ABSTRACT In-Vitro Studies of Staphylococcus aureus and Klebsiella pneumoniae from Clinical Specimens of Various hospitals Hyderabad

ABSTRACT
In-Vitro Studies of Staphylococcus aureus and Klebsiella pneumoniae from Clinical Specimens of Various hospitals Hyderabad,Sindh,Pakistan Region
Staphylococcus aureus a major human pathogen capable of causing a wide range of infections, from relatively mild skin infections such as folliculitis and furunculosis to life-threatening conditions, including sepsis, deep abscesses, pneumonia, osteomyelitis, blood stream infections . Klebsiella pneumoniae major cause of nosocomial infections world wide.Commonly found as a commensal bacterium, that has potential to cause a wide range of infections, including soft tissue, wound, and urinary tract infections (UTIs) and respiratory tract infections, The infections caused by both test organisms were emerged increasing frequency among the males and females. This research work presents an In-vitro study of both the bacteria. 111 samples of pus, skin swabs and urine were examined to collect various isolates for their prevelance of infection in males and females and their antibiotic pattern.The laboratory media showed beta hemolysis on blood agar whereas yellow colonies on manitol salt agar plates by S. aureus with mannitol fermentation. K. pneumoniae showed gray, mucoid, large colonies, mucoid and purple sticky colonies on blood agar; MacConkey’s agar and Eosine methylene blue agar plates respectively. Klebsiella isolates showed large dome shaped colonies on blood agar and lactose fermenting mucoid colonies on MacConkey agar.
During lab diagnosis, the Staphylococus aureusisolates showed gram reaction (+ve), cocci in shape, grape like clusters, arranged in bunches, non-sporing in nature and Klebsiella pneumonia were observed as gram negative, short, plump, straight rods were seen. Staphylococcus aureus showed catalase, coagulase, methyle red, Voges Proskauer, citrate, urease, nitrate reduction, gel hydrolysis (+ve) whereas oxidase, indole, H2S, gas production in TSI and phenylalanine deaminase (-ve). On the other hand K. pneumoniae showed catalase, citrate, Voges Praskauer, urease, nitrate reduction (+ve) whereas oxidase, indole, methyle red, gel liquification, H2S production were (-ve).
Various antibiotics such as tested for sensitivity and resistance of S.aureus and Klebsiella pneumoniae on MH agar plates. It was observed that Staphylococcus aureus are more sensitive to Tobramycin, Gentamycin (98%), Chloramphenicol, (97%), Clindamycin and Kanamycin (95%) and resistant to Methicillin (100%), Oxacillin (99%) and Erythrocin (99%) respectively.The sensitivity pattern of Klebsiella pneumonia revealed that the test isolates showed greater sensitivity on Cephalosporin, Clavulanic acid (97%), Imipenim (95%), Meropenim (88%), Amoxicillin (85%), Cefotaxime (83%) and resistance on Vancomycin (95%), Amikanin (90%), Chloramphenicol (85%), Fosfomycin and Gentamycin (80%) respectively.
