ABSTRACT
Mycobacterium canettii, which has a smooth colony morphology, is the tuberculous organism retaining the most genetic traits from the putative last common ancestor of the rough-morphology Mycobacterium tuberculosis complex. To explore whether M. canettii can infect individuals by the oral route, mice were fed phosphate-buffered saline or 106M. canettii mycobacteria and sacrificed over a 28-day experiment. While no M. canettii was detected in negative controls, M. canettii-infected mice yielded granuloma-like lesions for 4/4 lungs at days 14 and 28 postinoculation (p.i.) and positive PCR detection of M. canettii for 5/8 mesenteric lymph nodes at days 1 and 3 p.i. and 5/6 pooled stools collected from day 1 to day 28 p.i. Smooth M. canettii colonies grew from 68% of lungs and 36% of spleens and cervical lymph nodes but fewer than 20% of axillary lymph nodes, livers, brown fat samples, kidneys, or blood samples throughout the 28-day experiment. Ready translocation in mice after digestive tract challenge demonstrates the potential of ingested M. canettii organisms to relocate to distant organs and lungs. The demonstration of this relocation supports the possibility that populations may be infected by environmental M. canettii.
INTRODUCTION
Mycobacterium canettii is a rare member of the Mycobacterium tuberculosis complex (MTBC) that is responsible for fewer than 100 reported cases of tuberculosis in patients with exposure to the Horn of Africa, with a few exceptions (1). In the MTBC, M. canettii is most closely related to the vanished ancestor (1–5). M. canettii is uniquely characterized by a large (4.48 ± 0.05 Mb) and mosaic genome with traces of intraspecies horizontal gene transfer (HGT) (3, 5, 6), including M. tuberculosis-specific deletion 1 (TbD1), which is deleted in “modern” M. tuberculosis lineages (7). In culture, M. canettii appears as large and cordless mycobacteria, and it forms smooth colonies (3, 8, 9). Previous studies suggested that smooth M. canettii may be less well transmitted in aerosols than rough M. tuberculosis (9, 10). Preliminary investigations indicated that cultured M. canettii was less hydrophobic than M. tuberculosis (11, 12). Since higher hydrophobicity was linked to enhanced aerosol performance for nontuberculous mycobacteria (13), it was suggested that the relatively low hydrophobicity of M. canettii could be responsible for its lack of aerosol transmission (11). M. canettii-infected patients present with pulmonary and lymphatic forms of tuberculosis (1). The absence of properly designed studies limits interpretation of the current lack of evidence of interhuman transmission (9). This supports the existence of an unknown environmental or animal reservoir and suggests that M. canettii is poorly adapted to any specific host, behaving as an opportunist rather than an obligate pathogen (9). Published cases indicate that in Djibouti, cervical lymphadenitis is significantly more prevalent during M. canettii infection than during M. tuberculosis infection (1). Also, M. canettii was reported to cause esophageal tuberculosis (14), mesenteric lymph node tuberculosis (15), and ascites (16), and several isolates have been cultured in gastric fluid (14). These clinical observations led to our hypothesis that the digestive tract is a possible portal of entry of M. canettii (1). We developed a murine model to explore this hypothesis from an etiological perspective to contribute to a better understanding of M. canettii infections.
RESULTS
Clinical outcomes of challenged mice.In the first step, we observed that the M. canettii strain CIP 140010059T (STB-A) (5) investigated here resisted a 2-h exposure to a pH level of 2 to 5, chosen to mimic the low pHs encountered in the murine stomach and intestines (17). The number of CFU obtained following acid exposure (3.11 × 104 ± 7.85 × 103 CFU) was not statistically different from that obtained for mycobacterial suspension in phosphate-buffered saline (PBS) (3.76 × 104 ± 3.94 × 103 CFU) (P = 0.19). Mice challenged with M. canettii by orogastric inoculation exhibited no spontaneous death or symptoms of discomfort throughout the 28-day experiment. There was no difference in mean body weight at baseline between infected animals (20.98 ± 2.93 g) and controls (21.46 ± 3.30 g) (P = 0.1). We observed a significant gain in body weight from the first week until the end of the experiment for controls (P = 0.04) and infected mice (P = 0.006).
