INTRODUCTION:

Proteus mirabilis is an opportunistic pathogen that is widely distributed in nature and is a part of the normal flora of the human intestinal tract. It is a member of the family Enterobactereaceae. P. mirabilis causes 90% of all Proteus infections in humans and is a major causative agent of urinary tract infection^1,². The infection risk is particularly higher in catheterized patients or patients with structural abnormalities of the urinary tract^3-5.

It is also common to find P. mirabilis bacilli in immunocompromised individuals like diabetic individuals suffering from diabetic foot ulcer⁶ and in infected burn wounds⁷.

The excessive and misuse of antibiotics in treating bacterial infections have resulted in the emergence of resistant strains that are difficult to manage⁸. There are different mechanisms by which bacteria can resist the antibiotics. These mechanisms include production of antibiotic inactivating enzymes, mutations and changing the drug target, decreased cell membrane permeability and efflux pumps^9-11.

Multidrug resistance (MDR) can be defined as the resistance of a pathogen to at least one agent in three or more antibiotic classes^12,13. The emergence of MDR pathogens makes the antimicrobial treatment ineffective and helps in the spread of persistent infections¹⁴.

The aim of this study was to determine and compare the antimicrobial resistance profile on the basis of the source of the isolates. P.mirabilis isolates were collected from three different sources; urinary tract infection, burn wound infection and diabetic foot ulcer.

MATERIALS AND METHODS:

Media and chemicals:

Mac Conkey’s agar, nutrient agar, tryptone soya broth, triple sugar iron (TSI) agar, Simmon's Citrate agarand Mueller Hinton broth were the products of Oxoid (Hampshire, UK).Other chemicals were of pharmaceutical grade. Antibacterial disks were purchased from Oxoid, Hamphsire, England. The disks were Ampicillin (AM 10µg), Piperacillin (PRL 100 µg), Ampicillin-Sulbactam (SAM 20 µg), Amoxicillin-clavulanic acid(AMC 30 µg), Piperacillin-Tazobactam (TPZ 110 µg), Cefepime (FEB 30 µg), Cefoperazone (CEP 75 µg), Ceftazidime (CAZ 30 µg), Cefotaxime (CTX 30 µg), Aztreonam (ATM 10 µg), Imipenem (IPM 10 µg), Tobramycin (TOB 10 µg), Gentamycin (CN 10 µg), Amikacin (AK 30 µg), Tetracyclin (TE 30µg), Ciprofloxacin (CIP 5µg), Levofloxacin (LEV 5µg), Gatifloxacin (GAT 5µg), Sulphamethoxazole Trimethoprim (SXT 5µg) and Chloramphenicol (C 30µg).

Bacterial isolates:

A total of 45 clinical isolates of Proteus mirabilis were used in this study. They included 15 isolates obtained from diabetic foot ulcer patients admitted to the Surgery department in Zagazig University Hospital, 15 isolates collected from patients with urinary tract infection admitted to the Urology department in Zagazig University Hospital and 15 isolates from infected burn patients admitted to the burns department in Hehia General Hospital and Al-Ahrar Hospital in Zagazig.

Phenotypic identification of the isolates

The isolates were identified morphologically and biochemically according to Konemanet al.¹⁵

Antibiotic susceptibility testing

The susceptibility of isolates to different antibiotics was determined by using Kirby-Bauer Disk Diffusion Susceptibility Test according to Clinical Laboratory Standards Institute (CLSI)¹⁶. An overnight culture in Mueller Hinton broth was prepared for each isolate, and the turbidity was adjusted to match that of 0.5 McFarland Standard by dilution with sterile saline. The prepared suspensions should be used within 15 minutes by a sterile cotton swab into each inoculum, and removing the excess inoculum from the swab on the inside wall of the tube. Each swab was streaked over the entire surface and around the rim of the Mueller Hinton agar plate and the plates were dried before applying the antibiotic discs. The plates were incubated and the inhibition zones are measured. The results were recorded as resistant, intermediate or susceptible according to CLSI¹⁷.

RESULTS:

Identification of Proteus mirabilis:

Proteus mirabilis isolates were identified as Gram-negative rods. On Macconkey’s agar, they produced lactose non-fermenting colonies and showed swarming on nutrient agar. Moreover, they were urease and indole positive and they could produce hydrogen sulphide when grown in triple sugar iron agar.

