomatis IFUs At the different C. trachomatis IFU amounts, azithromycin and doxycycline MICs in HeLa cells did not differ significantly (P = 0.36 and 0.45, respectively), as well as doxycycline MICs in Caco-2 cells (P = 0.82). On the contrary, MICs of azithromycin in Caco-2 cells were higher, with the increasing level of C. trachomatis IFUs (P < 0.0001). Detailed results are shown in Figure 1. Figure 1 View largeDownload slide Correlation between C. trachomatis IFUs and MICs of AZM and DOX in different cell lines. Experiments were conducted both in HeLa cells (left) and in Caco-2 cells (right). Three different amounts of C. trachomatis IFUs, expressed as log, were used: 5 × 102, 5 × 103 and 5 × 104 IFUs. MIC values are expressed as mean ± SEM. AZM, azithromycin; DOX, doxycycline. Figure 1 View largeDownload slide Correlation between C. trachomatis IFUs and MICs of AZM and DOX in different cell lines. Experiments were conducted both in HeLa cells (left) and in Caco-2 cells (right). Three different amounts of C. trachomatis IFUs, expressed as log, were used: 5 × 102, 5 × 103 and 5 × 104 IFUs. MIC values are expressed as mean ± SEM. AZM, azithromycin; DOX, doxycycline. Discussion C. trachomatis rectal infections represent one of the commonest sexually transmitted infections in the MSM population, with increasing detection rates also in women.3,4 Although current treatment guidelines recommend either azithromycin or doxycycline, there are increasing concerns about treatment failure with azithromycin in the case of rectal localizations.6–9 Considering that the reasons behind the lack of azithromycin efficacy for rectal C. trachomatis infection are poorly understood, the aim of this study was to evaluate if a different antimicrobial susceptibility was present on the basis of the different cell targets, in a simplified model of the infection both at the urogenital site and at the rectal level. For this purpose, we tested the antimicrobial drugs recommended for C. trachomatis infection treatment (azithromycin, doxycycline, erythromycin, levofloxacin) against 20 C. trachomatis strains, belonging to different genovars, using epithelial cells of endocervical and of colorectal origin. When tested in HeLa cells, C. trachomatis strains showed MIC and MBC values comparable to those reported recently by a nationwide surveillance in Japan.15 Azithromycin showed a greater activity against C. trachomatis strains compared with doxycycline, as already stated.16 Moreover, we found that the antimicrobial susceptibility levels were comparable for D through to G genovars. Although Zheng et al.16 found a 2–4-fold MIC difference between C. trachomatis genovars, we confirmed the homogeneous data of antimicrobial susceptibility reported by Donati et al.14 Interestingly, all the C. trachomatis strains, regardless of the genovar, showed higher MIC and MBC values of macrolides in Caco-2 cells compared with HeLa cells, in contrast to doxycycline and levofloxacin. In addition, looking for a correlation between MICs and C. trachomatis IFU amounts, we noticed that azithromycin MICs seemed to be significantly higher with increasing levels of EBs on Caco-2 cells. Similarly, Suchland et al.17 demonstrated that MIC/MBC values of macrolides can be affected by the cell line used, whereas in the presence of tetracycline, doxycycline and ofloxacin, Chlamydia susceptibility was comparable with all the cell lines. Moreover, in line with our findings, they found no difference in doxycycline MICs, when the inoculum ranged between 300 and 300 000 IFUs/well.17 It is known that many laboratory conditions, such as pH, temperature, nutrients present in the media, polarity of the cell type and cytokine secretion by infected cells, may affect the ability of a particular antimicrobial to penetrate intracellularly and exert its action, leading to variations in C. trachomatis susceptibility.18 Even though a different activity of azithromycin in Caco-2 cells and HeLa cells cannot be totally ruled out (e.g. different cell permeability), it has been shown that high and sustained concentrations of azithromycin are found in rectal tissue following a single 1 g dose, suggesting that inadequate concentrations are unlikely to cause treatment failure.19 Besides the intracellular uptake of the antimicrobials, additional variables can influence the results of Chlamydia susceptibility testing, such as the interval between the establishment of the infection and the drug administration, or the endpoint used for defining the MIC values.17 Moreover, factors, such as the long-term storage of chlamydial isolates, the multiple culture