M. canettii disseminates in mice after digestive tract challenge.To examine the dissemination of M. canettii, the organs and peritoneums of M. canettii-challenged mice and control mice were cultured. Obtained colonies were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and real-time PCR targeting the M. canettii TbD1 region in the presence of negative controls. Cultures of samples from negative-control mice remained negative for M. canettii. Furthermore, cultures of eight collected lungs from M. canettii-infected mice euthanized immediately after inoculation remained negative. Considering both series of experiments, 25 of 28 infected mice (89%) had at least one organ with viable M. canettii, corresponding to 64/294 (22%) culture-positive samples (Table 1). Mouse gender was not significantly associated with the number of infected mice or culture-positive samples (14/16 males versus 11/12 females and 33/174 samples from males versus 31/120 samples from females; P = 0.15). At day 1 postinoculation (p.i.), 2/4 animals from the first series of experiments had culture-positive organs (positive lungs and blood for animal 1 and positive spleen and liver for animal 3). In the second series, three animals (2b, 3b, and 4b) had culture-positive lungs, and one animal (1b) had a culture-positive spleen. Esotracheal lymph nodes examined in the second series gave positive cultures in all cases (Table 1). In the first series, M. canettii cultures were obtained at day 3 p.i. for 4/4 animals, including 3/4 lung samples (mice 5, 7, and 8), 1/4 brown fat samples (mouse 6), and 1/4 liver samples (mouse 7). In the second series, positive cultures from lungs (1/4) and cervical lymph nodes (2/4) were observed for mouse 5b and animals 6b and 8b, respectively. On day 7 p.i. and day 14 p.i., 8/8 lungs were culture positive, in association with a culture-positive spleen (5/8 samples), cervical lymph nodes (4/8 samples), axillary lymph nodes (2/8 samples), or liver, kidneys, or blood (1/8 samples [each]). During dissection of the lungs from mouse 15, we observed macroscopically hypertrophic esotracheal lymph nodes. After this observation, sampling of this tissue was performed. Esotracheal lymph nodes yielded positive cultures for 2/2 sampled animals at day 14 p.i. (mice 15 and 16). At day 28 p.i., cultures were positive for 4/4 samples of cervical lymph nodes, 3/4 samples of the lungs, spleen, or tracheal lymph nodes, 2/4 samples of axillary lymph nodes, and 1/4 samples of the liver, brown fat, blood, or kidneys. All cultured M. canettii isolates retained a smooth colony morphology. Throughout the 28-day experiment, cultures were negative for all white fat and peritoneum samples from M. canettii-challenged mice (Table 1). Microbial contamination prevented any isolation of M. canettii from mesenteric lymph nodes and stools.
Culture results for samples from mice challenged with M. canettiia
Quantification of M. canettii organisms in culture-positive tissues.In order to quantify the M. canettii loads in infected tissues, quantitative real-time PCR (qPCR) was performed on homogenates of culture-positive tissues, all lungs, mesenteric lymph nodes, and pooled stools. While the negative controls and nine culture-negative lungs remained negative, the M. canettii TbD1 region was detected in 30/64 (47%) culture-positive tissues, including 16/19 (84%) lungs, 5/9 (44%) esotracheal lymph nodes, 4/10 (40%) cervical lymph nodes, 4/10 (40%) spleens, and 1/4 (25%) livers (Fig. 1; see Table S1 in the supplemental material). For mesenteric lymph nodes, the M. canettii TbD1 region was detected in 5/8 (62%) samples from 3/4 and 2/4 challenged mice at days 1 and 3 p.i., respectively (Table S1).
Dot plot representation of M. canettii burdens (number of mycobacteria divided by number of cells) in tissues growing M. canettii, as extrapolated from real-time PCR calibration curves. (A) Results obtained for lymphoid tissues from M. canettii-challenged mice. (B) Results of the nonparametric Mann-Whitney rank sum test to compare M. canettii burdens in the lungs of inoculated mice throughout the 28-day experiment. A statistically significant increase in bacterial load in infected lungs was noted throughout the 28-day experiment. Asterisks indicate statistical significance (*, P ≤ 0.05; **, P ≤ 0.01).