Antibiotic resistance profile:

The antibiotic resistance profile showed varying degrees of resistance to different antibiotics (Table 1). Complete resistance was found with each of ampicillin and tetracycline. High resistance was exhibited with cefepime (75.56%). The resistance was intermediate against ceftazidime (51.11%), cefotaxime (48.89%), sulfamethoxazole-trimethoprim and amoxicillin-clavulinic(46.67% each), chloramphenicol (31.11%) and cefoperazone, aztreonam and ampicillin-sulbactam (28.89% each). Low resistance was found with piperacillin (22.22%). These low resistance rates were also shown against tested aminoglycosides and fluoroquinolones. Thus, rates of resistance were 11.11% for each of gentamicin and amikacin and 13.33% for tobramycin, respectively, while the resistance rates were 17.78% for ciprofloxacin and 15.56% for each of levofloxacin and gatifloxacin. Very little resistance was found to imipenem (4.44%), while no resistance was exhibited against piperacillin- tazobactam was found.

Table 1.Antibiotic resistance profile of Proteus mirabilis isolated from diabetic foot, urinary tract and burn wound infections.

	Number (%) of resistant isolates
	DFI		UTI		BWI
Ampicillin	15(100)	15(100)		15(100)
Piperacillin	6(40)	0(0)		4(26.67)
Ampicillin-sulbactam	7(46.67)	0(0)		6(40)
Amoxicillin-clavulinic	7(46.67)	2(13.33)		12(80)
Piperacillin-tazobactam	0(0)	0(0)		0(0)
Cefepime	10(66.67)	11(73.33)		13(86.67)
cefoperazone	6(40)	1(6.67)		6(40)
Ceftazidime	5(33.33)	5(33.33)		13(86.67)
Cefotaxime	6(40)	4(26.67)		12(80)
Aztreonam	3(20)	4(26.67)		6(40)
Imipenem	0(0)	0(0)		2(13.33)
Tobramycin	2(13.33)	1(6.67)		3(20)
Gentamicin	0(0)	0(0)		5(33.33)
Amikacin	0(0)	0(0)		5(33.33)
Tetracycline	15(100)	15(100)		15(100)
Ciprofloxacin	2(13.33)	0(0)		6(40)
Levofloxacin	2(13.33)	0(0)		5(33.33)
Gatifloxacin	2(13.33)	0(0)		5(33.33)
Sulphamethoxazole-trimethoprim	7(46.67)	1(6.67)		13(86.67)
Chloramphenicol	3(20)	1(6.67)		10(66.67)

DFI, diabetic foot infection; UTI, urinary tract infection, BWI, burn wound infection

DISCUSSION:

P.mirabilis is an opportunistic bacterium that constitutes a part of human normal flora and is also widely distributed in nature. However, under certain circumstances it can turn into a virulent pathogen and produce infections in different tissues¹. It is a common etiologic agent of chronic UTI, cystitis, pyelonephritis, prostatitis, diabetic foot ulcers, multi-infection wounds^{6, 18-20}.

This study was performed to investigate the antimicrobial resistance of P. mirabilis isolated from three different common sources and to determine if there is a difference between the resistance profiles between these sources.

Different antibiotics were tested that are commonly used in treatment of P. mirabilis. The isolates were highly sensitive to imipenem and piperacillin-tazobactam. Low resistance was found to aminoglycosides and fluoroquinolones in addition to aztreonam and piperacillin. The cephalosporinsceftazidime, cefotaxime and cefoperazone showed intermediate activity. Similar results were observed against amoxicillin-clavulinic acid, ampicillin-sulbactam, sulfamethoxazole-trimethoprim and chloramphenicol. The resistance was high to cefepime and complete with ampicillin and tetracycline.

The resistance pattern showed variation among different sources. Generally, burn wound isolates showed the highest resistance rates (52%) followed by diabetic foot isolates (31%), while urinary tract isolates were the least resistant (20%). High resistance was found with cefepime only in isolates from urinary tract infections and no diabetic foot isolate was highly resistant to any of the tested antibiotics. However, such resistance was observed with amoxicillin-clavulinic acid, cefepime, ceftazidime, cefotaxime and sulphamethoxazole-trimethoprim in burn wound isolates.

Intermediate resistance was shown by diabetic foot isolates against piperacillin, ampicillin-sulbactam, amoxicillin-clavulinic acid, cefepime, cefoperazone, ceftazidime, cefotaxime and sulphamethoxazole-trimethoprim. Urinary tract isolates showed intermediate resistance to aztreonam, ceftazidime and cefotaxime only, while burn wound isolates showed similar resistance to piperacillin, ampicillin-sulbactam, cefoperazone, cefotaxime, aztreonam, gentamicin, amikacin, ciprofloxacin, levofloxacin, gatifloxacin and chloramphenicol.

