INTRODUCTION:

Antibiotic sources can include; microorganisms, semisynthetic and synthetic. Antibiotics from microorganisms can be obtained from bacteria like Bacillus spp, Streptomyces spp, etc. (e.g., Erythromycin, Chloramphenicol and Tetracyclines) and from culture extracts and filtrates of fungi (e.g., Cephalosporins and Penicillins). Semi-synthesis of an antibiotic includes fermentation of part of the molecule using the suitable microorganism and after that product is modified by a chemical process ¹_. Antibiotics have a selective toxicity which means that have a high toxicity for bacteria and a low toxicity for host cells². Many of bacteria display a multidrug- resistant phenotype (MDR)^3-5. Among this bacteria methicillin resistant Staphylococcus aureus (MRSA),

Vancomycin resistant Staphylococcus aureus (VRSA) and Pseudomonas aeruginosa ^6-10. Antibiotics resistance can be controlled by appropriate using of antibiotics agents, prudent infection control, using of alternative treatment and continued monitoring¹¹. The aim of the present study was to investigate the resistant of bacterial strains isolated from clinical samples to commercial antibiotics.

MATERIAL AND METHODS:

Sample processing:

Three Pus samples and one vaginal swab were collected aseptically from the operated patients and were transported immediately to the Microbiology lab, government hospital, Aurangabad, India. The pathogens were identified by standard laboratory procedures including Gram’s staining, motility, colony characters and biochemical reactions¹².

Antibiotic susceptibility testing:

Bacterial isolates were subjected to antibiotic sensitivity testing by Kirby-Bauer disk diffusion method^13-17. The Zone of inhibition interpretation was considered as: > 18mm (Sensitive), 13-17mm (Intermediate), < 13mm (Resistant). Following antibiotics were used:

For Gram-positive isolates:

Cephalothin (CEP - 30mcg), Gentamicin (GEN -10 mcg), Oxacillin (OX – 1mcg), Clindamycin (CD – 2 mcg), Erythromycin (E – 15mcg), Ampicillin (AMP – 10 mcg), Levofloxacin (LE – 5mcg), Vancomycin (VA – 30 mcg), Chloramphenicol (C – 25mcg), Erythromycin (E –5 mcg), Fusidic acid (FC – 10mcg), Methicillin (MET – 10 mcg), Novobiocin (NV – 5mcg), Penicillin - G (P – 1 unit), Streptomycin (S – 10mcg), Tetracycline (TE – 25 mcg) and Kanamycin (K – 30mcg).

For Gram-negative isolates:

Tobramycin (TOB -10mcg), Moxifloxacin (MO – 5 mcg), Ofloxacin (OF – 5mcg), Sparfloxacin (SPX – 5 mcg), Levofloxacin (LE – 5mcg), Imipenem (IPM – 10 mcg), Meropenem (MRP – 10mcg), Ciprofloxacin (CIP – 5mcg), Co-Trimoxazole (COT – 25mcg), Cefuroxime (CXM – 30mcg), Gentamicin (GEN – 10mcg), Ampicillin (AMP – 10mcg), Ceftriaxone (CTR – 30mcg), Amikacin (AK – 30mcg), Cephalothin (CEP – 30mcg), Ciprofloxacin (CIP – 5mcg) and Kanamycin (K – 30mcg).

For Pseudomonas Species:

Gentamicin (GEN – 10mcg), Amikacin (AK – 30mcg), Cefoperazone/Sulbactam (CFS – 75/30mcg), Amoxyclav (AMC –10mcg), Ceftriaxone (CTR –30mcg), Ceftizoxime (CZX – 30mcg), Ceftazidime (CAZ – 30 mcg), Cefpodoxime (CPD – 30mcg), Ampicillin/ Sulbactam (A/S – 10/10mcg), Piperacillin/Tazobactam (PIT – 100/10mcg), Ticarcillin/Clavulanic acid (TCC – 75/10 mcg), Carbenicillin (CB – 100 mcg), Cephalothin (CEP – 30mcg), Cefuroxime (CXM – 30mcg), Cefotaxime (CTX – 30mcg), Cefoperazone (CPZ – 75 mcg) and Kanamycin (K – 30mcg). All antibiotics used in this study have been purchase from Hi media, Mumbai, India.