Quantification of the number of mycobacteria per 106 mouse cells by qPCR showed that mesenteric lymph nodes represented the most loaded site early in the experiment, with 93 ± 121 mycobacteria/106 cells at day 1 p.i. and 360 ± 199 mycobacteria/106 cells at day 3 p.i. For the same periods, the mycobacterial burden was lower in the lungs, with 108 ± 187 mycobacteria/106 cells at day 1 p.i. and 50 ± 86 mycobacteria/106 cells at day 3 p.i. Esotracheal nodes and spleens had respective loads of 43 and 12.5 mycobacteria/106 cells at day 1 p.i. However, at day 28 p.i., esotracheal nodes and lungs were the most loaded sites, with respective burdens of 2.68 × 103 ± 1.68 × 103 mycobacteria/106 cells and 1 × 103 ± 9.34 × 102 mycobacteria/106 cells, against lower loads for the cervical lymph nodes (94 ± 108 mycobacteria/106 cells), spleen (47 ± 32 mycobacteria/106 cells), and liver (45 mycobacteria/106 cells). Throughout the 28-day experiment, a statistically significant increase in bacterial loads in infected lungs was noted (P = 0.009 for day 1 versus day 28, P = 0.02 for day 3 versus day 28, and P = 0.03 for day 14 versus day 28) (Fig. 1B). For infected spleens, the bacterial loads between day 1 and day 28 p.i. were relatively stable (around 12 to 82 mycobacteria/106 cells). For cervical lymph nodes, a low load of 4 mycobacteria/106 cells was detected in one positive sample at day 7 p.i. The mycobacterial burden in this tissue was relatively more important later, with a bacterial load of 514 mycobacteria/106 cells at day 14 p.i. for one positive sample and a mean of 94 ± 108 mycobacteria/106 cells for two positive samples at day 28 p.i. The detection of M. canettii in stool appeared early (10 mycobacteria/100 mg at days 1 and 2 p.i.) and persisted throughout the experiment, with a tendency to increase at day 28, to 33 mycobacteria/100 mg stool (Table S2).
Translocated M. canettii organisms induce lung pathology.The spleen weight remained stable throughout the experiments, without any significant differences between infected animals (0.085 ± 0.01 g) and controls (0.083 ± 0.008 g) (P = 0.63). No lesions were observed in any tissues sampled from controls. In contrast, for M. canettii-challenged mice, there was clear pathology in the lungs from day 7 p.i. until day 28 p.i. For these animals, gross pathology indicated bilateral and globally symmetric congestion of the lungs, dominant in the posterior and upper areas, with patchy dark-red zones. Pathological examination was performed on organs sampled in the first series of experiments. While control mouse lungs remained normal until day 28 p.i., microscopic examination of infected mouse lungs showed a normal aspect at day 1 p.i., light inflammatory infiltrates at day 7 p.i., and major lesions in 4/4 lungs sampled at days 14 and 28 p.i. Lesions were characterized by peribronchiolitis, perivasculitis with alveolitis, and granuloma-like lesions—defined as a limited area of nodular inflammatory lesions comprised of lymphocytes and macrophages (Fig. 2A). For diseased lungs and granuloma-like lesions, Ziehl-Neelsen staining disclosed a multibacillary status, with numerous organisms within cells with a macrophage morphology (Fig. 2A). In addition to those in the lungs, moderate lesions were observed in one liver dedicated to histological examination at day 28 p.i. for an M. canettii-challenged mouse (Fig. 2B). Microscopic examination showed three small granuloma-like lesions in the same sample, while livers of control mice remained normal. No lesions were observed in axillary and cervical lymph nodes, spleens, kidneys, or white and brown fat throughout the 28-day experiment. In the analyzed histological sections, no acid-fast bacilli were detected by Ziehl-Neelsen staining. Pathology and microbial load data obtained at day 14 and day 28 p.i. are summarized in Table 2.
Representative pathological findings for the lungs and livers of M. canettii-infected mice challenged at day 28 postinoculation. (A) Photomicrographs of hematoxylin-eosin staining of normal lungs from an uninfected mouse and M. canettii-infected lungs with clear alveolitis and granuloma-like lesions. Ziehl-Neelsen staining showed a multibacillary status, with intracellular organisms within cells with a macrophage morphology. (B) A photomicrograph of the liver from an M. canettii-infected mouse shows small granuloma-like lesions, while no lesions were observed in livers from control mice.
