Antibiotic Sensitivity against Proteus Mirabilis Isolated from Pus Samples in Tertiary Care Hospital of Quetta, Pakistan

Proteus is an important microorganism that have been isolated from wound infections causing severe complications. This study was aimed to isolate and determine the antibiotic sensitivity profile against isolates. A total of 50 wound and pus samples were collected from patients admitted in BMC Hospital, Quetta and processed in Pathobiology Laboratory of Center for Advance Studies of Vaccinology and Biotechnology (CASVAB) University of Baluchistan. All the samples were inoculated onto Brain Heart infusion (BHI), MacConkey agar, Cysteine-Lactose-Electrolyte-Deficien (CLED) and Blood agar and incubated at 37 C, overnight. The suspected colonies were identified by gram stain and different biochemical tests and also confirmed by local made Quick Test Strip 12 (QTS 12). Among the total 50 samples 8 were found positive for Proteus mirabilis. Antimicrobial susceptibility of Proteus mirabilis against different antibiotic was evaluated by Kirby-Baur Method. Four different antibiotics were tested and ampicillin was found highly susceptible (75%), followed by Gentamicin (50%), Ceftazidime (40%) and ciprofloxacin (40%). This study shows the incidence of Proteus mirabilis in wound infection and best possible antimicrobial therapies against its infection. ARTICLE INFORMATION Received: 12.12.2018 Revised: 03.03.2019 Accepted: 15.03.2019 DOI: 10.31580/pjmls.v2i1.1065


INTRODUCTION
Genus Proteus is a member of family Enterobactericeae. It is a Gram-negative rod in gram staining. Many species are member of genus Proteus i.e. Proteus mirabilis, Proteus hauseri, , Proteus penneri and Proteus vulgaris. This genus is usually Saprophytic in nature but the Many Member of this genus are opportunistic pathogens ie Proteus mirabilis, Proteus penneri and Proteus vulgaris. These are involved in septic diseases, nosocomial and genito urinary tract infections (1). Proteus is part of normal flora of Gastrointestinal tract. It is wide spread in nature and is 3 rd major causitive agent of nosocomial (hospital acquired infections) (2,3).
Usually it is not a pathogenic organism by it self but play role in seconday bacterial infection and create problems when reaches in urinary tact or other uncommon places. P. mirabalis is involved in many urinary tract problem in thousands of patients along with other oportunistic bacteria (4).
It can be identified on blood agar by production of concentric bracelets due to its crawling association and indole production is another distinguishing character of P. mirabilus from other proteus species (5).
Proteus mirabilis is Gram-negative, facultative anaerobic bacilli. It is characteristicaaly urease positive and shows swarming motility movement on blood agar. About 90% of all Proteus contaminations in humans are due to Proteus mirabalis. In many other infection the presence of Proteus spp. is notable such as ear, nose and throat (ENT) infection, respiratory tract, skin burns and gastroenteritis problems (1). Number of biochemical tests like indole , citrate , Motility, Oxidase, phenylalanine deaminase etc can be used for identification of Proteus species (6). A focused study on Proteus mirabilis causing nosocomial infection with emphasis on subclinical type is lacking in Quetta. It is therefore important to assess the effect of the Proteus mirabilis in case of pus infectious in Quetta and is necessary to estimate loss attributed to the disease. This study aims to isolateat Proteus spp. from Pus infection samples and to characterize it biochemically and evaluate antibiotic sensitivity profile.

Study area and Population
The study was performed at (post graduate laboratory) Centre for Advanced Studies in Vaccinology and Biotechnology (CASVAB) Brewery Road during June-Dec, 2016. A verbal consent was taken from 50 indoor patients that were included in this study.

Sample collection and media inoculation
A total of 50 pus samples were collected aseptically from wounds of indoor patients using swabs. All the samples streaked on Brain heart infusion (Oxoid, United Kingdom) agar, Blood agar base (Oxoid, United Kingdom) and CLED agar (Oxoid, United Kingdom).
The specimen were inoculated initially on BHI broth and on next day shifted on Blood agar and Mac-Conkey agar and and Cysteine-Lactose-Electrolyte-Deficient (CLED) agar for identification of proteus characters and incubated at 37ºC for 24 hrs. Different characteristics features of colonies were also noted such as, shape, size, color, hemolytic nature and pigmentation pattern.