passaging of strains and the different adaptation on cell lines, can affect the growth characteristics and the fitness and the virulence of chlamydial isolates, thus leading to variations in antimicrobial susceptibility levels.17,18 Although it is impossible to rule out that some of these factors could have affected our results, important points of our experimental design should be highlighted: (i) the main objective of this study was to compare the MICs/MBCs in two different cell lines, rather than to obtain absolute values; (ii) all the strains were processed in parallel in the two cell lines; and (iii) all the C. trachomatis strains included in the study were not previously adapted to grow in HeLa cells and Caco-2 cells, allowing to avoid imbalances due to the bacterial fitness in a particular cell line. Considering all the aspects mentioned above, we hypothesized that our findings could be ascribed to the peculiar replication model of C. trachomatis in the different epithelial cells. Effectively, in drug-free controls, C. trachomatis strains showed a significantly higher infectivity in Caco-2 cells compared with HeLa cells, as suggested by a considerable increase of inclusion number. Our results are in line with those previously reported for animal strains, showing an increase in number and inclusion size when Chlamydia suis and Chlamydia pecorum were cultivated in Caco-2 cells, opposite to other epithelial cells.20 The higher azithromycin MICs found in Caco-2 cells, together with the positive correlation between MICs and C. trachomatis IFUs, could explain azithromycin treatment failure for C. trachomatis infections at the rectal site, where higher bacterial loads have been found.10 We are fully aware that cell lines cannot sufficiently mimic the in vivo pathogen–host interactions and only the use of more complex and advanced models (e.g. three-dimensional polarized cell-line models, various tissue engineered anatomical constructs) will shed light on C. trachomatis behaviour in the rectal site. Anyway, to our knowledge, this is the first report evaluating the antimicrobial susceptibility of C. trachomatis in a cell line of colorectal origin and it could be of aid to understand the biological basis of azithromycin treatment failure for C. trachomatis rectal infections. Acknowledgements We would like to thank Maria Battaglia for providing excellent technical support during this study. Funding This study was supported by internal funding (RFO 2014 to A. M.) Transparency declarations None to declare. References 1 ECDC. Sexually Transmitted Infections in Europe 2013 . Stockholm: ECDC, 2015. PubMed PubMed  2 Menon S, Timms P, Allan JA et al.   Human and pathogen factors associated with Chlamydia trachomatis-related infertility in women. Clin Microbiol Rev  2015; 28: 969– 85. Google Scholar CrossRef Search ADS PubMed  3 Foschi C, Nardini P, Banzola N et al.   Chlamydia trachomatis infection prevalence and serovar distribution in a high-density urban area in the north of Italy. J Med Microbiol  2016; 65: 510– 20. Google Scholar CrossRef Search ADS PubMed  4 van Liere GA, Hoebe CJ, Niekamp AM et al.   Standard symptom- and sexual history-based testing misses anorectal Chlamydia trachomatis and Neisseria gonorrhoeae infections in swingers and men who have sex with men. Sex Transm Dis  2013; 40: 285– 9. Google Scholar CrossRef Search ADS PubMed  5 Workowski KA, Bolan GA; Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep  2015; 64: 1– 137. Google Scholar CrossRef Search ADS PubMed  6 Lanjouw E, Ouburg S, de Vries HJ et al.   2015 European guideline on the management of Chlamydia trachomatis infections. Int J STD AIDS  2016; 27: 333– 48. Google Scholar CrossRef Search ADS PubMed  7 Khosropour CM, Dombrowski JC, Barbee LA et al.   Comparing azithromycin and doxycycline for the treatment of rectal chlamydial infection: a retrospective cohort study. Sex Transm Dis  2014; 41: 79– 85. Google Scholar CrossRef Search ADS PubMed  8 Drummond F, Ryder N, Wand H et al.   Is azithromycin adequate treatment for asymptomatic rectal chlamydia? Int J STD AIDS  2011; 22: 478– 80. Google Scholar CrossRef Search ADS PubMed  9 Lau A, Kong F, Fairley CK et al.   Treatment efficacy of azithromycin 1 g single dose versus doxycycline 100 mg twice daily for 7 days for the treatment of rectal chlamydia among men who have sex with men—a double-blind randomised controlled trial protocol. BMC Infect Dis  2017; 17: 35. Google Scholar CrossRef Search ADS PubMed  10 Kong FY, Tabrizi SN, Fairley CK et al.   