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Journal of Biological Sciences

Year: 2017 | Volume: 17 | Issue: 4 | Page No.: 171-184
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Research Article

Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever

Ahmed Abduljabbar Jaloob Aljanaby Ahmed Abduljabbar Jaloob Aljanaby's LiveDNA and Ali Reyadh Medhat

ABSTRACT

Background and Objective: In developing countries such as Iraq, every year there are thousands peoples infected with typhoid fever caused by Salmonella typhi. These infections became difficult to treat by different types of antimicrobials. Therefore, the main aim of this study was to investigate the ability of S. typhi strains isolated from blood of inpatients and outpatients infected with typhoid fever to produce some antimicrobial resistance associated-genes. Materials and Methods: Disc diffusion method was used to investigate the ability of S. typhi to resistance of 12 antibiotics and PCR technique were used to investigate the prevalence of 13 antimicrobials resistance genes. Fisher's exact test was used for the comparison between samples of the study and then analyzed with graph pad prism version 5. Results: The results proved that 61.53 and 51.28% of S. typhi strains were resistant to ampicillin and chloramphenicol, respectively and all strains (100%) were susceptible to ceftriaxone 30 μg. Among 13 antimicrobial resistance genes, the most prevalent was floR (74.35%), while there was no prevalence of blaCMY-2, Cat3 and strA-strB genes. Conclusion: The floR, Cat1 and pse-1 genes were the most prevalent in S. typhi strains isolated from blood of inpatients with typhoid fever more than those isolated from blood of outpatients.
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Received: January 16, 2017;   Accepted: March 10, 2017;   Published: April 15, 2017
Copyright: © 2017. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

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INTRODUCTION

Typhoid fever or enteric fever remains the one of the most recurrent health problems worldwide and there are no advanced methods to prevent this infection except by using of antimicrobial. Typhoid fever is a most important systemic disease worldwide. Typhoid is the only remaining enteric disease for which the rates of mortality have not been declining and it is common in areas with inadequate hygiene and sanitation1.

Salmonella enterica serovar typhi (S. typhi) is the main causative agent of enteric fever which is members of the family of Enterobacteriaceae and it is the most important Gram-negative motile bacterium cause typhoid fever in Iraq and other developing countries2. The rate of morbidity of typhoid fever over than 21 million cases and some of this cases lead to death per year worldwide3. The recurrent use of the same antimicrobials against infections with typhoid fever led to an increase in multidrug resistance S. typhi and other bacterial species4. In recent years, S. typhi became more resistance to different antimicrobials such as chloramphenicol, ampicillin, sulfonamides and tetracycline and became resist to another different types of antimicrobials and therefore, called multidrug-resistant (MDR)5. Multidrug resistant S. typhi is defined as S. typhi isolates which are resistant to three different classes of antimicrobials6. The use of first-line antimicrobial for the treatment of typhoid fever such as chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole had been recommended to be replaced with other antimicrobials such as ceftriaxone and cefotaxime7.

Different studies showed that there were many prevalence of antimicrobials resistance-associated genes like, β-lactamase, ampicillin, chloramphenicol, aminoglycosides in both clinical and non-clinical Salmonella enterica isolates8,9. This study advance new knowledge by investigation the prevalence of new antimicrobial resistance associated genes in S. typhi strains.

In Iraq, there is no study focusing on isolation and investigation about antimicrobial resistance-genes in S. typhi strains isolated from clinical and non-clinical sources. Therefore, the main aim of this study was to investigate the prevalence of thirteen antimicrobial resistance associated-genes in S. typhi strains isolated from blood of inpatients and outpatients infected with typhoid fever to provide a clear image about the relationship between virulence of hospital S. typhi isolates and nonhospital S. typhi isolates.

MATERIALS AND METHODS

Salmonella typhi isolates, collection, culture and identification: During the period from June, 2016 to February, 2017 a total 187 blood samples were collected from inpatients (97 samples) and outpatients (90 samples) clinically suspected infected with typhoid fever in Al-Najaf Hospital in AL-Najaf Covernorate, Iraq. By sterile syringes (Hamilton, China), Five milliliter of blood samples were collected in sterile inoculated bottles (Oxoid,UK) containing sterile of 45 mL brain heart infusion broth (Oxoid,UK) and incubated aerobically at 37°C for 7 days with continuous shaking every day. Streaked by sterile loop (Himedia, India) on blood agar (Oxoid,UK), MacConkey agar (Oxoid,UK) and Salmonella-Shigella agar (SS agar) (Oxoid,UK) plates10. All grown colonies on the surface of agar plates were identified as S. typhi according to standard bacteriological tests according to MacFaddin11 such as, pale colonies on MacConkey agar surface, black colonies on SS agar surface, Gram-negative, motile, Alkaline/Acid, without gas and weak production of H2S according to Triple Sugar Iron test, cultivation on Chrome agar medium (Orientation Company-France) and all isolates were identified by using Vitek2® system (BioMerieux®-France).