Merged pathology and microbial load dataa
DISCUSSION
Mice challenged with ingested M. canettii organisms that resist the low pH encountered in the murine stomach and intestines (17) develop lung tuberculosis. This observation, authenticated by the negative controls in each experiment and the firm identification of growing organisms, was reproduced in both males and females, eliminating any potential sex-related bias. A few previously published models of M. canettii infection have relied on subcutaneous and intramuscular inoculation of guinea pigs (18) and intratracheal or intranasal aerosol inoculation of mice (5, 19, 20). However, these models did not mimic clinical situations, as there is no epidemiological or clinical evidence of patients becoming infected via the parenteral or respiratory route. Accordingly, the limited number of M. canettii infections in regions of the Horn of Africa where the organism is endemic and the absence of reported human-to-human transmission do not favor the hypothesis of a widespread respiratory disease (1, 9). Scarce epidemiological and clinical observations may suggest contaminated drinking water and food as potential sources, with an oral route of entry (1). On these bases, the present study aimed to explore the hypothesis of digestive tract infection by M. canettii.
In this model, positivity of lungs at days 1 to 3 p.i. was interpreted as resulting from the rapid dissemination of M. canettii from the digestive tract via a lymphatic route. This was supported by the detection of M. canettii in the mesenteric, esotracheal, and cervical lymph nodes soon afterwards. The gavage procedure was performed on animals that were awake, and no cough or dyspnea events were observed. Accordingly, lungs collected from mice euthanized immediately after inoculation remained sterile. Procedure-induced lung contamination is therefore improbable. Further culture positivity of the liver and spleen suggested a secondary blood dissemination of M. canettii. The correlation of microscopic observation of pathological lesions, including granuloma-like lesions, in infected lungs with a high burden of mycobacteria supports the installation of M. canettii in this organ and suggests that lungs are primary targets of M. canettii. Conversely, the absence of a detectable inflammatory response in spleens, lymphoid organs, fats, and kidneys correlates with the fact that mycobacteria were not detected or were detected at low burdens in these organs.
The dissemination of bacteria from the intestines has been described extensively and defined as translocation (21). We propose that M. canettii strain STB-A should be designated an additional pathogen that can translocate within the patient. However, the other smooth tubercle bacillus clonal groups (STB-B to STB-M) (3, 5) might behave differently when infecting individuals by the oral route, and the data reported here cannot be extrapolated for the other members of the MTBC. Indeed, the specificity of the results reported here will have to be assessed in further experiments. The observation that rodents infected by the digestive route with either M. canettii or M. bovis (22) all develop pulmonary tuberculosis indicates that this route of infection may be a common feature. M. bovis infection is an example of human tuberculosis contracted through the oral route by consumption of cattle products, particularly unpasteurized contaminated milk (23). The ease of humans contracting tuberculosis from hunted animals is also illustrated by the case of a 1,000-year-old pre-Colombian South American population being infected with Mycobacterium pinnipedii from sea mammals (24). Currently, it is not known if M. pinnipedii is transmissible between humans. However, the occurrence of digestive tract infection by M. tuberculosis remains unclear, since previous gavage experiments yielded contradictory results (25, 26). Therefore, in the absence of definitive results, M. tuberculosis could not be used as a control in our study focusing strictly on M. canettii. Moreover, developing an experimental model of digestive tract infection by M. tuberculosis has no acknowledged epidemiological and clinical basis: it is well established that M. tuberculosis infection is an airborne disease in the vast majority of cases (27), with few cases of inoculation tuberculosis (28) and no reported cases of digestive tract infection by M. tuberculosis.
It would be of great interest to know how the limited number of human cases of M. canettii infection in East Africa occurred. In order to determine the origins of M. canettii infections in the Horn of Africa and elsewhere, intensive investigations of environmental and animal sources are mandatory. The type of reservoir harboring M. canettii in the Horn of Africa remains unclear, with perhaps unspecified animals maintaining an equilibrium with an environmental source. Recently, it was shown that M. canettii was able to persist in soil much longer than M. tuberculosis could (29), so ongoing exchanges between the environment and animals are possibly favored. In addition, thermal inactivation experiments showed that M. canettii survives at temperatures of up to 45°C, which is compatible with an environmental reservoir in the Horn of Africa but suggests that cooked drinks and foods are unlikely sources of contamination (30).
M. canettii is the earliest recognizable associate member of the MTBC, and this taxon plays a pivotal role (2–5, 11). Indeed, given the likely evolutionary pathway from environmental Mycobacterium kansasii to M. canettii (4, 11, 31–33), a vital step in the mammalian evolution of modern virulent members of the MTBC from environmental mycobacteria is the dissemination of a potential ancestral intestinal pathogen, such as M. canettii, to other organs, particularly the lungs. The present results show that M. canettii is efficiently transferred from the intestine to the lungs in mice. Although the experimental data reported herein obviously cannot be extrapolated directly to human disease, they nevertheless support further clinical investigations to assess the potential role of a digestive route of contamination with M. canettii in exposed populations.