RESULTS AND DISCUSSION:

Bacterial species which isolated from samples are listed in Table 1. Only four bacterial species were identified.

Table-1. Type of clinical samples with identified bacterial strains.

Type of sample	Bacterial species (identified)
Pus	Staphylococcus aureus
Pus	Pseudomonas aeruginosa
Pus	Proteus mirabilis
Vaginal swab	Klebsiella pneumoniae

Antibiotic susceptibility testing:

Antibiograms for S. aureus, P. aeruginosa, P. mirabilis and K. pneumoniae are shown in Figure -1.

Figure 1. Antibiotic susceptibility testing of S. aureus (A), P. aeruginosa (B), P. mirabilis (C) and K. pneumoniae (D).

Antibiotics are medicines used to treat and prevent bacterial infection. Due to overuse and misuse of antibiotics, many of bacterial strains developed resistance mechanisms to many types of antibiotics which will increase medical costs, mortality and longer hospital stays. Although, there are many new antibiotics but the resistance of bacteria to antibiotics still increasing around the world¹⁸. In this study, four bacterial strains have been studied for their antibiotic resistant pattern. S. aureus was resistant to the majority of antibiotics (Table-2). S. aureus was only sensitive to Vancomycin (19.0 mm) and showed intermediate sensitivity to Cephalothin (17.0mm) and Clindamycin (15.0 mm), While P. aeruginosa showed intermediate sensitivity to Gentamycin (14.0 mm) and Cefpodoxime (16.0 mm) and was resistant to all other antibiotics (Table-3).

Antibiotic resistant pattern of P. mirabilis and K. pneumoniae showed that P. mirabilis was resistant to Tobramycin, Co-Trimoxazole, Cefuroxime, Ampicillin, Ceftriaxone, Cephalothin and Kanamycin (0.0.mm) and only have intermediate sensitivity to Moxifloxacin (16.0 mm) and was sensitive to the rest of antibiotics. K. pneumoniae was resistant to Tobramycin, Co-Trimoxazole, Cefuroxime, Ampicillin, Ceftriaxone, Cephalothin and Kanamycin (0.0mm) and showed intermediate sensitivity only to Moxifloxacin (16.0mm). K. pneumoniae was sensitive to all other antibiotics (Table-4).

Table 2. Antibiotic resistant pattern of S. aureus.

Antibiotics	*S. aureus* (Inhibition zone) (mm)	Interpretative Criteria
CEP -30 mcg	17.0	I
GEN -10 mcg	0.0	R
OX -1 mcg	0.0	R
CD - 2 mcg	15.0	I
E -15 mcg	11.0	R
AMP -10 mcg	11.0	R
LE - 5 mcg	10.0	R
VA - 30 mcg	19.0	S
C - 25 mcg	0.0	R
E - 5 mcg	0.0	R
FC - 10 mcg	0.0	R
MET - 10 mcg	0.0	R
NV - 5 mcg	0.0	R
P - 1 unit	0.0	R
S - 10 mcg	0.0	R
TE - 25 mcg	0.0	R
K - 30 mcg	8.0	R

Table 3. Antibiotic resistant pattern of P. aeruginosa.

Antibiotics	*P. aeruginosa* (Inhibition zone) (mm)	Interpretative Criteria
GEN – 10 mcg	14.0	I
AK – 30 mcg	11.0	R
CFS – 75/30 mcg	0.0	R
AMC – 10 mcg	0.0	R
CTR – 30mcg	0.0	R
CZX – 30 mcg	0.0	R
CAZ – 30 mcg	0.0	R
CPD – 30 mcg	16.0	I
A/S – 10/10 mcg	0.0	R
PIT – 100/10 mcg	0.0	R
TCC – 75/10 mcg	0.0	R
CB – 100 mcg	0.0	R
CEP – 30 mcg	0.0	R
CXM – 30 mcg	0.0	R
CTX – 30 mcg	0.0	R
CPZ – 75 mcg	0.0	R
K – 30 mcg	6.0	R

Zones of inhibition of: > 18mm (Sensitive), 13-17mm (Intermediate), < 13mm (Resistant).

