Plasmid Pattern of Antibiotic Resistance in Beta-Lactamase Producing Staphylococcus aureus Strains Isolated from Hospitals in Kerman, Iran

M.R. Shakibaie PhD,* S. Mansouri PhD,** S. Hakak MSc***

*Department of Genetics, Center for Research, ** Department of Microbiology, Kerman University of Medical Sciences, Kerman, Iran, *** Department of Microbiology, Azad University of Qum, Qum, Iran

Introduction

The genus S. aureus encompasses gram-positive, catalase-positive, non-motile cocci that cause a variety of human infections in all age groups.¹ It is the major causative agent in surgical wound infection and epidemic skin disease in newborn infants.² S. aureus infection may also be superimposed on superficial dermatologic diseases such as eczema, pediculosis and mycosis fungoides.³ Several investigations have been conducted to study the antimicrobial resistance pattern of S. aureus and it has been shown that the organism is resistant to ß-lactam antibiotics, aminoglycosides and macrolides.^4,5 The antibiotic resistance of S. aureus has been debated in recent years: for example, MIC to penicillins encountered in more than one million strains tested in various hospitals in the USA, indicate that resistance has increased from 17% to more than 90%.⁶ Vancomycin is the drug of choice against S. aureus when ß-lactams are inappropriate. However, because of poor tissue diffusion and moderate bactericidal activity, vancomycin is often combined with rifampicin for deep-seated infections.⁷ Once antibiotic therapy is instituted, the sensitive strains will be inhibited but the resistant strains (mutated or plasmid bearing) will survive and cause chronic infections.⁸ Plasmid-mediated antibiotic resistance in S. aureus has been reported by various authors.^8-10 Little information on this issue is available in Iran, hence this study was instigated to investigate¹¹ the antibiotic resistance and plasmid content of ß-lactamase-producing hospital isolates of S. aureus.

Materials and Methods

Antibiotics and media:

Antibiotic discs including, penicillin G (PG), ampicillin (Ap), amoxycillin (Am), cloxacillin (Clox), cephalexin (CT), ceftizoxime (CAZ), cefotaxime (CTX), cefazolin (Cf), kanamycin (Km), gentamicin (Gm), amikacin (Ak), chloramphenicol (Cm), tetracycline (Te), vancomycin (V), rifampin (Rif), and nalidixic acid (Nal) were purchased from Padtan Teb (Iran). Antibiotic powders were obtained from Hakim and Chimidarou Companies (Iran). Tris-hydrochloride (Tris-Hcl), agarose, glucose, chloroform, phenol, sodiumdodecylsulfate (SDS) and boric acid were from E-Merk Co.(Germany). Media like Müller-Hinton agar and/or broth, and nutrient broth were form Hi-medias, and ethidium bromide was received from Sigma Co. (USA).

Bacterial strains:

Forty strains of S. aureus were isolated from Kerman-Darman, Baahonar, Shafa and Kashanie hospitals in the city of Kerman, Iran. The identity of the strains was confirmed by various biochemical tests as described previously.³

Antibiotic resistance testing:

The sensitivity of the isolates to antibiotics were determined by disc diffusion break-point assay⁵, and minimum inhibitory concentration (MIC) for all the strains were measured by micro-dilution test in 2 ml Müller-Hinton broth using the two-fold dilution method.⁵ The MIC was defined as the lowest concentration of the antibiotic that would inhibit the growth (visible turbidity) after 18-24 hours. The bacterial inoculum was 10⁵-10⁶ cells/ml. The results were compared with S. aureus ATCC2913 (methicillin susceptible) supplied from the Reference Laboratory, Kerman, Iran.

ß-lactamase test:

Existence of ß-lactamase enzyme in each isolate was determined by iodometric method.¹²

Plasmid isolation and agarose gel electrophoresis:

Plasmid extraction was carried out by alkaline lysis technique.¹³ Agarose gel 0.7% (wt/vol) was prepared and 60 m l of DNA preparation were loaded into each well. Electrophoresis was conducted for 4h. at 60V, and the gel was stained by 0.5 m g/ml ethidium bromide. A plasmid DNA band was observed with UV transilluminator (Upland, model CA9178 USA) and photographed.