The sensitivity percentage revealed that S. aureus is 64% sensitive and 29% resistant to test antibiotics whereas K. pneumonia showed 50% sensitivity and 37.5% resistance to the test antibiotics

INTRODUCTION
Staphylococcus aureus has been demonstrated to be a major human pathogen capable of causing a wide range of infections, from relatively mild skin infections such as folliculitis and furunculosis to life-threatening conditions, including sepsis, deep abscesses, pneumonia, osteomyelitis, blood stream infections and infective endocarditis through both toxin-mediated and non-toxin-mediated mechanisms (Van Belkum et al., 2009; Moreillon et al., 2005; Lowy, 2012).The first case of methicillin-resistant S. aureus (MRSA) was described in the United Kingdom in 1961. (Saikia et al., 2009).Staphylococcus aureus resistant to methicillin (MRSA) is currently among the bacteria of global concern (R.Valaperta et al., 2010; R. H. Deurenberg et al., 2008). It is responsible for a broad range of community-associated MRSA infections, especially in young individuals soft tissue without risk factors (J.R. Mediavilla et al., 2012; C. A. Alvarez et al., 2010) MRSA are usually penicillinase producers and frequently multi drug resistant.( Garner JS et al., 1988 ) The most serious cases of MRSA resistance are those mediated by the mecA gene; these strains can simultaneously acquire pathogenicity genes that make them able to develop more aggressive infections (R.Valaperta et al., 2010). Drug of choice to treat infections caused by MRSA is vancomycin. It is a glycopeptide and has been used to treat infections caused by MRSA all over the world. ( Smith T.L et al., 2008)
Klebsiella pneumoniae is a common Gram-negative bacterium that has become a major cause of nosocomial infections worldwide.Commonly found as a commensal bacterium, K. pneumoniae has the potential to cause a wide range of infections, including soft tissue, wound, and urinary tract infections (UTIs) and respiratory tract infections, in particular in patients with a compromised immune system (Jarvis WR et al., 1985; Jones RN et al., 2010; Podschun R et al., 1998; Holt KE et al., 2015) Antimicrobial resistance in K. pneumoniae I increasing, particularly beta-lactamases and carbapenemases having been well-characterized as increasing the infection threat. This seriously antibiotic management problem is now frequently seeing both nosocomial and community associated infections. Infections caused by extensively drug-resistant (XDR) K. pneumoniae, (Mathers et al., 2015; Campos et al., 2016; Lee et al., 2016
Antibiotic resistance pattern in Pakistan
Antibiotic resistance is increasing in all regions of the world but in developing countries this rate is alarming primarily due to over-dosage of antibiotics. This over exposure to drug is making microbes stronger. In developing countries antibiotics are generally given for very long time and without proper culturing (Grover SS et al., 2004). Pakistan being a developing country is also facing the same problem. Cephalosporin was one of the most frequently prescribed antibiotics and an increase in its resistance has been reported, mainly due to over exposure to drug (Sheikh JM et al.,2007 ; Sheikh D et al.,2002). Pakistan has very high rate of the mortality and morbidity due to nosocomial infection primarily because of over-crowded hospitals with insufficient cleaning and disinfection (Shahid M et al., 2008).

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MATERIAL ; METHOD
Our area of research is based on the isolation and identification of gram positive (Staphylococcus aureus) and gram negative bacteria (Klebsiella pneumoniae) and their antibiotic resistance from clinical specimen. Our research work is mostly completed at microbiology Research Laboratory university of Sindh Jamshoro. A total of 111 samples of pus (23; Male 15, Females 08), skin swabs (35; Male 17, Female 18) and urine (58; Male 21 and Female 37) samples were processed during our study period.
Collection of Specimen
Various specimens were collected from various hospitals Such as Liaquat University of Medical ; health Science (LUMHS) Diagnostic and Research Laboratory Hyderabad, ISRA university Hospital, Maajee and Bhittai Hospital. These specimens were Urine, Pus, and Skin swabs and were examined in Department Lab.
Laboratory diagnosis of Specimens
All glass wares were decontaminated by autoclaving those at 121 C, 15 lbs/square inch for 20 minutes later these glass ware were washed with Liquid detergent ; rinsed with chromic acid and with water. Petri Plates were again autoclaved by wrapping paper for sterility. These were then oven-dried at 180 c for 1 hour
Preparation of Laboratory Media
All media were prepared by w/v concentration of the respective ingredients dissolved in 1 liter of distilled water. The media were sterilized in autoclave at 121°C, 15 lbs pressure for 20 minutes.
Preparation of Blood Agar
Nutrient agar powder was weighed and an agar medium was prepared by w/v concentration and poured in Erlenmeyer flask of 300 ml. distilled water, heated and sterilized by autoclave. After short time 7% sheep blood was added and mixed to form a blood agar. It was later poured under hygienic conditions in sterile Petri plates, dried, cooled and were left for 30 minutes for solidification. These blood agar plates were kept in incubator over night at 37°C for sterility test.
Pouring of media
All media were separately poured hygienically in laminar air flow chamber in sterile petri plates, plates were cooled for 20 to 30 minutes and kept incubator for 24 hours at 37 °C to determine sterility test.
Culturing
Using sterile wire loop, specimen were streaked over the surface of agar media plate for isolation of pathogens. Plates were covered, inverted and incubated over night at 37 °C for growth.
