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3-research journal-2010-bsr-iris.pmd

Antibiotic susceptibility and determination of Minimum Inhibitory
Concentration (MIC) of potent antibiotics used against Staphylococcus spp.
isolated from raw milk

Ankita Shrestha • Priyaragini
• Satyamvada Swayamprabha

: December 2010
: November 2011
Corresponding Author : Satyamvada Swayamprabha
Abstract : The uncontrolled use of antibiotics has led to the
Ampicillin + Cloxacillin was the most effective combination development of multiple antibiotic resistance, there by exhibiting 14.31% efficacy in phase II. The MIC values of rendering the treatment ineffective. In the present study, raw two antibiotics in pure form and three in combinations were milk samples were collected from different areas of Patna. determined by agar dilution and broth dilution methods. Out of the 12 isolates obtained, nine were identified as The MIC values ranged between 0.5 – 1.0 µg/L showing Staphylococcus species. The isolates were examined for comparable results throughout the dilution range. However, their susceptibilities by Bauer Kirby Disc Diffusion test slightly higher values were obtained for Amoxicillin + against ten antibiotics. Results showed that incidence of Erythromycin and Amoxicillin + Clavulanate i.e. > 1.0 µg/L. resistance to the antibiotics was quite high, as the maximum susceptibility obtained was only about 13.19%, Rifampicin Keywords: Antibiotic efficacy, Staphylococcus, MIC values.
and Tetracycline being the most ineffective in vitro. Amoxicillin and Cloxacillin were the most effective in phase I exhibiting 12.13% and 11.24% efficacy, respectively, while Ankita Shrestha
IMB-III year, Industrial Microbiology (Hons.),
Introduction :
Session:2008-2011, Patna Women’s College,Patna University, Patna, Bihar, India.
Milk is an excellent culture medium for different kinds of microorganisms, being high in moisture, Priyaragini
IMB-III year, Industrial Microbiology (Hons.),
nearly neutral in pH, and rich in nutrients like lactose, Session:2008-2011, Patna Women’s College, citrate, butter fat and proteins. Staphylococcus Patna University, Patna, Bihar, India.
species are known to be the common Satyamvada Swayamprabha
contaminants of milk which cause diseases like Assistant Professor, Dept. of Industrial Microbiology,Patna Women’s College, Bailey Road, Patna – 800 001, Mastitis or the inflammation of udder in cattle.
Ankita Shrestha et al. / IRIS – Journal for Young Scientists, 2011, pp. 58-65 chemotherapeutic agents of microbial origin, are Minimum inhibitory concentration is the lowest concentration that is able to inhibit growth of the Staphylococcus infections including mastitis bacteria. MIC’s are used by diagnostic laboratories, mainly to confirm resistance and as a research tool to determine the in-vitro activity of antibiotics. It is microbiological contamination and the degree of also used to decide the appropriate antibiotic handling can explain the fact that dairy products concentrations which can be administered at safe are frequently implicated in food-borne diseases.
clinical or subclinical levels (Andrews, 2006).
Among pathogenic bacteria, S.aureus is one of the Materials and Methods :
most abundant microorganisms isolated from raw The study was conducted in the Industrial milk, and also the microorganism most commonly Microbiology laboratory, Patna Women’s College isolated from bovine mastitis (Devriese et al. 1997).
and Indian Institute of Bioinformatics and The presence of S.aureus in asymptomatic food handlers is well documented and has been Sampling
attributed to contamination of dairy products.
Irregularities in storage time and temperature, and aseptically in glass vials from five different areas failures in the hygienic procedures during the of Patna, namely, Boring Road, Patliputra, production of dairy products, are factors that may Gardanibagh, Patel Nagar and Kankarbagh. The lead to high contamination with S.aureus.
Therefore, even industrialized dairy products can serial decimal dilutions of these milk samples were be sources of food intoxication, since the staphylococcal enterotoxin is not inactivated by supplemented with 0.05% (w/v) Tween-80 and pasteurization (Peterson and Shanholtzer, 1992).