Zones of inhibition of: > 18mm (Sensitive), 13-17mm (Intermediate),

< 13mm (Resistant).

Table 4. Antibiotic resistant pattern for P. mirabilis and K. pneumoniae.

Antibiotics	*P. mirabilis* Inhibition zone (mm)	Interpretative Criteria	*K. pneumoniae* Inhibition zone (mm)	Interpretative Criteria
TOB -10 mcg	11.0	R	0.0	R
MO – 5 mcg	16.0	I	16.0	I
OF – 5 mcg	23.0	S	20	S
SPX – 5 mcg	21.0	S	21.0	S
LE – 5 mcg	20.0	S	20.0	S
IPM – 10 mcg	20.0	S	20.0	S
MRP – 10 mcg	30.0	S	30.0	S
CIP – 5 mcg	22.0	S	25.0	S
CIP – 5 mcg	23.0	S	25.0	S
COT – 25 mcg	0.0	R	0.0	R
CXM– 30 mcg	0.0	R	0.0	R
GEN – 10 mcg	18.0	S	20.0	S
AMP – 10 mcg	0.0	R	0.0	R
CTR – 30mcg	0.0	R	0.0	R
AK – 30 mcg	18.0	S	18.0	S
CEP – 30 mcg	0.0	R	0.0	R
K – 30 mcg	0.0	R	8.0	R

Zones of inhibition of: > 18mm (Sensitive), 13-17mm (Intermediate), < 13mm (Resistant).

The results of this study were not similar to Nkang et al., 2009¹⁹ study, in which S. aureus was resistant to penicillin and chloramphenicol, P. aeruginosa was resistant only to rifampicin and vancomycin, K. pneumoniae was resistant to ampicillin and tetracycline. Only similar result was showed by P. mirabilis, which resistant to the most types of antibiotics (9 types). Another study indicated by Nutanbala et al., 2011²⁰, showed that all bacterial strains isolated from postoperative wounds infections showed a fewer resistant pattern in compare to our study. In which, S. aureus was resistant to oxacillin and penicillin, while P. aeruginosa was resistant to ceftazidime and meropenem. K. pneumoniae was resistant to ceftazidime, cefuroxime and cefdinir. P. mirabilis showed the highest resistant to cefdinir, cefuroxime, co- trimoxazole, ampicillin and sulbactam

Rama et al., 2012²¹, demonstrated that P. aeruginosa, K. pneumoniae and S. aureus which isolated from surgical wounds (Pus), showed a different resistant pattern from our results. In his study, S. aureus was only resistant to oxacillin and ofloxacin, while P. aeruginosa was resistant to pipercillin-tazobactam. Also, K. pneumoniae showed resistant to B- lactams, quinolones, B- lactam- B- lactamase and aztreonam.

Mwambete et al., 2015²², reported that P. mirabilis, K. pneumoniae, P. aeruginosa and S. aureus which also isolated from surgical wounds Pus showed a less resistant to antibiotics. S. aureus was resistant to Erythromycin, while P. aeruginosa and K. pneumonia were only resistant to Trimethoprim-sulfamethoxazole and P. mirabilis was resistant to Trimethoprim-sulfamethoxazole and Cefotetan. Also, Ravishankar et al., 2017²³ demonstrated that K. pneumonia (isolated from vaginal swabs) showed resistant to amoxicillin and less resistant pattern to ciprofloxacin, cotrimaxole and cefuroxime.

CONCLUSION:

In comparison of our study to the other studies, our isolates showed high resistant pattern to antibiotics which mean that bacterial strains develop more resistant mechanisms to the most commercial antibiotics. More researches are needed to investigate the spread of antibiotics resistance by bacterial strains isolated from clinical samples.

CONFLICTS OF INTEREST:

There are no conflicts of interest.

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Received on 26.09.2019 Modified on 29.11.2019

Research J. Pharm. and Tech. 2020; 13(8):3813-3816.

DOI: 10.5958/0974-360X.2020.00675.7