Isolation and maintenance of strains.
Isolated strains were grown in nutrient broth or nutrient agar and brain heart infusion agar. All plates and tubes were kept at 37°C for 18–24 hours. Pure culture was maintained on nutrient agar Brain heart infusion agar slants with glycerol and kept at 4°C for further analysis.
Gram Staining
Gram staining was done by making smear on clean grease free glass slide, smear was heat-fixed, flooded by primary stain crystal violet for 1 min. Later the smear was gently washed by tap water and Gram’s Iodine was used as chemical mordant for 1 min. The slide was again washed with water and ethanol 70% was used to decolorize the smear and then immediately wash with water. Slides were air-dried for short time and cover it with safranine for 1 minute. The slide is then washed from both sides with water and blotting may be done with filter paper. Using the 100X objective lens the morphology of the test strain was obtained
Antimicrobial Susceptibility Testing
Antimicrobial susceptibility was performed by Kirby-Bauer method of disc diffusion on pre-lawned culture on Mueller Hinton agar plates.

RESULTS
Total 111 samples of pus (23; Male 15, Females 08), skin swabs (35; Male 17, Female 18) and urine (58; Male 21 and Female 37) samples were processed during our study period. During the work Staphylococcus aureus isolates were mainly determined from skin swab and pus (09) whereas Klebsiella pneumoniae from pus (06) and urine (26) isolates and their percentage was determined (Table 1, Fig. 1-3).
Sample Total No. of samples Gender wise percentage
Male % Female %
Pus 111 15 13.5 08 7
Skin swab 17 15 18 16
Urine 21 19 37 33
Table No:1 Determination of the percentage of various samples from male and female patients
-47625624840Fig.-1. Determination of the percentage of various samples from male and female patients
Fig.-2. Determination of the percentage of clinical isolates (S. aureus) from varios samples from male and female patients
Fig.-3. Determination of the percentage of clinical isolates (K. pneumoniae) from varios samples of male and female patients
Colonial characters
-9525-4805680The laboratory media such ad blood agar, manitol salt agar, brain heart infusion agar, MacConkey’s agar and EMB agar, CLED agar were prepared according to the prescribed formulae (w/v) of Oxoid manual. The colonial observations of Staphylococci aureus were observed as mucoid, glossy, convex, color less colonies with beta hemolysis and yellow colonies on blood agar and manitol salt agar plates. On nutrient agar Staphylococcus aureus showed circular, pinhead colonies, convex with entire margins with a golden-brown color. On the other hand gram negative K. pneumoniae showed gray, mucoid, large colonies; deep pink to purplish colonies and dome shaped mucoid, purple sticky colonies on blood agar; MacConkey’s agar and Eosine methylene blue agar plates respectively. Klebsiella isolates showed large dome shaped colonies on blood agar and lactose fermenting mucoid colonies on MacConkey agar.
Morphological characters
During lab diagnosis, the Staphylococcus aureus showed gram reaction (+ve), cocci in shape, grape like clusters, arranged in bunches, non-sporing in nature. Morphologically Klebsiella pneumonia were observed as gram negative, short, plump, straight rods were seen.
-35991802432050The Antibiogram
Fourteen Antibiotics such as tested for sensitivity and resistance of S.aureus and Klebsiella pneumoniae on MH agar plates. It was observed that Staphylococcus aureus are more sensitive to Tobramycin, Gentamycin (98%), Chloramphenicol, (97%), Clindamycin and Kanamycin (95%) and resistant to Methicillin (100%), Oxacillin (99%) and Erythrocin (99%) respectively.
The sensitivity pattern of Klebsiella pneumonia revealed that the test isolates showed greater sensitivity on Cephalosporin, Clavulanic acid (97%), Imipenim (95%), Meropenim (88%), Amoxicillin (85%), Cefotaxime (83%) and resistance on Vancomycin (95%), Amikanin (90%), Chloramphenicol (85%), Fosfomycin and Gentamycin (80%) respectively.
The sensitivity percentage revealed that S. aureus is 64% sensitive and 29% resistant to test antibiotics whereas K. pneumonia showed 50% sensitivity and 37.5% resistance to the test antibiotics (Table 2-3, Fig. 4-6).