Biochemical Characterization
Suspected Proteus colonies were triple cloned and gram staining of the purified colonies was performed. Colonies showing swarming growth on blood agar were subjected to different Biochemical test like Indole, Methyl Red, Vogas prausker, Citrate utilization, Triple sugar iron, nitrate reduction, H2S gas production, Oxidase, Catalase and urease production test (7).

Quick Test Strip 12 (QTS 12)
For the identification of few oxidase positve and most oxidase nagative i.e Enterobacteicae the test is successful (8). QTS 12 before inoculation is shown in (Fig. 1)

Antibiotic Sensitivity Test (AST)
Different antibiotic discs were used against P. mirabilis isolates following Kirby-Bauer disk diffusion method. The plates were then incubated at 37 0 C for 24 hrs and the zone of inhibition was measured in accordance with Clinical and Laboratory Standards Institute (CLSI). Commercially available Mueller-Hinton agar (Oxoid, UK) media and antimicrobial discs (Oxoid, UK) were used for the assay.The inoculum was prepared and matched with 0.5 Mc Farland standard as proposed by (7).

RESULTS
Out of 50 clinical samples total of 8 isolates were confirmed as Proteus mirabilis by different biochemical tests as shown in (Table I).

Cultural Characteristics
The isolates were grown on different media. Proteus gave swarming growth on Blood agar (Fig. 3) and non-lactose fermented colorless pale colonies on Mac Conkey agar were produced.

Microscopy and Biochemical Characteristics
Microscopically the isolates were gram negative pink rods when stained with gram stain. All the 8 purified triple cloned isolates of Proteus mirabilis were subjected to different test like Indole, Methyl red, Vogas prausker, Citrate utilization, H2S gas production, Triple sugar iron, nitrate reduction, Oxidase, Catalase, urease reactions and results of all biochemical test are illustrated in (Table ll). Biochemical test results of QTS 12 are given in Fig 2 and Table lll.

Antibiotic Sensitivity of Proteus Isolates
All isolates (n=8) were tested for sensitivity to four different antibiotics. Proteus mirabilis showed high sensitivity against ampicillin (23 mm), followed by Gentamicin (18 mm), Ciprofloxacin (17 mm), and Ceftazidime (18 mm). Detail is given in Table ll.  (9). It is important to minimize its occurrence in infections that may further complicate the cases. Proteus mirabilis (n=8) were isolated in present study from wound infection. Different biochemical tests were applied for identification. Several other researcher also isolated the proteus spp from wound infection using such type of traditional techniques (10)(11)(12)(13).
Despite the literature cited no such work has been reported in the Quetta area. As Proteus species are found in multiple environmental habitats including long term care facilities and hospitals and important clinical infections are produced due to this organism. It is important to have the surveillance and estimation program in order to plan strategy to combat such infections.
Antibiotic sensitivity and resistivity profile was constructed using Kirby-Bauer disk diffusion method against P. mirabilis and data regarding measurment of zone of inhibition was collected according to Clinical and Laboratory Standards Institure (CLSI) protocol. AST was performed on Muller Hinton agar (Oxoid, UK) by using commercially available antimicrobial discs (14).
Highest inhibitory zone was shown by Ceftazidime (23 mm) followed by Gentamycin (18 mm) and Ciprofloxacin (17 mm) which confirms that these antibiotics can be used for clinical purposes for the treatment and management of wounds.
According to this study Gentamycine and Floroquinolones were found as sensitive and effective drugs in wound infection. In addition this study also gives the common profile of infected wounds regarding bacterial infection and give a guideline for treatment of wound infection due to proteus. Review of literature also prove gentamycine as sensitive drug against proteus infections (15).

CONCLUSION
These findinds highlights that Genatmycin and Quinolones can be used in the treatment of wound infections complicated with proteus organisms. This study serves as base line and may be helpful for the clinicians while managing the treatment against wound infections.