Higher organism load associated with failure of azithromycin to treat rectal chlamydia. Epidemiol Infect  2016; 144: 2587– 96. Google Scholar CrossRef Search ADS PubMed  11 Versteeg B, Himschoot M, van den Broek IV et al.   Urogenital Chlamydia trachomatis strain types, defined by high-resolution multilocus sequence typing, in relation to ethnicity and urogenital symptoms among a young screening population in Amsterdam, The Netherlands. Sex Transm Infect  2015; 91: 415– 22. Google Scholar CrossRef Search ADS PubMed  12 Marangoni A, Foschi C, Nardini P et al.   Chlamydia trachomatis serovar distribution and other sexually transmitted coinfections in subjects attending an STD outpatients clinic in Italy. New Microbiol  2012; 35: 215– 9. Google Scholar PubMed  13 Marangoni A, Fiorino E, Gilardi F et al.   Chlamydia pneumoniae acute liver infection affects hepatic cholesterol and triglyceride metabolism in mice. Atherosclerosis  2015; 241: 471– 9. Google Scholar CrossRef Search ADS PubMed  14 Donati M, Di Francesco A, D'Antuono A et al.   In vitro activities of several antimicrobial agents against recently isolated and genotyped Chlamydia trachomatis urogenital serovars D through K. Antimicrob Agents Chemother  2010; 54: 5379– 80. Google Scholar CrossRef Search ADS PubMed  15 Takahashi S, Hamasuna R, Yasuda M et al.   Nationwide surveillance of the antimicrobial susceptibility of Chlamydia trachomatis from male urethritis in Japan. J Infect Chemother  2016; 22: 581– 6. Google Scholar CrossRef Search ADS PubMed  16 Zheng H, Xue Y, Bai S et al.   Association of the in vitro susceptibility of clinical isolates of Chlamydia trachomatis with serovar and duration of antibiotic exposure. Sex Transm Dis  2015; 42: 115– 9. Google Scholar CrossRef Search ADS PubMed  17 Suchland RJ, Geisler WM, Stamm WE. Methodologies and cell lines used for antimicrobial susceptibility testing of Chlamydia spp. Antimicrob Agents Chemother  2003; 47: 636– 42. Google Scholar CrossRef Search ADS PubMed  18 Wang SA, Papp JR, Stamm WE et al.   Evaluation of antimicrobial resistance and treatment failures for Chlamydia trachomatis: a meeting report. J Infect Dis  2005; 191: 917– 23. Google Scholar CrossRef Search ADS PubMed  19 Kong FY, Rupasinghe TW, Simpson JA et al.   Pharmacokinetics of a single 1g dose of azithromycin in rectal tissue in men. PLoS One  2017; 12: e0174372. Google Scholar CrossRef Search ADS PubMed  20 Schiller I, Schifferli A, Gysling P et al.   Growth characteristics of porcine chlamydial strains in different cell culture systems and comparison with ovine and avian chlamydial strains. Vet J  2004; 168: 74– 80. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. 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Chlamydia trachomatis antimicrobial susceptibility in colorectal and endocervical cells

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Abstract

Abstract Background Rectal Chlamydia trachomatis infections represent one of the most common sexually transmitted infections in the MSM population. Although current treatment guidelines suggest the use of either azithromycin or doxycycline, several clinical studies reported on azithromycin treatment failures in the case of rectal C. trachomatis localizations. In this context, the biological reasons behind the lack of azithromycin efficacy for C. trachomatis infections at the rectal level are still poorly understood. Objectives To evaluate the in vitro antimicrobial susceptibility of several C. trachomatis strains in two different cell lines, mimicking the urogenital localization and the rectal site of infection. Methods The susceptibility to macrolides (i.e. azithromycin and erythromycin), doxycycline and levofloxacin was assessed for 20 C. trachomatis strains, belonging to the most frequently reported genovars (D, E, F and G), both in human endocervical cells (HeLa cells) and in colorectal cells (Caco-2 cells). Moreover, a correlation between MIC values and C. trachomatis bacterial load was investigated in both cell lines. Results For all the C. trachomatis strains, regardless of the genovar, macrolides showed higher MIC and MBC values (2-fold dilutions) in Caco-2 cells compared with HeLa cells, whereas for doxycycline and levofloxacin, no significant differences were found between the two cell lines. Moreover, azithromycin MICs were significantly higher with increasing levels of C. trachomatis elementary bodies on Caco-2 cells. Conclusions The higher azithromycin MICs observed in colorectal cells, together with the positive correlation between MICs and C. trachomatis loads found, could explain azithromycin treatment failure for C. trachomatis infections at the rectal site. Introduction Chlamydia