Name of the Antibiotic Susceptibility Pattern
Resistance % Sensitivity % Intermediate
%
Cefoxitine 78 20 2
Vancomycin 11 89 0
Methicillin 100 00 00
Oxacillin 99 00 1
Lenizolid 08 91 1
Clindamycin 05 95 0
Rifampin 07 91 2
Ciprofloxacin 18 80 2
Chloramphenicol 03 97 0
Erythromycin 99 00 1
Tetracycline 30 68 2
Gentamycin 01 98 1
Kanamycin 02 95 3
Tobramycin 00 98 2
Table-2 Determination of antibiogram of
Staphylococcus aureus
-104775150495
Fig.-4. Determination of antibiogram (%) of S. aureus on various antibiotics
Name of the Antibiotic Susceptibility Pattern
Resistance % Sensitivity % Intermediate
%
Amoxicillin 15 85 00
Cefotaxime 16 83 01
Imipenem 05 95 00
Gentamicin 80 14 06
Amikacin 90 02 08
Tetracycline 44 55 01
Ciprofloxacin 25 68 07
Nalidixic acid 75 20 05
Chloramphenicol 85 14 01
Fosfomycin 80 19 01
Vancomycin 95 02 03
Chloramphenicol 12 85 03
Cephalosporins 02 97 01
Clavulanic acid 03 97 00
Meropenem 11 88 01
Ampicillin 11 85 04
-55880422275Table-3 Determination of antibiogram of Klebsiella pneumonia
Fig.-5. Determination of antibiogram (%) of K. pneumoniae on various antibiotics
-10477519050Fig.-6 Determination of total percentage of antibiotic sensitivity pattern of Staphylococcus aureus and Klebsiella pneumoniae
DISCUSSIONS
This research work is entirely done to get the basic information of the Staphylococcus aureus and Klebsiella pneumonia from the clinical specimens of various patients of the Hyderabad. During experiments the plates and other glassware, steel ware were decontaminated by autoclave in order to prevent contamination during the lab. Investigations. Oven dry technique was applied to eliminate un-necessary moisture from the laboratory wares. Various media were prepared by w/v methods to get the exact concentration of the nutrients in the respective media to the test isolates.
A total of 111 samples of pus (23; Male 15, Females 08), skin swabs (35; Male 17, Female 18) and urine (58; Male 21 and Female 37) samples were processed during our study period. During the work Staphylococcus aureus isolates were mainly determined from skin swab and pus (09) whereas Klebsiella pneumoniae from pus (06) and urine (26) isolates. The variation in positivity of specimens in male and female patients may be due to the exposure to the specific environment and the immune system, rational use of drugs and food habits. Staphylococci are mainly isolated from skin swabs and pus due to the fact that they are more frequently grown bacteria in hospital environment such as operation theaters, medico-legal wards and the habitant of hospital staff as carriers, which could be transferred through handling of surgical instruments and by direct contact. S. aureus is the most common bacteria that produce pus (Kumar, 2013). The most common pus producing bacteria are Staphylococcus aureus followed by Klebsiella spp.,
Growth of S. aureus on blood agar shows beta-hemolysis, which was characterized by a clear, transparent zone surrounding the colonies. Staphylococcus aureus growth is well documented on Mannitol Salt Agar because of the ability of a bacterium to grow in a 7.5% salt environment and the fermentation of mannitol as a food source and produce acidic byproducts of fermentation that will lower the pH of the media. Growth of Klebsiella on Eosin Methylene Blue Agar selectively due to the inhibition of the growth of Gram+ bacteria. The enhanced cell walls of Gram-negative bacteria protect these bacteria from the dye in the EMB plates. The dye is able to enter the cells of Gram positive bacteria and kill them. MacConkey Agar demonstrates the ability of a gram negative bacterium to metabolize Lactose. It contains crystal violet dye and bile salts, both of which inhibit the growth of most gram-positive bacteria. On MacConkey agar Klebsiella shows dark pinkish colonies that may be due to the fermentation of lactose producing acid
