Increasing Antibiotic Resistant Pattern in Clinical Bacterial Isolates, From Tertiary Care Hospital, Hayatabad Medical Complex, Peshawar, Pakistan.cdr

Bacterial infections are spreading worldwide especially in the developing countries. Most clinical pathogens have evolved mechanisms of resistance due to which most antibiotics are less or not effective to restrict their growth. Current study aims on finding the prevalence of antibiotics resistance in clinical isolates. Material and Methods: Total (n=753) clinical specimens were collected, among them, total (n=105) bacteria were identified on the basis of standard culture characteristics and biochemical tests and their antibiotics resistance pattern were determined. Results: Higher incidence of multidrug resistance bacteria were found in patients aged above 50 years and were prevalent in OPD, emergency and gynea wards. The dominant bacterial species were gram negative i.e. Escherichia coli (29%), Staphylococcus aureus (19%), Pseudomonas aeruginosa (13.33%), Acinetobacter species (5.71%), whereas, gram negative isolates were Staphylococcus epidermidis (9.52%), Streptococcus specie (5.71%), and Enterococcus faecium. Antibiotics like amoxicillin/clavulanic acid, cefuroxime and sulphamatoxazole/trimethoprim were resistant to 64.61%, 63.07% and 61.53% of gram negative bacteria respectively while ciprofloxacin, doxycycline and fusidic acid were resistant to 70%, 52.5% and 52.5% gram positive bacteria respectively. The most susceptible antibiotics against gram negative were sulbactum/cefoperazone and amikacin while to gram positive were linezolid, chloramphenicol and rifampicin. Conclusion: Current study revealed increasing antibiotic resistance pattern that need intimidate focus on surveillance of antibiotics resistance regularly and to ensure long lasting efficacy of antibiotics.
 

Bacterial infections are spreading worldwide especially in the developing countries. Most clinical pathogens have evolved mechanismsof resistance due to which most antibiotics are less or not effective to restrict their growth. Objective: To nd the prevalence of antibiotics resistance in clinical isolates. Methods: Total (n=753) clinical specimens were collected, among them, total (n=105) bacteria were identi ed on the basis of standard culture characteristics and biochemical tests and their antibiotics resistance pattern were determined Results: Higher incidence of multidrug resistance bacteria were found in patients agedabove 50 years and were prevalent in OPD, emergency and gynae wards. The dominant bacterial species were gram negative, Escherichia coli (29%), Staphylococcus aureus (19%), Pseudomonas aeruginosa (13.33%), Acinetobacter species (5.71%), whereas, gram negative isolates were Staphylococcus epidermidis (9.52%), Streptococcus specie (5.71%), and Enterococcus faecium. Antibiotics like amoxicillin/ clavulanic acid, cefuroxime and sulphamatoxazole/ trimethoprim were resistant to 64.61%, 63.07% and 61.53% of gram-negative bacteria respectively while cipro oxacin, doxycycline and fusidic acid were resistant to 70%, 52.5% and 52.5% gram positive bacteria respectively. The most susceptible antibiotics against gram negative were sulbactum/ cefoperazone and amikacin while to gram positive were linezolid, chloramphenicol and rifampicin Conclusion: Current study revealed increasing antibiotic resistance pattern that need intimidate focus on surveillance of antibiotics resistance regularly and to ensure long lasting e cacy of antibiotics.
were considered on their way to eradication [3]. However, extensive antibiotics usage has upheld the antibioticresistant pathogens. Resistance spreading promptly, predominantly in hospitals, where different bacteria may come in close contact with each other and providing the environment for distributing the resistant genes with other bacteria [4,5]. Bacterial infections due to both gram positive and negative bacteria have caused huge causalities. Most gram negative pathogens such as Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. make hindrance in treatment by developing different strategies [6]. The use of antibiotics in any environment produces selection forces that favor the survival of antibiotic resistant pathogens [7]. Vatopoulos and Kalapothaki, (1999) [8] reported that Escherichia coli, K. pneumoniae, Enterobacter spp, P. aeruginosa, Acinetobacter baumanii and Staphylococcus aureus have shown resistivity patterns to different pathogens. And these pathogens have been isolated from nosocomial or outpatient in most parts of the world because of their rate of isolation, pathogenicity and virulence [8]. It is obvious that we are at a critical time in the history of medicines, where the genetic variations and acquirements from environmental sources by bacteria may leave us with no lifesaving therapeutics options [9]. Keeping in view the emergence of resistance in bacterial pathogens and due to limited options of treatment, present study was designed to nd the prevalence of antibiotics resistant bacteria in tertiary care hospital sittings and to design strategies to control the spread of resistant bacteria.