trachomatis genovars D through to K represent the agents of the most common bacterial sexually transmitted infection worldwide.1 Urogenital C. trachomatis infections (i.e. urethritis and cervicitis) are often asymptomatic and, if left untreated, can lead to several complications, including pelvic inflammatory disease, tubal infertility and epididymo-orchitis.2 Besides urogenital localizations, C. trachomatis can be found at extra-genital sites, such as pharyngeal and rectal mucosa, particularly in the MSM population.3 In considering the frequent asymptomatic nature of extra-genital C. trachomatis infections, they can act as an important reservoir for further transmission.4 In the absence of a chlamydial vaccine, public health control of C. trachomatis infections relies primarily on effective, accessible and affordable antimicrobial treatment, combined with appropriate prevention, diagnostics and epidemiological surveillance. In this context, international guidelines suggest, as recommended regimens for urogenital C. trachomatis infections, the use of either 1 g of azithromycin orally in a single dose or 100 mg of doxycycline orally twice a day for 7 days. Erythromycin and quinolones (i.e. levofloxacin or ofloxacin) represent alternative regimens.5,6 For rectal infections, several non-randomized clinical studies showed higher efficacy rates for doxycycline (98.8%–100%) than for azithromycin (74%–87%) at this anatomical site.6–8 Considering the important limitations of these studies and the low quality of data supporting the superiority of doxycycline over azithromycin, both regimens continue to be recommended as first-line for treating rectal infections.6 Randomized controlled trials will probably allow to better compare the efficacy of azithromycin versus doxycycline for the treatment of rectal infections.9 Although recently it has been suggested that azithromycin treatment failure can be associated with high bacterial loads,10 the reasons behind its failure in rectal C. trachomatis localizations are still poorly understood. To understand better the biological basis of these findings, the aim of this study was to evaluate the in vitro antimicrobial susceptibility of several C. trachomatis strains in two different cell lines, providing a simplified model of both the urogenital localization and the rectal site of infection. In particular, the susceptibility to macrolides (azithromycin and erythromycin), doxycycline and levofloxacin was assessed for 20 C. trachomatis strains, belonging to the most frequently reported genovars (D, E, F and G),3,11 both in human endocervical cells (HeLa cells) and in colorectal cells (Caco-2 cells). Moreover, a correlation between MIC values and C. trachomatis bacterial load was investigated in both cell lines. Materials and methods C. trachomatis strains and genotyping A total of 20 C. trachomatis strains were tested. Of these, four were reference strains: serovar E strain Bour (ATCC®-VR-348B), serovar D strain UW-3/Cx (ATCC®-VR-885), serovar F strain IC-Cal-3 (ATCC®-VR-346) and serovar G strain UW-57/Cx (ATCC®-VR-878). The remaining 16 were randomly selected from a broad collection of strains isolated during the years 2005 and 2008 in the Microbiology Unit, S. Orsola Hospital, Bologna (Italy). In particular, these strains were recovered from urethral swabs (n = 9) of male patients with non-gonococcal urethritis and from cervical swabs (n = 7) of women with cervicitis.12 At the time of C. trachomatis isolation, all the patients were not under antibiotic treatment and most of them (70%) were symptomatic, complaining about various urogenital disorders (dysuria, genital discharge and dyspareunia). When C. trachomatis infection was diagnosed the subjects were treated with doxycycline twice a day for 7 days. After the isolation, C. trachomatis strains were propagated for ∼2/3 weeks in LLC-MK2 cells (ATCC® CCL-7TM). Afterwards, C. trachomatis elementary bodies (EBs) were purified from cell debris and reticulate bodies by Renografin density gradient centrifugation,13 harvested in sucrose–phosphate–glutamate (SPG) buffer and stored at −80 °C until use. The infectivity titre of each C. trachomatis strain was determined by a serial dilution method, inoculating suitable dilutions into susceptible cell cultures and calculating the number of inclusions inside the host cells. The infectivity titre of EBs was expressed as the number of inclusion-forming units (IFUs)/mL. C. trachomatis molecular genotyping was performed for the clinically isolated strains by omp1 gene sequencing, as previously described.12 Globally, six genovar E strains, five