0.1% (w/v) MgCl .6H O. These dilutions were prepared in duplicates and then transferred to Antibiotics are used to treat diseases of cattle Mannitol salt agar (Dubey and Maheshwari, 2006).
and as preservatives for milk. The uncontrolled use All plates were incubated at 37ºC for 48 hours.
of antibiotics has led to the development of multiple antibiotic resistance, there by rendering the Biochemical Characterization and
treatment ineffective. Thus, there is an urgent need Identification
to study and uncover the recent trends in resistance Typical colonies grown on MSA plates were identified as Staphylococcus species using the Consumption of milk having significant amount following tests: Gram staining, IMViC tests, of antibiotic without proper antibiotic flush, results Carbohydrate (dextrose, sucrose and lactose) in accumulation of these antibiotics in humans.
fermentation, Gelatinase production, Amylase Moreover, several resistant Staphylococcus may production and Catalase production. The isolates also enter the body and can cause infections like were also tested for production of Coagulase by impetigo, cellulitis, food poisoning, scalded skin the Slide Coagulase test and furthur grouped as α, syndrome, toxic shock etc. MIC can reveal the β and γ hemolytic by performing Hemolysis on lowest concentrations, which leads to a reduction in the antibiotic load passing on from the milk to Antibiotic susceptibility and determination of Minimum Inhibitory Concentration (MIC) of potent antibiotics used againstStaphylococcus spp. isolated from raw milk Antibiotic susceptibility test (AST) by Bauer
Preparation and loading of antibiotic discs
Kirby Disc Diffusion Technique
Discs of equal size and uniform shape were punched out of a Whatman filter paper No. 1 and antimicrobial disc diffusion susceptibility testing of sterilized. The discs were applied by means of a common, rapidly growing bacteria by the Bauer- sterile forceps, strictly under aseptic conditions.
Kirby method (Barry et al. 1992) as standardized The discs were deposited onto the plates so that by the National Committee for Clinical Laboratory the centers were at least 24 mm apart. Placing Standards. The tested antibiotics were: Amoxicillin, discs adjacent to one another was avoided. After Erythromycin, Rifampicin, Tetracycline and discs have been placed on the agar, they were Cloxacillin. Further, following combinations of tapped gently with the sterile forceps to make antibiotics were also tested: Amoxicillin +
complete contact with the medium surface. A Clavulanate, Amoxicillin + Erythromycin, Rifampicin control plate without any antibiotic was also prepared for each isolate. All steps were performed + Erythromycin, Tetracyclin + Cloxacillin and in duplicates (Isenberg, 1988). Within 15 minutes Cloxacillin + Ampicillin (Andrews 2006). Stock after the discs were applied; the plates were solutions of the antibiotics were prepared in the inverted and incubated at 35°C. Plates were recommended solvents and three dilutions i.e. 50, examined after 24 hours of incubation.
100 and 150 µg/ml were prepared from the stocks.
Inhibition Zone measurement
The disc diffusion test was done for each isolate on Mueller-Hinton agar. For this, 25 ml of The measuring scale was held on the back of the inverted plate over a black, non-reflecting medium was poured into sterile Petri dishes to a background, and the more obvious margin was depth of 4 mm on a level surface to make the depth measured to determine the zone diameter. Growth of the medium uniform and left at 37ºC temperature within the apparent zone of inhibition was indicative overnight to check sterility (Barry et al. 1992). For inoculum preparation 5 ml Tryptic Soy broth was dispensed in 15 ml culture tubes and sterilized by Determination of Minimum Inhibitory
autoclaving at 121°C for 15 minutes.
Concentration (MIC) of potent antibiotics
Minimum inhibitory concentration (MIC) is The tubes were cooled and kept in an incubator defined as the lowest concentration of antimicrobial for 24 hours at 35°C to check sterility . Then, each that will inhibit the visible growth of a micro- isolate was inoculated in the sterilized tubes organism after overnight incubation, and minimum containing the medium, and placed in an incubator bactericidal concentration (MBC) is the lowest overnight at 35°C. An antibiotic free control was also concentration of antimicrobial that will prevent the growth of an organism after sub-culture on to Inoculation of plates
antibiotic free media (Andrews, 2006). MICs areused by diagnostic laboratories, mainly to confirm 100µL of each isolate suspension was pipetted resistance, but most often as a research tool to out through a micropipette and placed on the plates.
determine the in-vitro activity of new antimicrobials, Then, the inoculum was spread evenly over the and data from such studies have been used to surface of the medium using a glass spreader.
determine MIC breakpoints (James, 1990).