MATERIALS AND METHODS
Ethics statement.The experimental protocol, registered by the Ministère de l'Enseignement Supérieur et de la Recherche (reference number 2015092415474605), was approved by the Institutional Animal Care and Use Committee of Aix-Marseille University (C2EA-14), France. Mice were handled according to the rules of Décret 2013-118, 7 February 2013, France. All procedures on animals were performed in accordance with the European law and agreed with ARRIVE (“Animal Research: Reporting In Vivo Experiments”) guidelines (http://www.nc3rs.org.uk ). Animals were euthanized using blood sampling via intracardiac puncture under deep volatile anesthesia (sevoflurane), followed by cervical dislocation. All experiments were performed in a biosafety level 3 laboratory of the Faculté de Médecine, Aix-Marseille University.
Strains, culture conditions, and preparation of inocula. M. canettii strain CIP 140010059T (STB-A) (5) was cultured in Middlebrook 7H10 medium (Becton Dickinson, Le Pont de Claix, France) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (Becton Dickinson). Prior to infection, mycobacteria were resuspended in phosphate-buffered saline (PBS), vortexed for 10 min with sterile glass balls, and centrifuged for 1 min at 300 × g to remove clumps. The supernatant was dispersed by expelling the suspension 10 times through a sterile 25-gauge needle attached to a 1-ml syringe. Calibration was performed according to McFarland standards and confirmed by counting mycobacteria after Ziehl-Neelsen staining. We then assayed the in vitro resistance of M. canettii to low-pH conditions. A 100-μl M. canettii suspension containing 106 mycobacteria was mixed with 900 μl of pH-modified PBS solution at pH 2 to 5. Acid challenge was carried out for 2 h at 37°C. Next, PBS (pH 6.8) was added to result in a 10-fold sample volume. Samples were serially diluted and plated onto Middlebrook 7H10 agar supplemented with 10% OADC (Becton Dickinson) to assess viability during challenge by scoring the number of CFU.
General procedures and mouse challenge.A total of 46 8-week-old BALB/cByj mice (25 males and 21 females; Charles River Laboratories, L'Arbresle, Lyon, France) were housed in individual plastic cages (two or three animals per cage) placed in an isolator with free access to water and a standard diet. Mice were randomly allocated to the control group (five males and five females) or the M. canettii-infected group (20 males and 16 females). For each mouse, 200 μl of sterile PBS or mycobacterial suspension adjusted to 106M. canettii organisms in PBS was administered by gavage, using sterile steel 20-gauge feeding needles for rodents, with ball tips (Instech Laboratories, Inc., Philadelphia, PA, USA) to ensure soft placement. To assess any possibility of procedure-induced lung contamination, eight M. canettii-infected mice were euthanized immediately after inoculation, and the lungs were collected and cultured. After 1 month of incubation, cultures remained negative. For the remaining mice, animals were transferred back to their cages after challenge and housed in a safety cabinet with food and water provided ad libitum. All mice were observed daily for signs of discomfort and respiratory distress.
Clinical outcomes.Each animal was weighed before challenge, on the first day postinfection (p.i.), and then every 2 days. In the first series of experiments, aimed to document dissemination of M. canettii, 30 mice were challenged at different time intervals: four infected mice (two males and two females) and two control mice (one male and one female) were euthanized at 1, 3, 7, 14, and 28 days p.i. Blood sampling was performed by transparietal intracardiac puncture and aliquoted for culture and PCR. Stools as well as miscellaneous organs were sampled, including the cervical, axillary, and esotracheal lymph nodes, lungs, spleen, liver, kidneys, and brown and white fats. The peritoneum was swabbed. Stools from mice challenged the same day were pooled. Spleens were immediately weighed to account for spleen weight loss or gain. All organs were stored at −80°C for microbial load experiments. Two animals of each group were randomly assigned to have a part of the organs fixed in 4% formalin for Ziehl-Neelsen staining and histopathology. The one or two collected esotracheal lymph nodes were too small to be divided, so only culture and PCR were performed. In the second series of experiments, eight additional mice (six males and two females) were challenged under the same conditions. The second series aimed to precisely determine the translocation of the bacteria from the digestive tract during the early periods. In order to do this, four animals were euthanized at day 1 p.i. (two males and two females) and day 3 p.i. (four males). In addition to the series 1 sampling scheme, esotracheal and mesenteric lymph nodes were sampled at early times.
