Microorganisms have developed unlimited resistance to antibiotics since their introduction into clinical practice. The emergence of antibiotic-resistant strains among these organisms has resulted in a major public health concern worldwide (Mosland et al., 2006).
Essentially there are two methods of Antimicrobial Susceptibility Testing (AST): Agar diffusion methods and antimicrobial dilution tests. Quantitative methods include agar dilution and broth dilution and the commercially available E-test Minimum Inhibitory Concentration (MIC) strips. The MIC is the minimum inhibitory concentration of antibiotic that inhibits growth of the organism (King and Brown , 2001). Qualitative methods include disk diffusion test which is a qualitative method since the organism is classified as susceptible (S), intermediate (I), or resistant (R) to the antibiotic (NCCLS, 1997). Several antimicrobials, such as sultrim, oxytetracycline, enrofloxacin and florfenicol are manufactured by different Iranian companies.
Since some of these products are exported to regional countries, to have an idea about the quality and performance of these products, the current study was undertaken to evaluate the susceptibilities of E. coli K12 to 5 reference antimicrobial agents as control, using MIC method and their products produced by Iranian manufacturers using the disk diffusion method (NCCLS, 1997).
MATERIALS AND METHODS
The study was conducted through October 2006 to March 2007 in Microbiology Lab. of the Faculty of Veterinary Medicine of Shahrekord University in Shahrekord, Iran.
The minimum inhibitory concentration was selected as the reference method for this study (Saiman et al., 1999).
Sultrim, oxytetracycline, enrofloxacin and florfenicol from different manufacturers were the agents assayed. (Table 1).
The following drugs were obtained from ICN biochemical's (sulfadiazine + trimetoprime = sultrim, oxytetracycline), glaxo (enrofloxacin) and padtanteb (florfenicol). The MICs of these drugs were assayed and used as control for their relative products. MICs were determined by a two- fold serial dilution method using Mueller-Hinton broth according to the National Committee for Clinical Laboratory Standards recommendations (NCCLS, 1997). One milliliter of 200 μg mL-1 concentration of each drug was added to first serial tubes of tested antimicrobials. The MIC endpoint was the lowest concentration of an antimicrobial that completely visibly inhibited the growth of E. coli K12 in a tube.
For disk diffusion, concentrations were in accordance with the NCCLS (1997)
recommendations. The ranges of concentrations tested by the disks for each of
the tested drugs were 23.75, 30, 5 and 30 μg for Sultrim, oxytetracycline,
enrofloxacin and florfenicol, respectively.
The same concentrations were used for relative products of mentioned antimicrobials produced locally.
Testing strain was E. coli RTCC 2310 (K12) from Razi institute incubated over night in TSB (Tryptonic Soy Broth), at 37 degree centigrade and then adapted with 0.5 McFarland turbidity, adjusted to final concentration of 1.5x108 colony-forming units mL-1 according to Carter and Chengappa (1991) method.
The plates were then incubated for 20 h at 37°C after which interpretation was performed using a ruler for measuring inhibition diameters according to the criteria recommended by NCCLS (2002a).
RESULTS AND DISCUSSION
MIC of the E. coli K12 to tested antimicrobials for Sultrim, Oxytetracycline, Enrofloxacin and Florfenicol were 1.5625, 0.7812, 6.25 and 3.125 μg mL-1 respectively. Based on these MICs E. coli K12 was resistant to Enrofloxacin but sensitive to others. Zone diameter of reference Enrofloxacin (21 mm) and its products (19- 21 mm) are very near and are near optimum. For sultrim, oxytetracycline and florfenicol zone diameter of reference drugs were 18, 25 and 24 mm respectively, while for their products ranges between 17-20, 22-26 and 22-24 mm, respectively. For Sultrim products these results seems to be the optimum but for others are linear across the range. The results for disk diffusion of these drugs and their products are summarized in Table 1, as it appeared the results for DD of the above antimicrobials and their local products are just as the results of MICs of reference drugs.
The aim of this study was to compare the reference group of antimicrobials and their locally produced products against E. coli K12 a representative of gram negative bacteria to have an idea about the quality and performance of these products.
Animal species-bacterial pathogen breakpoints and zone interpretive criteria for in vitro susceptibility testing were as outlined in the NCCLS (2001).
Our data indicate that E. coli K12 is resistant to enrofloxacin (MIC value of 6.25 μg mL-1) by both MIC and DD evaluation. No remarkable difference between reference and local products of this drug were observed. It therefore seems likely that the reduced susceptibility to fluoroquinolones of this bacteria is sufficient to allow it to survive antibiotic challenge in chickens or animal infections.
Based on MIC of reference florfenicol (3.125 μg mL-1) and DD
inhibition zone diameter (NCCLS, 2002b) of related products (22-24 mm), Table
1, tested E. coli was sensitive to this synthetic antibiotic and
florfenicol commercial products seem to have acceptable anti E. coli effect.
However there are no approved NCCLS breakpoints for MIC of E. coli currently
available, the resistance breakpoint of Florfenicol for other gram negative
bacteria (i.e., Pasteurella multocida) is suggested from 8 μg mL-1
(David et al., 2000) to 0.12-4 μg mL-1 (Sung et al.,
2005). In an earlier study, Rajaian et al. (2003) examined the concentration
of active ingredient of several common veterinary antimicrobial products by
disk diffusion method using Staphylococcus aureus as tested bacteria.
They reported high quality of oxytetracycline products from Iranian manufacturer
and 10 to 100% impurities in other antimicrobial products. In this study the
data for Oxytetracycline products are in across the range and so in agree with
that study but for other products are not in agree. There are some differences
between susceptibilities of E. coli and S. aureus to different
antimicrobials and this can be one explanation for this difference.
|| Tested antimicrobial agents by disk diffusion method (NCCLS,
2002), their purity and relative sources
|S = Sensitive, NS = Non Sensitive
In our study E. coli K12 was used as a representative of gram negative
bacteria, however all gram negative bacteria have not the same antimicrobial
susceptibility patterns, more over this pattern for gram positive bacteria may
be completely different. So a complete study on some gram positives and some
other gram negative bacteria is essential to achieve a satisfying idea about
these products. But as a pilot study we can conclude that, all products of tested
antimicrobials seem to be in synergy with their reference MIC evaluation against
E. coli K12 and can be supposed to be reliable.