Antimicrobial susceptibility testing: This test was done by the method of Kirby-Bauer according to the Clinical Laboratory Standards Institute (CLSI.)12. Antimicrobial susceptibility and resistance was determined by strain growth zone diameter according to CLSI guidelines12. All the following 12 antimicrobial discs were provided from (Bioanalyse-Turkey) were used in this study: Ampicillin (AM)10 μg, amoxicillin (AX) 25 μg, cefotaxime (CTX) 30 μg, ceftriaxone (CRO) 30 μg, gentamicin (CN) 10 μg, amikacin (AK) 30 μg, tobramycin (TM) 10 μg, streptomycin (S) 10 μg, tetracycline (TE) 30 μg, ciprofloxacin (CIP) 5 μg, chloramphenicol (C) 30 μg, sulfonamide(SSS) 300 μg. Escherichia coli ATCC 25922 strain was used as controls.

DNA extraction: DNA was exacted according to the method of Aljanaby and Alfaham13.

Molecular detection of antimicrobials resistance-associated genes: Polymerase Chain Reaction Protocol (PCR) was used to detect 13 antimicrobials resistance-associated genes. All PCR products were loaded on a 1% (w/v) agarose gel with 0.5 mg mL–1 safe stain and were analyzed by gel electrophoresis.

Table 1: Sequencing of primers used in PCR for 13 antimicrobials resistance-associated genes of S. typhi
Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever

All primers used in this study are listed in Table 1 and all PCR thermo cycling conditions are listed in Table 2.

Statistical analysis: Statistical analysis was performed with graph pad prism version 5 software. Fisher's exact test20 was used for the comparison between samples of the study. p-values less than the 0.05 level of significance were considered statistically significant.

RESULTS

Total isolates and S. typhi strains: The result proved that, out of total 187 blood samples there were 39 S. typhi strains isolated from inpatients (22 strains, 11.76% ) and outpatients (17 strains, 9.09%) (Fig. 1, 2).

Antimicrobial susceptibility testing: The results of the present study proved that the antimicrobial resistance rates of the 93 strains of ampicillin were high. The moderate resistance rate was observed for chloramphenicol and amoxicillin, while the lowest resistance rate was observed for cefotaxime and ceftriaxone (Fig. 3, Table 3). On the other hand, the results demonstrated that there were 17 strains (43.58%) were MDR (Table 4). Phenotypic resistance profile of 39 S. typhi strains are given in Table 5.

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 1:
Black colonies of S. typhi on SS agar surface after 24 h at 37°C of incubation

Molecular detection of antimicrobials resistance-associated genes: The results of the current study proved that out of the 39 S. typhi strains there were 18 strains (46.15%) positive for pse-1 gene, 6 strains (15.38%) were positive for blaTEM gene, 5 strains (12.82%) were positive for blaSHV gene, 24 strains (61.53%) were positive for Cat1 gene, 8 strains (20.51%) were positive for Cat2 gene and 4 strains (10.25%) were positive for cmlA gene, 29 strains (74.35%) were positive for floR gene (Fig. 4-9).

Table 2: Thermo cycling conditions of PCR for 13 antimicrobials resistance-associated genes of S. typhi
Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever

Table 3: Antibiotics sensitivity test of 39 S. typhi strains isolated from blood of patients with typhoid fever
Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
AM: Ampicillin 10 μg , AX: Amoxicillin 25 μg, CTX: Cefotaxime 30 μg, CRO: Ceftriaxone 30 μg , S: Streptomycin 10 μg, CN: Gentamicin 15 μg, AK: Amikacin 30 μg, TM: Tobramycin 10 μg, TE: Tetracycline 30 UI, SSS: Sulfonamide 300 μg, CIP: Ciprofloxacin 5 μg, C: Chloramphenicol 30 μg

Table 4:
Numbers and percentage of multidrug resistance S. typhi strains isolated from blood of patients infected with typhoid
Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
MDR: Multidrug resistance, XDR: Extensive drug resistance, PDR: Pandrug resistance,*p<0.05