Collection of samples and specimens:
In this study total 753 specimen samples were collected from patients who were admitted or visited tertiary care hospital Peshawar. Clinical history, informative details and demographic features like age and gender of each patient were noted. Different clinical culture and sensitivity test samples were taken from urine, wound (pus) swab, sputum samples, blood etc. and were transported on ice bag and glycerol stock to the Department of Microbiology, Kohat University of science and technology for further analysis. This research was conducted from May, 2017 to October, 2017. Bacteriological analysis: The samples were streaked on culture media for isolation and growth. Then all the samples except urine samples were streaked on MacConkey to differentiate between gram positive and gram negative bacteria and also to examine lactose and non-lactose fermenting bacteria. Urine samples were streaked on Cystine Lactose Electrolyte-de cient (CLED) medium. Blood agar plates were used to differentiate fastidious bacteria especially streptococcus species. Hemolysis were observed, alpha hemolysis showed, Streptococcus aureus while beta and gamma hemolysis indicated Streptococcus pneumoniae. All the cultures were incubated for 24hours [8]. For morphological identi cation the isolates were grown on agar media and their growth patterns, shape and color were observed. Gram staining was performed to further differentiate bacteria based on staining and shape. Biochemical tests: For biochemical characterization tests like catalase, triple sugar ion (TSI), motility, indole, urease and oxidase were performed and according to Bergey's Manual of systematic Bacteriology, on the basis of morphological, physiological and biochemical features, characterization was done[10]. Antibiotic susceptibility patterns: By using Kirby-Bauer disc diffusion method [11], following the recommendation 12 of the clinical and laboratory standard institute the antibiotic resistivity of bacterial isolates was assisted. With addition of 5% sheep blood, Mullen-Hinton agar was prepared. A suspension of bacteria was made and then streaked on agar plate. Antibiotic disks were applied and o then incubated at 37 C for 24 to 48 hours. , nitrofurantoin and fosfomycin were used.All these antibiotic disks were purchased from Oxoid, UK. After 48 hours of incubation, the diameters of zone of inhibition around the antibiotic disks were measured by using graduated ruler and interpreted the results according to CLSI guidelines [20].
Essential oil and standard drug were tested against Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Pseudomonas aeruginosa (P.aeruginosa) and Escherichia coli (E. coli). Cumin essential oil showed signi cant antibacterial activity against both grampositive and gram-negative bacterial strains. Standard drug data showed that it was effective against S .aureus and least against E.coli which is a resistant gram negative strain.   Among the total of 105 bacterial isolates, 61.90% (n=65) were gram negative and38.1%(n=40) were gram positive. The biochemical testsare given in Table 3 used for the identi cation process. E. coli was the most frequently found gram negative bacteria whereas S. aureus was most frequently found gram positive bacteria. Overall frequency of E.coli (29%) was high, followed by S.aureus (19.4%), and P. aeruginosa (13.33%). Among less frequently found bacteria, the Acinetobacter specie (5.71%), Enterobacter, Coliforn and Proteus mirabellis (3.80%) and Providencia species (2.85%) were gram negative whereas, S.epidermidis (9.52%), Streptococcus species (5.71%), and Enterococcus faecium (3.80%) were gram positive as shown in Table 4    Alekshun MN, Levy SB. Molecular mechanisms of a n t i b a c t e r i a l m u l t i d r u g r e s i s t a n c e . C e l l . 2 0 0 7 ; 1 2 8 ( [4] [5] [6]  Infectious diseases are the result of host invasion with a pathogen. The detection, treatment and prevention of human diseases are the challenges physicians, pharmacists and microbiologists are facing. In our total studied clinical samples 38% urine, 32% wound and 30% swabs samples were collected from patients. Similar specimen samples were also studied by Khurshid et al., (2002) [13] at Ayub Medical College, Pakistan. Gram negative pathogens were more prevalent in our study which is correspondence with a study conducted at tertiary general hospital China where 59% gram negative isolates were observed [14]. In our study the majority of infections are due to E. coli29%followed byS.aureus19.04%. In another study it is reported that S.aureusas 18.5% and E. coli as 16.7% isolated from nosocomial infection patients [15]. The most susceptible antibiotic against gram negative bacteria were AK and SCF while against gram positive were LZD, C and RIF. Similar studies on antibiotic susceptibility pro ling were conducted by Sekhar, et al (2014) [16] in India where all gram positive isolates were sensitive to DO while gram negative to AK, SCF, and MEM and George et al (2018) [17] who reported IMP as most effective antibiotic against gram negative while VA and CL were effective against gram positive. Antibiotics are

D I S C U S S I O N
notorious to put selective pressure on antibiotic susceptible bacteria and boost the development of antibiotics resistant. It is therefore a possible reason of little disagreement with other studies [18]. Moreover, high antibiotic resistance rates were observed in gram negative bacteria against CE, AMC and SXT while in gram positive against CIP and DO. In a previous pilot study resistance against AMC was observed in gram negative isolates while against PEN to gram positive isolates [19]. The changes in bacterial resistance pro le are may be due to the close relationship of bacteria with each other and their easy acquirement of genes in different environments [20].

C O N C L U S I O N S
It is concluded that the most prevalent pathogens in our study were E.coli (29%), S.aureus (19.4%) and P. aeruginosa (13.33%) and higher incidence were observed in urine specimens, male and old age people. The AK and SCF in gram negative while LZD, Cand RIF were found most susceptible while CE, AMC and SXT in gram negative and CIP and DO in gram positive were found most resistant antibiotics. So knowledge of the clinical, bacteriological nding as well as antibiotic susceptibility pro les are essential for choice of appropriate antibiotic with maximum effectiveness in correct management of the patient and to reduce the risk of complications.