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Comparative efficacy of some quinolones and doxycycline against chronic infection of Brucella melitensis 16M in BALB/c mice.

Introduction

Brucellosis remains the most common and serious problem in some parts of the world (Pappas et al., 2006). Ingestion of unpasteurized dairy products, as well as occupational exposure to infected animals are the major causes of brucellosis. Many Brucella species infect animals through direct contact. However, human could be infected when exposed to B. abortus, B. melitensis, or B. suis and the exhausting disease could become, over time, a chronic disease that affects several organs. In addition, some species could be used in a bioterrorist attack (Ariza et al., 2001).

In ruminants, no symptoms were seen after the first abortion; but these infected animals might develop a chronic disease, become chronic carrier, and Brucella could be found in milk and uterine discharges of these carriers during subsequent pregnancies (Diaz et al., 2006).

In humans, undulant fever recovers completely within three to twelve months with only few patients suffering from a chronic disease. However, relapses could be seen months after the initial symptoms. The most common complications occasionally seen in the undulant and chronic forms are arthritis, spondylitis, meningitis and chronic fatigue (Hendaus et al., 2015; Bosilkovski et al., 2007; Morata et al., 2003).

In mice, the most infected organs are spleen and liver which are rapidly colonised with Brucella in splenectomised mice. During the chronic phases, the higher Brucella colonisation of the spleen protects the liver from infection (Grillo et al., 2012). Therefore, spleen is the best target organ to study Brucella infections. Chronic steady phase, where bacterial counts were stable, commonly started at 21 days and lasting till 56-77 days post-infection. At this phase, the maximum number of CFU within spleen is recorded. On the contrary, a slow elimination of the bacteria from target organs is observed during the chronic declining phase which may last beyond 252 days (High et al., 2007). However, experiments on mice are not normally prolonged beyond 120 days. Large and prominent splenomegaly was rarely seen in ruminants (Carvalho-Neta et al., 2010) and the sequelae observed in chronic brucellosis in humans have not been reported in mice (Bosilkovski et al., 2007).

Although many regimens have been experimented and tested, treatment regimen approved by the World Health Organisation (1986) still recommended for the treatment of human brucellosis. However, the incidence of treatment failure and frequent relapses are still a major concern (Roushan et al., 2006). Moreover, quinolones utilisation as alternatives in the treatment of brucellosis did not improve the results (Lopez-Merino et al., 2004).

Currently the human chronic brucellosis is generally treated with antibiotic triple therapy. The combination of rifampin, doxycycline, and streptomycin often is used whereas in non-responded or complicated cases, quinolones could be added to the conventional treatment as a third agent (Falagas and Bliziotis, 2006).

The aim of the current study was to compare the short-term treatment efficacy of several quinolones alone or in combination with doxycycline in experimentally induced B. melitensis 16M infection in mice.

Materials and Methods

Bacteria. Brucella melitensis 16M strain was obtained from the Laboratory of Microbiology and Immunology URBM (University of Namur, Belgium) and grown in the Department of Molecular Biology and Biotechnology (Atomic Energy Commission of Syria), for 48 h in 2YT agar (peptone, 16 g/L; yeast extract, 10 g/L; NaCl, 5 g/L; agar, 13 g/L [GibcoBRL]) at 37 [degrees]C. Prior to dilution to appropriate concentrations of inoculates, bacteria were harvested into 20 mL of sterile phosphate-buffered saline (PBS) and the bacterial suspension was standardized to [10.sup.10] colony-forming units (CFU)/mL. The concentrations were determined retrospectively by enumeration of ten-fold dilutions of the inoculates on 2YT plates. All experiments with live Brucella were performed in biosafety level 2 facilities.

Antibiotics. Doxycycline (Sigma, St. Louis, USA), ciprofloxacin (Bayer, Istambul, Turkey), ofloxacin (Sigma), and levofloxacin (Sigma) were dissolved according to the manufacturer's recommendations to a working concentration of 8 mg/mL. Antibiotics were prepared freshly every day and sterilized through a 0.2 [micro]m filter.

Animals. The effect of quinolones against chronic Brucella infection was studied according to the method of Domingo et al. (1995) with some modifications. Briefly, between February and October 2012, 200 BALB/c mice were infected with [approximately equal to] [10.sup.6] CFU of B. melitensis 16M intraperitoneally. Thirty days later, the infected mice were divided into 8 groups of 25 mice each. The untreated control group received only the vehicle and was included in order to estimate the normal splenic infection during the course of this study, 225 days. At the 31st day, the seven other groups received one of the following treatment regimens: doxycycline (oral, 40 mg/kg every 12 h for 14 days); ciprofloxacin, ofloxacin or levofloxacin (oral, 40 mg/kg/day of body weight for 14 days); and one of doxycycline-ciproflox-acin, doxycycline-ofloxacin or doxycycline-levofloxacin combinations at the same doses mentioned above (i.e. doxycycline orally, 40 mg/kg every 12 h for 14 days; and quinolone orally, 40 mg/ kg/day of body weight for 14 days). The experimental procedures on mice and the facilities used to hold the experimental animals are in accordance to National (Real Decreto 233/1988, in BOE number 67) law. Batches of five mice were sacrificed by cervical dislocation, on 45, 90, 135, 180 and 225 days post-infection. At post-mortem, spleens were removed, weighted, homogenised in 3 mL of distilled water using a stomacher 80-Biomaster (Seward, England). Bacterial loads were determined following enumeration of ten-fold serial dilutions on 2YT agar plates (incubated for 3 days at 37 [degrees]C in air, with 10% C[O.sub.2]) using the spread plate technique. The average of three replicates of each dilution was calculated, and considered as the mean CFU/mL of each studied mouse.