genovar D strains, five genovar G strains and four genovar F strains were included in the study. Cell lines Experiments were conducted using both HeLa cells (ATCC® CCL-2TM), an epithelial cell line originating from a human cervix adenocarcinoma, and Caco-2 cells (ATCC® HTB-37TM), epithelial cells derived from a colorectal adenocarcinoma (kindly provided by Dr Elisa Michelini, FaBiT, University of Bologna, Italy). Cells were grown to confluent monolayers in individual tubes containing sterile coverslips in 5% CO2 at 37 °C. The cell lines were cultivated in DMEM (EuroClone, Pero, Italy), supplemented with 10% fetal bovine serum and 1% l-glutamine, without antibiotics. In the case of antimicrobial susceptibility testing, 1 mg/mL of cycloheximide was added to the medium. Antimicrobial susceptibility The antimicrobial drugs levofloxacin (GlaxoSmithKline, Verona, Italy), doxycycline, erythromycin and azithromycin (Sigma–Aldrich, Milan, Italy) were provided as powders and solubilized according to the manufacturers’ instructions. Antimicrobial susceptibility testing was performed in both HeLa cells and Caco-2 cells, as described elsewhere with slight modifications.14,15 The confluent cell monolayers were inoculated with a total of 5 × 103 IFUs of each C. trachomatis strain. In particular, a single SPG aliquot of purified EBs stored at −80 °C was thawed slowly in ice and vigorously vortexed. Afterwards, the stock solution was diluted with SPG to a concentration of 5 × 104 IFUs/mL and 100 μL (corresponding to 5 × 103 IFUs) was added to each of the individual tubes, containing 900 μL of antibiotic-free medium. After centrifugation at 1700 g for 1 h, the medium was removed and replaced with medium containing scalar concentrations of the different antimicrobial drugs. The concentrations tested for each antimicrobial ranged as follows: levofloxacin from 0.06 to 2 mg/L, doxycycline from 0.006 to 2 mg/L, erythromycin from 0.006 to 4 mg/L and azithromycin from 0.003 to 2 mg/L. After incubation at 37 °C for 48 h, infected monolayers were washed with PBS, fixed with methanol and stained with a monoclonal antibody against the chlamydial lipopolysaccharide antigen conjugated with fluorescein (Meridian, Cincinnati, OH, USA). The MIC was defined as the lowest concentration able to reduce the number of chlamydial inclusions >90%, compared with the level of drug-free controls. When evaluating the number of inclusions, both ‘aberrant’ inclusions (small but intensely bright inclusions lacking granularity) and ‘normal’ inclusions (large chlamydial vacuoles displaying a high degree of granularity) were taken into account. The MBC values were measured by aspirating the antibiotic-containing medium, washing the monolayer twice with PBS and re-incubating in antibiotic-free medium for 48 h at 37 °C. Afterwards, cell monolayers were fixed and stained as described before. The MBC was the lowest concentration of the drug reducing >90% of chlamydial inclusions after the re-incubation of monolayers in antimicrobial-free medium. Each experiment was run in triplicate. Correlation between MIC and C. trachomatis IFUs Azithromycin and doxycycline MICs were determined as previously described, using different amounts of C. trachomatis IFUs (5 × 102, 5 × 103 and 5 × 104 IFUs), both in HeLa cells and Caco-2 cells. Statistical analysis The mean number of IFUs/microscopic field at ×200 magnification ± SD was compared between Caco-2 cells and HeLa cells in drug-free controls, by means of a paired t-test. The antimicrobial MIC values, expressed as mean ± SEM, were compared between the three different amounts of C. trachomatis IFUs (5 × 102, 5 × 103 and 5 × 104 IFUs) in both cell lines, by means of a one-way analysis of variance (ANOVA) test. Statistical analysis was performed by using GraphPad Prism software (GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com). P < 0.05 was considered as statistically significant. Results Antimicrobial susceptibility In drug-free controls, a significantly higher number of chlamydial IFUs was noticed in Caco-2 cells compared with HeLa cells (mean number of IFUs/microscopic field at ×200 magnification ± SD: 195 ± 23.3 versus 117 ± 22.0; P < 0.0001). Table 1 shows the MIC and MBC values of the antimicrobial agents tested for the 20 C. trachomatis strains, subdivided on the basis of the different genovars. In both cell lines, except for levofloxacin, showing MICs comparable to MBCs, the other antimicrobial drugs were characterized by MBC values two to four times the MICs. No significant differences were noticed between different C. trachomatis genovars. Table 1 MIC and