S. aureus the resistance to the ?-lactam antibiotics is due to the inhibition of the cell enzyme which in this case would be ?-lactamase (Lowy, 2003). Due this inhibition, Cell wall synthesis slows down that brings down osmotic pressure and eventually kills the cell. This is due to the fact that blaZ gene; located on a transposable part of the large plasmid within the S. aureus bacteria cells that codes for penicillinase; does not allow for it to bind with ?-lactam ring. The mecA gene is located within the chromosome which allows the bacteria cell to transfer it to other cells. Methicillin resistant strands of S. aureus contain the mecA gene that may induce the methicillin resistance. These genes are regulated by two recombinase genes including the ccrA and the ccrB, which help express the MecA gene. Klebsiella on the other hand were also reported from pus as G-ve pathogen that may be transferred through the hospitalization and by using contaminated bed sheets, pillows and the use of contaminated catheters, drips or direct contact(microbewiki.kenyon.edu).The sensitivity and resistance pattern of the Klebsiella isolates have been associated with different types of infections. However the main importance of Klebsiella as a pathogen is in causing infections in hospitalized patients, the strains responsible are nearly always biochemically typical members of Klebsiella pneumoniae (Murray et al., 2005). Moreover, extensive use of broad spectrum antibiotics in hospitalized patients has led to both increased carriage of Klebsiella and the development of multidrug resistant strains like those of Extended Spectrum Beta Lactamases (Sikarwar et al., 2011).
Our results of S. aureus and Klebsiella pneumoniae isolates are in accordance with (Verma, 2012; Sharma et al., 2015; Bhat and Vasaikar, 2010; Rao et al., 2014).
Klebsiella pneumoniae is reported a most common carbapenem-resistant Enterobacteriaceae (CRE) species in the United States but its more utility emerged the resistance to CRE and nearly all available antibiotics (Gupta et al., 2011; Centers for Disease Control and Prevention, 2013; Shon et al., 2013; Tzouvelekis et al., 2012). This resistance is due to the enzymes that inactivate mechanism of resistance e.g. serine ?-lactamase KPC (Gupta et al., 2011). A more recently identified enzyme carbapenem-active metallo-?-lactamase NDM-1is also known for resistance (Tzouvelekis et al., 2012). Additionally alarmingly, blaNDM-1 is often found on large conjugative plasmids along with additional antibiotic resistance determinants (Chen et al., 2011). The recent spread of blaNDM-1 across a large geographic area has been remarkable and well documented (Yong et al., 2009; Chen et al., 2011; Huang et al., 2013; Castanheira et al., 2011;)
Non-carbapenemase mechanisms such as increasing efflux pump activity and altering the profile of outer membrane porins that control access of carbapenems to the cell wall (Pfeifer et al., 2010; Walsh et al., 2011).
Drug resistant Enterobacteriaceae Klebsiella pneumoniae are emerging as serious infectious agents. These strains can accumulate many antibiotic resistance genes. The gene for the broad-spectrum and carbapenem-active metallo-?-lactamase NDM-1 is rapidly spreading in Klebsiella pneumonia. The resistance is also reported by ATCC in 23 ?-lactams e.g. penicillins with or without inhibitors, cephalosporins, carbapenems and aztreonam, 05 fluoroquinolones, 03 aminoglycosides e.g. tobramycin, amikacin and gentamicin and 04 others tetracycline, tigecycline, nitrofurantoin, and trimethoprim / sulfamethoxazole (www.atcc.org)
CONCLUSIONS
It is concluded that the S. aureus and Klebsiella pneumonia are the pathogenic bacteria of human diseases. S. aureus is most frequently isolated microorganism both male and female (?male) as compared to Klebsiella pneumonia.
It is also concluded that females are more susceptible to urinary tract infections as compared to males where K. pneumonia is the potential pathogen (?females) isolated from both pus and urine specimens but no pathogen isolated from skin swabs of female patients.
The antibiograph of S.aureus showed 64% sensitivity and 29% resistance whereas K. pneumonia showed 50% sensitivity and 37.5% resistance to the test antibiotics. It was also concluded that some of the drugs are more effective whereas few others could be the alternate therapy to the S. aureus and K. pneumonia infections.