Table 5: Phenotypic resistance profile of 39 S. typhi strains isolated from blood of patients with typhoid fever
Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
*Total numbers (n) and percentage (%) of S. typhi strains that were resistant to 12 antibiotics, MDR: Multidrug resistance, +: Positive, -: Negative, No. (100%)

Also, the results proved that there were one strain (2.56%) positive for ant (3")-la gene, 19 strains (48.71%) were positive for tetA, 11 strains (28.20%) were positive for tetB (Fig. 10, 11). On the other hand, the results indicated that, there was no prevalence of bla CMY-2, Cat3 and strA-strB genes in any strain. The prevalence and distribution of antimicrobials resistance genes are given in Table 6, 7 and Fig. 12, 13.

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 2:
Brown-pale colonies of S. typhi on chrome agar surface after 24 h at 37°C of incubation

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 3: Numbers of S. typhi strains that were resistant to 12 antibiotics
 
AM: Ampicillin 10 μg, AMX: Amoxicillin 25 μg, CTX: Cefotaxime 30 μg, CRO: Ceftriaxone 30 μg, S: Streptomycin 10 μ,CN: Gentamicin 15 μg, AK: Amikacin 30 μg, TM: Tobramycin 10 μg, TE: Tetracycline 30 UI, SSS: Sulfonamide 300 μg, CIP: Ciprofloxacin 5 μg, C: Chloramphenicol 30 μg

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 4: PCR amplified products from extracted DNA of S. typhi strains
 
Amplified with diagnostic gene pse-1 show positive results at 419 bp. L: DNA molecular size marker. Lane 1-39: Samples numbers

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 5:PCR amplified products from extracted DNA of S. typhi
  Amplified with diagnostic gene blaTEM show positive results at 643 bp. L: DNA molecular size marker. Lane 1-17: Samples numbers

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 6: PCR amplified products from extracted DNA of S. typhi strains
 
Amplified with diagnostic gene blaSHV show positive results at 714 bp. L: DNA molecular size marker. Lane 1-17: Samples numbers

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 7:PCR amplified products from extracted DNA of S. typhi strains
 
Amplified with diagnostic gene Cat1 show positive results at 582 bp. L: DNA molecular size marker. Lane 1-39: Samples numbers

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 8:Multiplex PCR amplified products from extracted DNA of S. typhi strains
 
Amplified with diagnostic genes Cat2 and cmlA show positive results at 547 bp and 662 bp respectively. L: DNA molecular size marker. Lane 1-18: Samples numbers

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 9:PCR amplified products from extracted DNA of S. typhi strains
 
Amplified with diagnostic gene floR show positive results at 198 bp. L: DNA molecular size marker. Lane 1-35: Samples numbers

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 10:PCR amplified products from extracted DNA of S. typhi strains
 
Amplified with diagnostic gene ant (3")-la show positive results at 526 bp. L: DNA molecular size marker. Lane 1-19: Samples numbers

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 11:Multiplex PCR amplified products from extracted DNA of S. typhi strains
 
Amplified with diagnostic genes tetA and tetB show positive results at 210 bp and 659 bp respectively. L: DNA molecular size marker. Lane 1-29: Samples numbers

Table 6:
Numbers and percentages of 13 anti-microbials resistance-associated genes among 39 S. typhi strains isolated from patients infected with typhoid fever
Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
*p<0.05

Table 7:
Genotypic resistance profile of 39 S. typhi strains isolated from blood of patients with typhoid fever
Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Total numbers (n) and percentage (%) of S. typhi strains that were carriers of antimicrobial resistance genes (total: 13 genes)

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 12:
Total numbers of antimicrobials resistance genes among 39 S. typhi strains isolated from blood of total patients infected with typhoid fever

Image for - Prevalence of Some Antimicrobials Resistance Associated-genes in Salmonella typhi Isolated from Patients Infected with Typhoid Fever
Fig. 13:
Prevalence of 13 antimicrobials resistance genes among 39 S. typhi strains isolated from blood of inpatients and outpatients infected with typhoid fever

DISCUSSION

Salmonella typhi is one of the most drug-resistant Gram-negative bacteria cause typhoid fever and it is the one of the most major public health concern in developing Asian countries and a common cause of bloodstream infection in the Middle East20,21.