MBC values (mg/L) of different antimicrobial drugs for 20 C. trachomatis strains, belonging to different serovars C. trachomatis serovar (no. of strains)  Azithromycin   Erythromycin   Doxycycline   Levofloxacin   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  D (5)  0.03  0.06  0.125  0.25  0.06–0.125  0.125  0.25–0.5  0.5–1  0.06  0.125  0.06  0.125  0.25  0.25  0.25–0.5  0.25–0.5  E (6)  0.03  0.06–0.125  0.125  0.25–0.5  0.06–0.125  0.125  0.25  0.5  0.06–0.125  0.125–0.25  0.125  0.125–0.25  0.25  0.25  0.5  0.5  F (4)  0.03  0.06  0.125  0.25  0.06–0.125  0.125  0.25–0.5  0.5  0.125  0.25  0.125  0.25  0.25  0.25  0.25–0.5  0.25–0.5  G (5)  0.03–0.06  0.06–0.125  0.25  0.5  0.125  0.25  0.5  1  0.06–0.125  0.125–0.25  0.125  0.25  0.25  0.25  0.5  0.5  C. trachomatis serovar (no. of strains)  Azithromycin   Erythromycin   Doxycycline   Levofloxacin   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   HeLa cells   Caco-2 cells   MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  MIC  MBC  D (5)  0.03  0.06  0.125  0.25  0.06–0.125  0.125  0.25–0.5  0.5–1  0.06  0.125  0.06  0.125  0.25  0.25  0.25–0.5  0.25–0.5  E (6)  0.03  0.06–0.125  0.125  0.25–0.5  0.06–0.125  0.125  0.25  0.5  0.06–0.125  0.125–0.25  0.125  0.125–0.25  0.25  0.25  0.5  0.5  F (4)  0.03  0.06  0.125  0.25  0.06–0.125  0.125  0.25–0.5  0.5  0.125  0.25  0.125  0.25  0.25  0.25  0.25–0.5  0.25–0.5  G (5)  0.03–0.06  0.06–0.125  0.25  0.5  0.125  0.25  0.5  1  0.06–0.125  0.125–0.25  0.125  0.25  0.25  0.25  0.5  0.5  Antimicrobial susceptibility testing was performed both in endocervical cell line monolayers (HeLa cells) and in epithelial colorectal cells (Caco-2 cells). Globally, for all the strains, macrolides show higher MIC and MBC values (2-fold dilutions) in Caco-2 cells compared with HeLa cells, whereas for doxycycline and levofloxacin, no significant differences were found between the two cell lines. Correlation between MIC and C. trachomatis IFUs At the different C. trachomatis IFU amounts, azithromycin and doxycycline MICs in HeLa cells did not differ significantly (P = 0.36 and 0.45, respectively), as well as doxycycline MICs in Caco-2 cells (P = 0.82). On the contrary, MICs of azithromycin in Caco-2 cells were higher, with the increasing level of C. trachomatis IFUs (P < 0.0001). Detailed results are shown in Figure 1. Figure 1 View largeDownload slide Correlation between C. trachomatis IFUs and MICs of AZM and DOX in different cell lines. Experiments were conducted both in HeLa cells (left) and in Caco-2 cells (right). Three different amounts of C. trachomatis IFUs, expressed as log, were used: 5 × 102, 5 × 103 and 5 × 104 IFUs. MIC values are expressed as mean ± SEM. AZM, azithromycin; DOX, doxycycline. Figure 1 View largeDownload slide Correlation between C. trachomatis IFUs and MICs of AZM and DOX in different cell lines. Experiments were conducted both in HeLa cells (left) and in Caco-2 cells (right). Three different amounts of C. trachomatis IFUs, expressed as log, were used: 5 × 102, 5 × 103 and 5 × 104 IFUs. MIC values are expressed as mean ± SEM. AZM, azithromycin; DOX, doxycycline. Discussion C. trachomatis rectal infections represent one of the commonest sexually transmitted infections in the MSM population, with increasing detection rates also in women.3,4 Although current treatment guidelines recommend either azithromycin or doxycycline, there are increasing concerns about treatment failure with azithromycin in the case of rectal localizations.6–9 Considering that the reasons behind the lack of azithromycin efficacy for rectal C. trachomatis infection are poorly understood, the aim of this study was to evaluate if a different antimicrobial susceptibility was present on the basis of the different cell targets, in a simplified model of the infection both at the urogenital site and at the rectal level. For this purpose, we tested the antimicrobial drugs recommended for C. trachomatis infection treatment (azithromycin, doxycycline, erythromycin, levofloxacin) against 20 C. trachomatis strains, belonging to different genovars, using epithelial cells of endocervical and of colorectal origin. When tested in HeLa cells, C. trachomatis strains showed MIC and MBC values comparable to those reported recently by a nationwide surveillance in Japan.15 Azithromycin showed a greater activity against C. trachomatis strains compared with doxycycline, as already stated.16 Moreover, we found that the antimicrobial susceptibility levels were comparable for D through to G genovars. Although Zheng et al.16 found a 2–4-fold MIC difference between C. trachomatis