Ankita Shrestha et al. / IRIS – Journal for Young Scientists, 2011, pp. 58-65 Based on the results obtained from the Bauer Kirby antibiotic susceptibility test, the following antibiotics were found to be the most effective and hence selected for determining the MIC values: Erythromycin, Amoxicillin + Clavulanate and The following methods were used to determine From the stock 100 mg/L the following amounts Method I - Agar Dilution Method
Stock preparation:
The standard stock solutions were prepared Staphylococcus spp. To prepare a stock of No antibiotic was added to the vial labeled 0 concentration of 50 mg/ml, 500 mg powder from mg/L (antibiotic free growth control).
the capsule was directly weighed and dissolved in Preparation of inoculum:
10 ml of solvent. For preparing stock solution of antibiotics in combination 250 mg powder of each 5 ml tryptic soy broth was dispensed in 15 ml antibiotic was weighed and mixed in 10 ml of the culture tubes and sterilized by autoclaving at 121°C solvent. Further, a stock of concentration 10,000 for 15 minutes (Aneja 2004). The tubes were cooled mg/L was obtained by adding 2 ml of 50 mg/ml and kept in an incubator for 24 hours at 35°C to solution to 8 ml of distilled water. Further stock check sterility. Then, a loopful of each isolate was solutions, from the initial 10,000 mg/L solution, were inoculated in the sterilized tubes containing the prepared as follows :
medium, and placed in an incubator overnight at 1000 µL of 10,000 mg/L solution + 9 mL diluent = 1000 mg/L 100 µL of 10,000 mg/L solution + 9.9 mL diluent = 100 mg/L Inoculation and incubation:
Preparation of Antibiotic dilution range:
100µl of each isolate suspension was pipetted out through a micropipette and added onto the plates. Then, the inoculum was spread evenly over according to the target range. The target range of the surface of the medium using a glass spreader.
MIC was between 0.25 – 128 mg/L (Andrews, All plates were incubated under appropriate 2006). Thus, this dilution range was selected and incubation conditions i.e. at 37ºC for 16 – 18 hours.
prepared as follows: 11 sterilized glass vials were Thereafter, the plates were observed for growth.
labeled as follows: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, Method II – Broth Dilution Method
(A) Macrodilution
From the 10,000 mg/L stock, the following Preparation of antibiotic stocks and
amounts were dispensed with a micropipette: dilution:
Antibiotic ranges were prepared which were one step higher than the final dilution range required, Antibiotic susceptibility and determination of Minimum Inhibitory Concentration (MIC) of potent antibiotics used againstStaphylococcus spp. isolated from raw milk i.e. for a final dilution range of 0.25, 0.5, 1, 2, 4, 8, to solidify. After the agar has solidified wells were 16, 32, 64 and 128 mg/L, a range of 0.5, 1, 2, 4, 8, bore into the plates using a well borer.12 wells were 16, 32, 64, 128 and 256 mg/L were prepared (to prepared in each row. A total of 96 wells were compensate for the addition of an equal volume of prepared, 48 on each plate (for each antibiotic in inoculum). Sufficient 75 x 12 mm sterile capped duplicate).75 µ l of each antibiotic dilution was addedto two rows of wells, except for two wells in each tubes were arranged in two rows for each antibiotic, row.75 µ l of test organism was dispensed into one to cover the range of antibiotic dilutions chosen in row and 75 µ l of control into the second row of duplicates. The tubes were labeled with each dilution and 5 ml of Tryptic Soy broth was poured in uninoculated wells of antibiotic-free broth (the first each tube. 0.25 ml volume of each antibiotic dilution controls the adequacy of the broth to support the was added to the broth in tubes (Aneja 2004).
growth of the organism, the second is for check of Preparation of inoculum:
sterility).The plates were incubated at 37ºC for 18 Normal saline was prepared by dissolving 0.875 g NaCl in 100 ml distilled water and A 96 well sterile microtitre tray was labeled from autoclaved. 5 ml of saline was poured into 10 sterile 1 to 12 which represent the appropriate antibiotic glass vials. A loopful of each isolate was taken and dilutions and two controls. The contents of eachwell from the plates were pipetted out and placed added to the saline in each vial and vortexed to into the wells of the tray. Mixing of the contents was performed in the wells by gently flushing the Inoculation of tubes:
inoculum in and out of the pipetted tip four to fivetimes, avoiding splashing or creation of bubbles.
1 ml aliquot of test organism was added to The tray was covered with a sealing tape and gently each set of tubes and the contents of the tubes agitated. The endpoint reading was performed were mixed thoroughly. Two antibiotic free control tubes were also prepared as given in Table 1.
(i) Reference MIC endpoint reading (V)
Table 1: Indication and purpose of the control tubes
The broth microdilution wells that had not been Control type Inoculation with
agitated were read visually (V) with the aid of a reading mirror; the growth in each well was compared with that in the growth control (drug-free) well. A numerical score, which ranged from 0 to 4, was assigned to each well according to the scale recommended by National Committee for Clinical All tubes (including control) were incubated at Laboratory Standards (NCCLS) as shown in Table 35 - 37oC for 18 – 20 hr in air. Spectrophotometric reading of each sample was performed with a Table 2: Numerical scores as per NCCLS guidelines
spectrophotometer (Thermo) set at 492nm.