Low resistance was found with tobramycin and imipenem in burn wound isolates. On the other hand, diabetic foot isolates showed such resistance with aztreonam, tobramycin, ciprofloxacin, levofloxacin, gatifloxacin and chloramphenicol. Similar degree of resistance was observed in urinary tract isolates against amoxicillin-clavulinic acid, cefoperazone, tobramycin, sulphamethoxazole-trimethoprim and chloramphenicol.

In addition to piperacillin-tazobactam that was effective against all isolates, imipenem, gentamicin and amikacin were the most potent agents against diabetic foot isolates. Urinary tract isolates were completely sensitive to piperacillin, ampicillin-sulbactam, imipenem, gentamicin, amikacin, ciprofloxacin, levofloxacin and gatifloxacin.

The resistance of Proteus mirabilis to different antibiotics was variable in different studies. Thus, Mordi and Momoh²¹ found that Proteus mirabilis isolated from wound infections were completely resistant to tetracycline and highly resistant to chloramphenicol. They showed low resistance against gentamicin, while ciprofloxacin was effective against all isolates.

In another study performed by Manikandan and Amsath²², Proteus species isolated from wound infections showed low resistance to gentamicin, tobramycin, ciprofloxacin, ceftazidime and cefotaxime and were completely sensitive to amikacin. Al-Ali²³found that all Proteus mirabilis isolates isolated from burn infection were sensitive to imipenem and cefepime. Very little resistance was found against piperacillin-tazobactam, cefotaxime, ampicillin-sulbactam, aztreonam and ceftazidime. Low resistance was reported against ciprofloxacin, gentamicin and amikacin. Intermediate resistance against sulphamethoxazole-trimethoprim was also observed.

El-Tahawy²⁴ reported that Proteus mirabilis isolated from DFIs showed low resistance to amoxicillin-clavulinic acid, gentamicin, ceftazidime and amikacin and high resistance to sulphonamides. All isolates were sensitive to piperacillin, imipenem, ciprofloxacin, aztreonam and cefotaxime.

Shanmugamet al.⁶ reported that DFI P. mirabilis isolates were completely resistant to gentamicin and co-trimoxazole. High resistance was found with amikacin, cefotaxime and ciprofloxacin. Intermediate resistance was observed to aztreonam, cefepime and tobramycin.

Cunha et al.²⁵ found that urinary tract Proteus mirabilis isolates showed low resistance to amikacin, ampicillin-sulbactam and amoxicillin-clavulinic acid. Intermediate resistance was observed to gentamicin, cefepime, ciprofloxacin and sulphamethoxazole-trimethoprim.

Multidrug resistance (MDR) was also found with varying rates in isolates from different sources. MDR was more common in burn wound isolates (86.67%) than in diabetic foot isolates (53.33%) or urinary tract isolates (6.67%). Khawcharoenporn et al. (2013) reported a higher rate of MDR uropathogenic Proteus mirabilis isolates (15.63%).The frequency of multidrug resistant Proteus mirabilis isolated from diabetic foot infection was 14.4% in a study performed by Xavier et al.²⁶; this rate was lower than that found in this study. Also, a much lower rate of MDR burn infection Proteus mirabilis isolates (2.9%) was reported by Song et al.²⁷ The high rate of MDR isolates in this study may be due to the inappropriate use of broad spectrum antibiotics without sensitivity testing.

The higher resistance of burn wound isolates may be due to the lowered immune response resulting from severe burn wound inflammation in addition to prolonged antibiotic in accordance with previous studies by Church et al.⁷ (2000) which showed that burn wound provides an ideal environment for bacterial proliferation due to its protein rich nature. Moreover, the avascularity of the necrotic tissues causes an impaired delivery of both immune defenses and antibiotics. As a result, the bacteria can grow and multiply with maximum virulence.

Several factors contribute to the high antibiotic resistance of bacteria isolated from diabetic foot infections including the type and duration of previous antibiotic therapy, frequency of hospitalization and length of hospital stay.

CONCLUSION:

The source of P. mirabilis may affect its resistance to antibiotics. Special care may be taken with patients with immunosuppression such as burn wound and diabetic foot patients. The high resistance rates make proper antibiotic dispensing policy a must to minimize the emergence of resistance.

CONFLICT OF INTEREST:

There is no conflict of interest

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Received on 30.08.2017 Modified on 06.09.2017

Research J. Pharm. and Tech. 2018; 11(1): 249-252.

DOI: 10.5958/0974-360X.2018.00046.X