Effect of Combined Therapy on Colistin Resistant Pseudomonas aeruginosa : An in Vitro Study

Pseudomonas aeruginosa is known for its antibiotic resistance to the clinicians. The infections caused by this pathogen are hard to treat because of its highly versatile property to mutate and acquire drug resistance. Pseudomonas aeruginosa also possesses intrinsic property of resistance to certain antibiotics like tetracyclines. However; in a practice to overcome the problem of multi drug resistance, clinicians restored the use of some antibiotics that were pre-viously been used to treat the Pseudomonal infections; but they were discon-tinued because of its toxic effects. Colistin is an example of one such antibiotic. Use of Colistin was barred for its neurotoxicity. However in recent clinical trials, Colistin was reintroduced to fight with this superbug. Sadly in recent years, Pseudomonas aeruginosa developed resistance to Colistin as well. Therefore combined therapy is an alternate and suitable treatment to overcome the infections caused by multidrug-resistant Pseudomonas aeruginosa. The present study is an in vitro study; in which we tested synergy between two antibiotics namely streptomycin and Colistin on 29 clinical isolates of P. aeruginosa collected from hospitals in Jazan city KSA. 17.3% isolates Pseudomonal In addition it was also analyzed that the highest percentage of multi drug resistant P. aeruginosa strains where associated with and respiratory tract infections (24%). while 5.7% of urine and 4% of blood isolates showed multi drug resistance. Our findings differ with the findings conducted which it was documented that most of


Introduction
Antibiotic resistance is one of the major concerns in treating the bacterial infections around the world; Pseudomonas aeruginosa is among the fast-developing antibiotic-resistant bacterial species; this bacterium is a gram-negative, opportunistic pathogen and a significant cause of acute and chronic infections in patients with compromised host defenses. Pseudomonas aeruginosa is a leading cause of nosocomial infections, such as infections of urinary tract, surgical sites wounds, bacteremia and middle ear infections etc. [1]. This organism is highly susceptible to mutations and is continuously exhibiting resistance to a number of antibiotics. Standard antibiotic regimes against P. aeruginosa are increasingly becoming ineffective due to the rise in drug resistance [2]. The treatment of the infections caused by this pathogen is getting difficult day by day because of the emergence of (MDR) multi drug resistance strains [3] [4]. P. aeruginosa is popular for its antibiotic resistance against drugs such as carbapenems, fluoroquinolones and aminoglycosides [3]. Mechanisms responsible for multidrug resistance include restricted permeability to drugs, changes in efflux systems; drug inactivation and changes in targets [5]. Apart from being resistant to antibiotics the sub-inhibitory concentrations of antibiotics such as tetracyclines can increase the cytotoxicity of this bacteria by four folds; in addition to this, sub-inhibitory concentrations of antibiotics namely tobramycin and ciprofloxacin induce biofilm formation in Pseudomonas aeruginosa [6]. Many countries have reported the rise of antibiotic-resistant strains of P. aeroginosa; Among Gulf co-operation council (GCC) countries, 92.3% clinical isolates of P. aeroginosa from Saudi Arabia were reported to be resistant to antibiotics [7]. In order to fight with multidrug-resistant Pseudomonal infections Colistin is the drug of choice. Many studies have communicated Colistin treatment being successful; in patients with nosocomial infections, pulmonary infections and in cancer patients suffering from MDR Pseudomonas aerogonisa infections [8] [9] [10]. On the other hand resistance to Colistin has also been reported by many countries in clinical isolates of P. aeruginosa [11] [12]. However in situation of Colistin resistance, it requires some other alternative solution such as combined therapy to fight with the multidrug resistance strains of Pseudomonas aeruginosa. Combination therapies usually are effective in situation of antibiotic resistance. Studies have shown the synergistic action of Colistin with other drugs on MDR Pseudomonas aeroginosa [13] [14]. Therefore the present study is an attempt to deduce synergistic activity of Streptomycin and Colistin on Colistin resistant Pseu-

Materials & Methods
Incidence of MDR P. aeroginosa: To discover the incidence of multidrug resistance peudomonal infections in Jazan region. A total of 174 case reports on Pseudomonas aeroginosa were collected from microbiology lab of hospitals in Jazan city and their antibiotic sensitivity for four anti Pseudomonal drugs such as Pipercillin, cefepime, ceftazidime and gentamycin were studied. were re-identified by culturing on cetrimide agar to study pigmentation and colony morphology. Gram staining of the colonies was performed to study the morphology of isolates. Biochemical identification was done by performing oxidase test [15].
Determination of Colistin resistance: Colistin sensitivity test was performed by disc diffusion technique. The isolates and reference strain of Pseudomonas aeroginosa (control) was sub cultured on Muller hinton agar plates and from each 24 hrs subculture plates an inoculum of 0.5 Mcfarland unit turbid suspension was prepared respectively. A sterile swab was dipped into each suspension and was spread on sterile Muller hinton agar plates. After spreading the inoculum 10 µg Colistin disc was placed in the center of the plates. The plates were then incubated at 37˚C for 24 hrs. After incubation the plates were observed for zone of inhibition. The zone of inhibition was measured in millimeter (mm). The susceptibility or resistance interpretations were determined in accordance to National Committee for Clinical Laboratory Standards (NCCLS) standard breakpoints [16]. Determination of MIC (minimum inhibitory concentration): All the 29 resistant cultures were tested by minimum inhibitory concentration technique. The protocol for MIC is preparation of two fold dilution of each drug (streptomycin alone and Colistin alone) and combination of two drugs (streptomycin-Colistin combined) which were prepared in Muller hinton broth tubes (macrobroth dilution technique). The concentrations ranging from 500 µg•ml −1 -15.6 µg•ml −1 respectively was obtained [17].
Inoculum preparation for MIC: An inoculum adjusted to 0.5 McFarland turbidity was diluted to obtain a concentration of 5 × 10 5 CFU/mL i.e. 2.0 ml of the 0.5 McFarland suspension is diluted into 38 ml sterile water (1:20 dilution). Then 0.01 ml of the diluted suspension is delivered to each tube with Muller hinton broth with concentrations of antibiotics ranging from 500 µg•ml −1 -15.6 µg•ml −1 respectively. After inoculation the tubes were incubated at 37˚C for 18 hrs. Inoculums for reference strains from American type culture collection (ATCC) were also prepared and tested for MIC as positive controls while tubes, without any inoculum was considered as negative control. After incubation the tubes were observed for visual turbidity. The lowest concentration of antibiotic or combined that completely inhibits visual growth of bacteria (no turbidity) was recorded as MIC [16].
The synergistic effect of streptomycin and Colistin: The synergy of Colistin and streptomycin at different concentrations on Colistin resistant Pseudomonas aeroginosa was determined by FICI (fractional inhibitory concentration index). FICI is described as the sum of the MIC of each drug when used in combination divided by the MIC of the drug used alone.