Statistical analysis. Data were transformed into [log.sub.10] CFU. Differences in CFU between the treated and untreated groups were evaluated by student's t-test. The data was analysed by version 5.0 GraphPad Prism. P values of 0.05 or less were considered statistically significant.

Results and Discussion

The results show that doxycycline was effective at day 45 post-infection (p=0.03) when compared with the control. Doxycycline and ofloxacin were found the most effective at day 90 post-infection (p<0.0001 and p=0.005, respectively). Whereas, ciprofloxacin and levofloxacinwere less effective (p=0.011 and p=0.047, respectively). Only doxycycline was found effective at day 135 post-infection (p<0.001). In the same way, doxycycline was more effective than ciprofloxacin, ofloxacin and levofloxacin at day 90 post-challenge (p<0.0001, p<0.0005 and p<0.0001, respectively); and most effective than ciprofloxacin and ofloxacin at day 135 post-infection (p=0.04 and p=0.02, respectively) as shown in Fig. 1. Furthermore, Fig. 2 indicates that doxycycline-ciprofloxacin, doxycycline-ofloxacin and doxycycline-levofloxacin combinations were extremely efficient at day 45 post-challenge (p<0.0001 at all cases); and doxycycline-ciprofloxacin and doxycycline-ofloxacin combinations were very effective at day 90 post-challenge (p=0.0026 and p=0.0015, respectively).

Additionally, doxycycline-ciprofloxacin combination was very efficient at day 135 post-infection (p<0.0001), and doxycycline-ofloxacin combination was less effective (p=0.032). However, comparing with doxycycline, splenic CFU reduction was observed from day 45 post-infection when using all doxycycline-quinolone combinations (p<0.0001) comparing with doxycyclineciprofloxacin and doxycycline-ofloxacin combinations; and p=0.024 comparing with doxycycline-levofloxacin combination.

The success of any drug in the treatment of brucellosis depends on its ability to penetrate the phagocytic cell membrane and work within its acidic environment. The failure in the treatment of acute cases of brucellosis may lead to the development of relapse, focal complications or even chronic disease (Castano and Solera, 2009).

In human, most patients usually recover after one year of effective treatment while a few patients complain from focal or non-focal disease. Domingo et al. (1995) reviewed that relapses after the treatment with doxycycline alone for 21 days were higher than that noticed after the treatment with doxycycline for 45 days (29% versus 10-14%). In addition, ciprofloxacin at a 500 mg dose twice a day within 20 days in chronic stage of disease essentially reduced duration of local inflammatory processes of brucellosis with simultaneous treatment of the chronic infection focus, provides good proximate and distant outcomes of treatment (Pappas et al., 2005).

Whereas, in mice, treatment regimens with azithromycin 50 mg/kg/day for 10 days, doxycycline 50 mg/ kg/12 h for 21 days or doxycycline 50 mg/kg/12 h for 45 days, produced a significant reduction in the chronic infection. In addition, when doxycycline was administered for 45 days, it was possible to eradicate B. melitensis completely in comparison with the treatment for 21 days only (Domingo et al., 1995). In agreement with these results, present research results revealed that the short-term treatment with doxycycline for 14 days was more effective than all quinolones and almost completely eradicates the infection 135 days after infection. Similarly, Shasha et al. (1992) reported that the longer the treatment with doxycycline the better the results obtained.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

In addition, our results showed that ofloxacin, given orally for 14 days, was the most effective quinolone against chronic infection with B. melitensis 16M in murine model, and showed a significant reduction of the splenic infection at 90 days after infection. Moreover, doxycycline-quinolone combinations were more effective than the treatment with doxycycline alone since they could significantly reduce the splenic infection from day 45 post-infection. Unfortunately, these combinations could not completely eradicate the chronic infection.

Despite a few elevations in bacterial counts at 180 days post-infection comparing with that noticed at 135 days post-infection when doxycycline alone was applied; this elevation was not significant.

Conclusion

Different treatment methods were studied to control chronic human infection caused by Brucella. In conclusion, experiments on mice showed that short-term treatment with doxycycline was more effective than the treatment with quinolones. In addition, doxycyclinequinolones combinations were slightly more effective than doxycycline alone. However, treatment with doxycycline alone eliminates more number of bacteria than all other combinations after 135 days post-infection.

Acknowledgement

The authors would like to thank the Director General of the Atomic Energy Commission Syria (AECS) and the Head of the Molecular Biology and Biotechnology Department for their support.

References

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Mazen Safi *, Bassam Albalaa, Nermen Haj Mahmoud and Ayman Al-Mariri

Department of Molecular Biology and Biotechnology, Atomic Energy Commission, P.O. Box 6091, Damascus, Syria

(received April 8, 2015; revised December 3, 2015; accepted December 22, 2015)

* Author for correspondence; E-mail: ascientific@aec.org.sy
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Author:Safi, Mazen; Albalaa, Bassam; Mahmoud, Nermen Haj; Mariri, Ayman Al-
Publication:Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences
Article Type:Report
Date:Jul 1, 2016
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