The antimicrobials susceptibility test of 39 S. typhi strains proved that 61.53% of the strains were resistant to ampicillin, 51.28% were resistant to chloramphenicol, 46.15% were resistant to amoxicillin and 43.58% were resistant to tetracycline and sulfonamide. Most S. typhi strains were susceptible to tobramycin, streptomycin, amikacin, gentamicin, ciprofloxacin and cefotaxime and all S. typhi strains were susceptible to ceftriaxone (Fig. 3). Salmonella typhi strains isolated from blood of inpatients were more resistant to antimicrobials from those isolated from blood of outpatients (Table 4) and there were 17 strains (43.58%) were MDR most of them (14 strains, p<0.05) isolated from blood of inpatients (Table 5).

Multi-drug resistant S. typhi is endemic in Iraq and in some Asian countries such as Pakistan and India, also, MDR S. typhi has been reported in some parts of the world such as Finland, United Kingdom and United States22-24. Recently, the incidence of Multi-drug resistant S. typhi increased in the different countries worldwide25,26.

In developing countries, typhoid fever is a common infection in both urban and rural areas and there were over 120 million infections and more than 500000 annual deaths, worldwide27.

In Iraq and other developing countries, different antimicrobials such as ampicillin, chloramphenicol, amoxicillin and aminoglycosides were used as the first line antimicrobials typhoid fever caused by multidrug-resistant S. typhi and other type of salmonella therefore most Gram-negative bacteria including S. typhi developed new resistant against these antimicrobials27,28. In Nigeria, Fashae et al.29 proved that there were resistance rate ranged from 59-36% against ampicillin, trimethoprim, sulfamethoxazole and chloramphenicol. In previous years, multi-drug resistant S. typhi strains have been reported in different studies30-33. Recently, 986 S. typhi isolates were collected in Asia and Middle East, from Iraq, Jordan, Qatar, Egypt, Uzbekistan and Pakistan and were tested for antibiotic susceptibility to 5 antimicrobials using the Kirby-Bauer method and the results proved that 83% of strains were MDR in Iraq, 52% were MDR in Pakistan and 14% in Qatar, these S. typhi strains were resistance to chloramphenicol, trimethoprim-sulfamethoxazole and ampicillin27. In Kuwait, Dimitrov et al.34 found in his study that there were 50 clinical S. typhi strains (37%) were MDR . In Saudi Arabia, out of total 33 S. enterica strains there were 26 strain (78.78%) were MDR35. Also, in the current study, all S. typhi strains were susceptible to Ceftriaxone 30 μg, this result was observed in other Gulf countries such as, Kuwait and United Arab Emirates36. The results of the present study are in agreement with results by El-Tayeb et al.35, who proved that there were 2 S. enterica strains (6.1%) were resistance to third-generation cephalosporin such as cefotaxime. While the results are disagreement with Burke et al.37, who reported that 11% of the S. enterica strains were resistance to third-generation cephalosporin. Ceyssens et al.38 reported that there were high prevalence in quinolone resistance-associated genes in S. enterica strains.

Infected by multi-drug resistant S. typhi strains have rapidly increased over the past 25 years in Asia and the Middle East39,40. In this study, most of S. typhi strains isolated from blood of inpatients infected with typhoid fever were MDR while others strains isolated from blood of outpatients were non-MDR, this might be due to vertical transfer of antimicrobials resistance gene from other hospitalized Gram-negative bacteria such as blaSHV, blaTEM and blaCTX-M. Hassing et al.41 suggested that the presence of a mutation in some antimicrobials resistance-association genes are responsible for the emergence of this reduced susceptibility. Conjugative transfer of bacterial plasmids cause acquired antimicrobial resistance phenotypes between different bacteria42-44. Class I integrons may be localized in plasmids, which are mobile DNA elements that are important in the proliferation of MDR Gram-negative enteric bacteria and others45,46.

Molecular analysis of the 39 S. typhi strains shows there were high prevalence in floR gene (74.35%) followed by Cat1 gene (61.53%) and there were moderate prevalence in tetA gene (48.71%) and pse-1 gene (46.15%), while blaTEM, blaSHV, Cat2, cmlA, ant (3")-la, tet A, tet B genes were prevalent in low rates. On the other hand, there were no any prevalence in bla CMY-2, Cat3 and strA-strB genes (Table 7). And the results proved that S. typhi strains isolated from inpatients were carried antimicrobials resistance associated-genes more than those isolated from outpatients (p<0.05) (Table 7, Fig. 13). This results are in agreement with some previous studies2,3,47,48.