Results
Incidence of MDR P. aeruginosa (2017-2018): Upon analyzing the data collected, it was determined that out 173 clinical cases reported 96 isolates i.e. (55.5%) were multidrug-resistant P. aeroginosa strains and 77 (44.5%), isolates were found to be sensitive to routinely used anti Pseudomonal drugs as shown in Table 1, Figure 1.
Colistin Resistance: The Zone of inhibition diameters obtained following the Colistin susceptibility test it was found that all the 29 isolates tested were resistant to Colistin antibiotic. According to NCCLS breakpoints a Pseudomonas aeruginosa strain is sensitive to Colistin if the zone diameter is ≥10 mm. In our study on 29 isolates, the zone diameters obtained were <10 mm as shown in Table 2 & Figure 2 Synergistic activitity: Based on the FICI results obtained we found that the combination of Colistin and streptomycin showed synergistic killing of nineteen strains (55.1%) seven strains (20.6%) had partial synergistic action by the drugs. Similarly indifferent synergy was observed on five strains. Two strains showed additive synergy (6.8%) while two strains (3.4%) showed antagonism to the combined drugs as shown in Table 3.

Discussion
Treating multi drug resistance is the current major challenges for clinicians. It is reported that Pseudomonas aeruginosa is the most common non fermenting isolates in clinical samples in Saudi Arabia [20]. In the present study the incidence of Pseudomonas aeruginosa isolates causing infections were studied; it was found that 55.5% of the isolates were multi drug resistant; Among which   in the respective studied years [21]. In our study the respiratory specimens (sputum and throat swabs) showed the highest percentage of association (24%) of MDR P. aeruginosa strains while wound swabs where the second most common specimens that showed association with MDR P. aeruginosa strains. In recent years Colistin is been used extensively to treat multidrug resistance P. aeruginosa infections. In the present study firstly we evaluated the effect of Colistin on twenty nine clinical isolates of multi drug resistance Pseudomonas aeruginosa collected from different hospitals of Jazan region of Saudi Arabia. To our surprise all the 29 clinical isolates showed Colistin resistance which is in contrary to results stated in study carried out in King Khalid University Hospital, Riyadh, Saudi Arabia (2012). In this study the researchers stated that 93.9% P. aeruginosa strains were susceptible to Colistin [22]. Colistin resistance is due to acquisition of  mcr-1 gene in plasmid which was described by a study in china [23]. However E. coli strains harboring plasmids carrying mcr-1 gene have been reported from many parts of the world. For example in recent study in conducted in UAE found plasmids carrying mcr-1 gene in four strains of E. coli collected from Arabian peninsula [24]. Similarly in a study on hajj pilgrims demonstrated the presence of mcr-1 gene in 10 isolates of E. coli [25]. In another study on  Colistin is effective as supplemental therapy in patients with nosocomial pneumonia or trachea bronchitis resulting from MDR P. aeruginosa [8]. Hence Colistin may be included in the combination therapies to treat the infections caused by Colistin resistant MDR P. aeruginosa.
Our study is first of its kind to evaluate Colistin with streptomycin effect on MDR Pseudomonas aeruginosa. Streptomycin belongs to aminoglycosides and is used as anti tuberculous drugs. It inhibits protein synthesis in bacterial cell.
However researchers have demonstrated the bactericidal activity of streptomycin against Pseudomonas aeroginosa [19] [28] but recent studies has demonstrated the developing streptomycin resistance in Pseudomonas aeruginosa [29] [30].
Therefore our study suggest that Colistin and streptomycin combined therapy may prove to be effective in treating Pseudomonas aeruginosa infections caused by MDR strains and as well as MDR strains that are resistant to Colistin and streptomycin antibiotics. Our study is also the first study report the antagonism between streptomycin and Colistin (FICI = 32.5). Therefore we recommend that it's very essential to choose the antibiotic combinations carefully and the combination should be evaluated before administration.

Conclusion and Recommendations
Based on data obtained we conclude that multi drug resistance Pseudomonal infections are on rise in this region (55.5%). In addition most of the MDR strains