Numerical Indication
(B) Microdilution
suspensions were prepared as above. Tryptic Soy agar was prepared (Dubey and Maheswari, 2006).
It was poured into 150 mm petriplates and allowed Ankita Shrestha et al. / IRIS – Journal for Young Scientists, 2011, pp. 58-65 (ii) Visual MIC following agitation (VS)
Fig 1 and 2 show that incidence of resistance In order to assess alternative methods for the to the antibiotics was quite high as the maximum determination of MIC endpoints, the MIC for each susceptibility obtained was only about 14.31%, drug-organism pair was read visually following Rifampicin and tetracycline being the most ineffec- agitation of the microdilution trays. Agitation was tive in-vitro. Amoxicillin and Cloxacillin were the most accomplished by first sealing the tops of the trays effective in phase I exhibiting 12.13% and 11.24% with clear tape and then shaking gently until a efficacy respectively, while Ampicillin + Cloxacillin homogeneous suspension was obtained in each was the most effective combination in phase II ex- well. The MIC endpoints for the agitated trays (VS) hibiting 14.31% efficacy. Fig 3 shows the inhibition were defined exactly as described above for the Results and Discussion :
A total of 12 isolates were obtained from 10 raw milk samples. Out of the 12 isolates obtained,nine were identified as Staphylococcus species.
Out of nine, two isolates were coagulase positiveand 7 coagulase negative. Also, five were hemolyticand four non hemolytic. The isolates were examined Fig 3: Results of AST: Susceptibility to Amoxicillin
for their susceptibilities by Bauer Kirby Disc (left) and resistance to Rifampicin (right)
Diffusion test. The susceptibility patterns exhibitedby the isolates and the efficacy of the antibiotics Method I - Agar Dilution Method
antibiotics were recorded between 0.5-1.0 mg/L.
The MIC values were found to be slightly greater than those which have been evaluated in the Method II – Broth Dilution Method
For Broth Macrodilution the following optical Fig 1: Percentage efficacy of antibiotics in AST phase I
Fig 4: Optical density for Amoxicillin + Cloxacillin
Fig 2: Percentage efficacy of antibiotics in AST
by MIC Broth macrodilution method
Antibiotic susceptibility and determination of Minimum Inhibitory Concentration (MIC) of potent antibiotics used againstStaphylococcus spp. isolated from raw milk In the Broth dilution method, the optical density through the acquisition of mobile drug resistance showed a steep decrease even after a single doubling of dilution from 0.5 to 1.0 mg/L. This has Results obtained from the Bauer Kirby Disc been illustrated taking the example of the Diffusion test or the AST showed that Rifampicin combination Amoxicillin + Cloxacillin which yielded and Tetracycline were most ineffective while Amoxicillin, Cloxacillin and a combination of For broth microdilution the end point readings Ampicillin + Cloxacillin was most effective with were recorded and the numerical scores were 12.13%, 11.24% and 14.31% efficacy, respectively.
assigned according to the NCCLS guidelines (Table Results of MIC determination and a statistical and 2). Results obtained from both V and VS methods comparative analysis of the data obtained showed were in accordance with those of NCCLS 2000 as that Amoxicillin in pure form and even when present in combination with Erythromycin and Clavulanate may be the most effective antibiotics against susceptibility patterns. However, changes for certain combinations could not be traced and hence, further evaluations are needed before clinical trials. The methods used were practical Fig 5: Comparative results of MIC for Amoxicillin +
Cloxacillin: V (MIC) and VS (MIC after agetation)
for a clinical laboratory that chooses to perform endpoint readings
bactericidal activity testing assuring a high level of reproducibility between duplicate assays.
Conclusion :
Acknowledgement :
The present study pointed out that the milk We are grateful to Dr. Sister Doris D'Souza samples were contaminated with Staphylococcus A.C., Principal, Patna Women's College (PWC) spp. probably due to the ineffectiveness of and the Research Committee for providing facilities antibiotics used for clinical purposes. It can be said and financial support. We thank Prof. S. Bedi, Head, that using antibiotics to declare “all-out war” against Department of Industrial Microbiology, PWC, for bacteria like Staphylococcus spp. is a war that we taking keen interest in our research work.
cannot win. Moreover, the indiscriminate use of References :
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Staphylococcus aureus from bovine mastitis Antimicrob. Chemother. 25:209–216.
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