Production of extended-spectrum-lactamases enzymes lead to increase resistance to 3rd generation cephalosporins, is an ever increasing problem and is a cause of serious concern worldwide. These enzymes have been detected in many Gram-negative bacteria especially in the Enterobacteriaceae family including different serovars of Salmonella enterica47-49. In Iraq, Chloramphenicol was used as the first line to treatment typhoid fever caused by S. typhi and S. paratyphi, this recurrent use of the same antimicrobial led to limiting its therapeutic value, therefore, chloramphenicol associated-genes were high prevalence in this study.

In South African, Zishiri et al.7 reported that the pse-1 gene was prevalent with percentage 50% and tet A gene was 70%, tet B, sul 1 and sul 2 genes were exhibited 60% while ant (3")-la gene was prevalent with percentage 80%. The pse-1 gene is responsible for resistance to β-lactamase was detected in 46.15% of the total S. typhi strains found to be resistant to ampicillin and amoxicillin. Llanes et al.50 proved that resistance to β-lactam is due to the production of the pse-1 enzyme. Glenn et al.51 reported that Salmonella was carried the pse-1 gene and it is one of the most prevalent β-lactamases. One of the most important aminoglycoside resistance association-genes is the ant (3")-la and it has been detected in many pathogenic bacteria such as K. pneumoniae and p.aeruginosa but there is no enough information about the prevalence of this gene in Salmonella, especially in Iraq. In this study there was only one S.typhi strain was carried this gene. In this study, none of S. typhi strains were found to be positive for Extended-spectrum beta-lactamases by a Double-disk synergy test.

This result is quite agreement with Elumalai et al.2, who suggested a possible reason for this result is that bla-TEM gene don’t has the ability to hydrolysis 3rd and 4th generation cephalosporins while has the ability to hydrolysis penicillins and may be 1st generation cephalosporins, therefore which could not be detected by double-disk synergy test.

Overproduction and mutation in beta-lactamases is due to the continuous exposure of bacterial strains to different types of β-lactams and lead to enables these bacteria to resist antimicrobials duo to hydrolysis different β-lactam antibiotics such as penicillins and cephalosporins2,52.

The blaTEM genes are found mainly in clinical isolates of K. pneumoniae and E. coli. The first blaTEM variant with increased activity against extended spectrum cephalosporins was blaTEM-3. Th e BlaSHV-2 isolated from K. pneumoniae in 1983, showed transferable resistance to cephalosporins such as cefotaxime53. Pathogenic bacteria that cause different nosocomial infections such as K. pneumoniae, P. aeruginosa and S. aureus have long been known to produce β-lactamase13. These enzymes have spread to other bacteria such as S. typhi and S. paratyphi that previously lacked to these enzymes54. In this study, tetA and tetB genes were detected in 48.71 and 28.20%, respectively, in total isolates. The mechanism of energy-dependent membrane-associated efflux proteins is encoded by tetA and tetB genes55. But Nde and Logue56 suggested that there were another mechanisms restraint against tetracycline such as enzymatic inactivation and ribosomal protection may be including in this study. In this study, by phenotypic test a total of 39 S. typhi strains were resistant to streptomycin with percentage 23.07% but the identity of the gene strA-strB was not found, this may be due there are many genes other than strA-strB responsible for streptomycin resistant and it is very difficult to cover all antimicrobials association- genes in the present study. Also, in the current study, there was no fully compatibility between phenotypic and molecular in antimicrobials resistance results.

The antimicrobials resistant associated-genes were also detected in some susceptible and resistant isolates, this might be due to that some antimicrobial-resistant genes are silent in S. typhi strains under some circumstances. These silent genes can spread to other bacteria under continuous use of antimicrobials. Finally, the main recommendations in this study are: Avoid recurrent use the same antimicrobials such as ampicillin and chloramphenicol to treated typhoid fever, study the prevalence of other antimicrobials resistance genes in S. typhi strains and make more precautionary measures to different bacterial infections especially in hospitals.

CONCLUSION

Chloramphenicol and ampicillin resistance associated-genes (floR, Cat1 and pse-1) were more prevalent in S. typhi strains isolated from blood of inpatients with typhoid fever more than those isolated from blood of outpatients.

SIGNIFICANCE STATEMENTS

This study discovers that most S. typhi strains isolated from hospital patients became highly resistant to antimicrobials especially against chloramphenicol and ampicillin. This study will help the researcher to use the ceftriaxone to treat patients with typhoid fever and create a good image about resistance associated-genes in clinical S. typhi strains. On the other hand, the results of this study proved that S. typhi strains isolated from hospitals patients were more virulent than those isolated from non-hospital patients.

ACKNOWLEDGMENT

We are grateful to staff of Medical Laboratory in Al-Najaf Hospital for providing the clinical samples.

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