genovars, we confirmed the homogeneous data of antimicrobial susceptibility reported by Donati et al.14 Interestingly, all the C. trachomatis strains, regardless of the genovar, showed higher MIC and MBC values of macrolides in Caco-2 cells compared with HeLa cells, in contrast to doxycycline and levofloxacin. In addition, looking for a correlation between MICs and C. trachomatis IFU amounts, we noticed that azithromycin MICs seemed to be significantly higher with increasing levels of EBs on Caco-2 cells. Similarly, Suchland et al.17 demonstrated that MIC/MBC values of macrolides can be affected by the cell line used, whereas in the presence of tetracycline, doxycycline and ofloxacin, Chlamydia susceptibility was comparable with all the cell lines. Moreover, in line with our findings, they found no difference in doxycycline MICs, when the inoculum ranged between 300 and 300 000 IFUs/well.17 It is known that many laboratory conditions, such as pH, temperature, nutrients present in the media, polarity of the cell type and cytokine secretion by infected cells, may affect the ability of a particular antimicrobial to penetrate intracellularly and exert its action, leading to variations in C. trachomatis susceptibility.18 Even though a different activity of azithromycin in Caco-2 cells and HeLa cells cannot be totally ruled out (e.g. different cell permeability), it has been shown that high and sustained concentrations of azithromycin are found in rectal tissue following a single 1 g dose, suggesting that inadequate concentrations are unlikely to cause treatment failure.19 Besides the intracellular uptake of the antimicrobials, additional variables can influence the results of Chlamydia susceptibility testing, such as the interval between the establishment of the infection and the drug administration, or the endpoint used for defining the MIC values.17 Moreover, factors, such as the long-term storage of chlamydial isolates, the multiple culture passaging of strains and the different adaptation on cell lines, can affect the growth characteristics and the fitness and the virulence of chlamydial isolates, thus leading to variations in antimicrobial susceptibility levels.17,18 Although it is impossible to rule out that some of these factors could have affected our results, important points of our experimental design should be highlighted: (i) the main objective of this study was to compare the MICs/MBCs in two different cell lines, rather than to obtain absolute values; (ii) all the strains were processed in parallel in the two cell lines; and (iii) all the C. trachomatis strains included in the study were not previously adapted to grow in HeLa cells and Caco-2 cells, allowing to avoid imbalances due to the bacterial fitness in a particular cell line. Considering all the aspects mentioned above, we hypothesized that our findings could be ascribed to the peculiar replication model of C. trachomatis in the different epithelial cells. Effectively, in drug-free controls, C. trachomatis strains showed a significantly higher infectivity in Caco-2 cells compared with HeLa cells, as suggested by a considerable increase of inclusion number. Our results are in line with those previously reported for animal strains, showing an increase in number and inclusion size when Chlamydia suis and Chlamydia pecorum were cultivated in Caco-2 cells, opposite to other epithelial cells.20 The higher azithromycin MICs found in Caco-2 cells, together with the positive correlation between MICs and C. trachomatis IFUs, could explain azithromycin treatment failure for C. trachomatis infections at the rectal site, where higher bacterial loads have been found.10 We are fully aware that cell lines cannot sufficiently mimic the in vivo pathogen–host interactions and only the use of more complex and advanced models (e.g. three-dimensional polarized cell-line models, various tissue engineered anatomical constructs) will shed light on C. trachomatis behaviour in the rectal site. Anyway, to our knowledge, this is the first report evaluating the antimicrobial susceptibility of C. trachomatis in a cell line of colorectal origin and it could be of aid to understand the biological basis of azithromycin treatment failure for C. trachomatis rectal infections. Acknowledgements We would like to thank Maria Battaglia for providing excellent technical support during this study. Funding This study was supported by internal funding (RFO 2014 to A. M.) Transparency declarations None to declare. References 1 ECDC. Sexually Transmitted Infections in Europe 2013 . Stockholm: ECDC, 2015. PubMed PubMed  2 Menon S, Timms P, Allan JA et al.   