Mycobacterial culture.Stored organs were thawed, ground in sterile PBS, and then cultured in Middlebrook 7H10 medium with OADC (Becton Dickinson). The inoculated medium was examined weekly by naked eye for mycobacterial colonies for up to 4 weeks. Colonies were confirmed by MALDI-TOF MS (34) and by a real-time PCR system targeting the M. canettii TbD1 region.
PCR-based experiments.Molecular detection was used to confirm the identification of cultured colonies and to quantify the M. canettii mycobacterial load in tissues. For positive cultures, colonies were inactivated in 200 μl PBS by 1 h of incubation at 56°C with 200 μl G2 buffer and 20 μl proteinase K (20 mg/ml) and then broken with glass powder by use of a FastPrep instrument (MP Biomedical Europe, Illkirch, France) at a speed of 6.5 m/s for 90 s. DNA was extracted using an EZ1 DNA tissue kit (Qiagen, Hilden, Germany). Quantitative real-time PCR (qPCR) was performed using CFX96 qPCR and incorporating Takyon qPCR reagents (Eurogentec, Liege, Belgium). The M. canettii-specific primers and probe targeting the TbD1 region were designed with the following sequences: TbD1_ Forward, 5′-CAAAGGAACCGCGAAAGTTA-3′; TbD1_ Reverse, 5′-ACCGTGATAAGCACCAGGAC-3′; and TbD1_Probe, 5′-6-carboxyfluorescein (FAM)-TCGCGGTGATGTTGCTCTTCG-3′. Colonies were identified using TbD1 PCR in the presence of negative controls. For quantification, all lungs, mesenteric lymph nodes, and stools as well as homogenates of tissues growing M. canettii were tested. Aliquots of 150 μl were incubated overnight at 56°C with 150 μl of G2 buffer mixed with 15 μl proteinase K (20 mg/ml). After two cycles of mechanical lysis (45 s), total DNA was then extracted using an EZ1 DNA tissue kit. DNA extraction from stools was performed using a QIAamp DNA stool kit (Qiagen, Hilden, Germany). Two qPCRs were performed, targeting M. canettii TbD1 and the mouse hydroxymethylbilane synthase housekeeping gene (HMBS) for normalization, as previously described (35). For both systems, the qPCR conditions were 50°C for 2 min and 95°C for 5 min followed by 40 cycles of 95°C for 1 s and 60°C for 35 s and then a final cycle of 45°C for 30 s. Negative controls consisted of DNAs extracted from organs and stools from PBS-challenged mice. In addition, a standard curve for M. canettii TbD1 qPCR was generated using extracted DNAs from 10-fold serial dilutions of M. canettii suspensions (107 CFU/ml to 1 CFU/ml). Simultaneously, L929 mouse cells were calibrated from 106 cells to 1 cell per 200 μl, and then suspensions were extracted and quantified using qPCR targeting the HMBS gene to generate a calibration curve. The density of mycobacteria (expressed as the number of mycobacteria per cell) was tabulated by dividing the number of mycobacterial DNA copies by the number of mouse cell DNA copies (see Table S3 in the supplemental material).
Pathological examination.Organs were fixed with 4% buffered formalin and embedded in paraffin. Serial sections (3 μm) of these specimens were obtained for routine hematoxylin-eosin-saffron and Ziehl-Neelsen staining. In all pathological examinations, tissues collected from PBS-challenged mice were used as negative controls.
Statistical analyses.Statistics were performed using SigmaPlot 13, Systat Software Inc. The data were expressed as means ± standard deviations (SD). Student's t test or the Mann-Whitney rank sum test was used for intergroup comparisons, with P values of <0.05 regarded as significant.
ACKNOWLEDGMENTS
This study was financially supported by URMITE, IHU Méditerranée Infection, Marseille, France, and by the A*MIDEX project (grant ANR-11-IDEX-0001-02), funded by the Investissements d'Avenir French government program, managed by the French National Research Agency (ANR).
We declare that we have no competing interests.
FOOTNOTES
- Received 25 July 2017.
- Returned for modification 9 August 2017.
- Accepted 14 September 2017.
- Accepted manuscript posted online 18 September 2017.
Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00507-17 .
- Copyright © 2017 American Society for Microbiology.