Human and pathogen factors associated with Chlamydia trachomatis-related infertility in women. Clin Microbiol Rev  2015; 28: 969– 85. Google Scholar CrossRef Search ADS PubMed  3 Foschi C, Nardini P, Banzola N et al.   Chlamydia trachomatis infection prevalence and serovar distribution in a high-density urban area in the north of Italy. J Med Microbiol  2016; 65: 510– 20. Google Scholar CrossRef Search ADS PubMed  4 van Liere GA, Hoebe CJ, Niekamp AM et al.   Standard symptom- and sexual history-based testing misses anorectal Chlamydia trachomatis and Neisseria gonorrhoeae infections in swingers and men who have sex with men. Sex Transm Dis  2013; 40: 285– 9. Google Scholar CrossRef Search ADS PubMed  5 Workowski KA, Bolan GA; Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep  2015; 64: 1– 137. Google Scholar CrossRef Search ADS PubMed  6 Lanjouw E, Ouburg S, de Vries HJ et al.   2015 European guideline on the management of Chlamydia trachomatis infections. Int J STD AIDS  2016; 27: 333– 48. Google Scholar CrossRef Search ADS PubMed  7 Khosropour CM, Dombrowski JC, Barbee LA et al.   Comparing azithromycin and doxycycline for the treatment of rectal chlamydial infection: a retrospective cohort study. Sex Transm Dis  2014; 41: 79– 85. Google Scholar CrossRef Search ADS PubMed  8 Drummond F, Ryder N, Wand H et al.   Is azithromycin adequate treatment for asymptomatic rectal chlamydia? Int J STD AIDS  2011; 22: 478– 80. Google Scholar CrossRef Search ADS PubMed  9 Lau A, Kong F, Fairley CK et al.   Treatment efficacy of azithromycin 1 g single dose versus doxycycline 100 mg twice daily for 7 days for the treatment of rectal chlamydia among men who have sex with men—a double-blind randomised controlled trial protocol. BMC Infect Dis  2017; 17: 35. Google Scholar CrossRef Search ADS PubMed  10 Kong FY, Tabrizi SN, Fairley CK et al.   Higher organism load associated with failure of azithromycin to treat rectal chlamydia. Epidemiol Infect  2016; 144: 2587– 96. Google Scholar CrossRef Search ADS PubMed  11 Versteeg B, Himschoot M, van den Broek IV et al.   Urogenital Chlamydia trachomatis strain types, defined by high-resolution multilocus sequence typing, in relation to ethnicity and urogenital symptoms among a young screening population in Amsterdam, The Netherlands. Sex Transm Infect  2015; 91: 415– 22. Google Scholar CrossRef Search ADS PubMed  12 Marangoni A, Foschi C, Nardini P et al.   Chlamydia trachomatis serovar distribution and other sexually transmitted coinfections in subjects attending an STD outpatients clinic in Italy. New Microbiol  2012; 35: 215– 9. Google Scholar PubMed  13 Marangoni A, Fiorino E, Gilardi F et al.   Chlamydia pneumoniae acute liver infection affects hepatic cholesterol and triglyceride metabolism in mice. Atherosclerosis  2015; 241: 471– 9. Google Scholar CrossRef Search ADS PubMed  14 Donati M, Di Francesco A, D'Antuono A et al.   In vitro activities of several antimicrobial agents against recently isolated and genotyped Chlamydia trachomatis urogenital serovars D through K. Antimicrob Agents Chemother  2010; 54: 5379– 80. Google Scholar CrossRef Search ADS PubMed  15 Takahashi S, Hamasuna R, Yasuda M et al.   Nationwide surveillance of the antimicrobial susceptibility of Chlamydia trachomatis from male urethritis in Japan. J Infect Chemother  2016; 22: 581– 6. Google Scholar CrossRef Search ADS PubMed  16 Zheng H, Xue Y, Bai S et al.   Association of the in vitro susceptibility of clinical isolates of Chlamydia trachomatis with serovar and duration of antibiotic exposure. Sex Transm Dis  2015; 42: 115– 9. Google Scholar CrossRef Search ADS PubMed  17 Suchland RJ, Geisler WM, Stamm WE. Methodologies and cell lines used for antimicrobial susceptibility testing of Chlamydia spp. Antimicrob Agents Chemother  2003; 47: 636– 42. Google Scholar CrossRef Search ADS PubMed  18 Wang SA, Papp JR, Stamm WE et al.   Evaluation of antimicrobial resistance and treatment failures for Chlamydia trachomatis: a meeting report. J Infect Dis  2005; 191: 917– 23. Google Scholar CrossRef Search ADS PubMed  19 Kong FY, Rupasinghe TW, Simpson JA et al.   Pharmacokinetics of a single 1g dose of azithromycin in rectal tissue in men. PLoS One  2017; 12: e0174372. Google Scholar CrossRef Search ADS PubMed  20 Schiller I, Schifferli A, Gysling P et al.   Growth characteristics of porcine chlamydial strains in different cell culture systems and comparison with ovine and avian chlamydial strains. Vet J  2004; 168: 74– 80. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Feb 1, 2018

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