Curing of ß-lactamase production:

The ß-lactamase producing cultures of S. aureus were grown overnight in 5 ml L-B containing 0.25-16 m g/ml ´ MIC ethidium bromide and acridines. Subsequently, the cultures were diluted and spread on N-agar plate to give 100 colonies/plate. ß-lactamase deficient organisms were then detected by penicillin-iodine agar overlay method.¹³

Results

The antibiotic resistance of the 40 strains of S. aureus isolated from patients hospitalized in Kerman is shown in Table 1 and Fig. 1. On the basis of multimodal distribution, the strains were categorized into sensitive (MIC 1 m g/ml), moderately sensitive (MIC 4 m g/ml) and resistant (MIC 16 m g/ml), according to the indicated breakpoint.

The isolates were highly resistant to penicillins (average MIC 128 m g/ml). MIC to rifampin and amikac in 95% of the isolates was comparatively low (1 m g/ml). Fifty-five percent of the isolates showed a MIC of less than 16 m g/ml to cefazoline and cephalexin (Table 1), while 75% exhibited varied sensitivity to chloramphenicol and erythromycin. However, 57% were highly resistant to the third generation cephalosporins like cefotaxime and ceftizoxime (MIC > 32 m g/ml). Similarly, 22% of the isolates showed a MIC of greater than 16 m g/ml to gentamicin and tetracycline (Table 1).

The ß-lactamase production test was positive for 57% of the strains of S. aureus, especially those isolated from Kerman-Darman Hospital, while none of the 10 isolates of Kashani hospital produced the enzyme. Furthermore, the ß-lactamase-cured cells did not exhibit any enzyme activity.

Plasmid isolation and agarose gel electrophoresis of some isolated strains are shown in Fig. 2., which indicates that strains 5, 12, 14, 22, 25, 27, 35 and 37 were encoded by plasmid. These strains were also ß-lactamase producers.

Discussion

From the above data it can be generally concluded that S. aureus isolates are relatively resistant to commonly-used antibiotics. This is mainly because of the extensive application of antibiotics and the selective environment in hospitals.

The resistance to the third generation cephalosporins is an important feature in this investigation and was observed among the ß-lactamase producing isolates. Similarly, plasmid isolation revealed the existence of at least one plasmid band in these strains which were similar to each other. This indicates that the resistance pattern may have originated from one source (organisms) and spread to other hospitals by the hands of the medical staff.

The prevalence of penicillin and cephalosporin- resistant clinical isolates of S. aureus has increased rapidly and most of the isolates are ß-lactamase producers.¹⁴ The ß-lactamase elaborated by S. aureus hydrolyses a wide variety of ß-lactam compounds including aminopenicillins, carboxypenicillins, ureidopenicillins and cephalosporins.¹⁴ In most strains, the enzyme is encoded by plasmid but can also be found on the chromosome.¹⁰

Worldwide, many strains of S. aureus are already resistant to all antibiotics except vancomycin¹⁴ and thus the organism has progressed one step closer to becoming an unstoppable killer.

Although many factors may be operative as to whether the bacteria acquired in the hospital setting or community will become insensitive to an antibiotic, the two main determinants appear to be the prevalence of the resistant genes and the extent of the antibiotic use.⁵

In Iran, because of a high rate in self-medication of antibiotics, the antibiotic resistance to S. aureus is now emerging in hospitals. As little information is available regarding the source and spread of antibiotic resistant genes among the strains, it is hoped that this study will pave the way to achieve this goal.

Acknowledgements

This study was supported by the Research Council of the Kerman University of Medical Sciences, Kerman, Iran. Our sincere thanks are due to the staff of the Center for Research for their help during this investigation.

References

1 Boyce JM. Nosocomial staphylococcal infections. Ann Intern Med 1981;95:241-5.

2 Baldwin JN, Rheins MS, Sylvester RF. Staphylococcal infections in newborn infants. Ann J Dis Chid 1990;94:107-12.

3 Kloos WE, Bannerman TL. Staphylococcus and micrococcus. In: Murray PR, Baron EJ, Faller MA, et al. (ed.). Manual of Clinical Microbiology. 6th ed. ASM: 1995:282-98.

4 Maple PA, Hamilton-Miller J, Barunfitt W. World wide antibiotic resistance in methicillin resistant Staphylococcus aureus. Lancet 1989;ii:537-40.

5 Atkinson BA, Lorian V. Antimicrobial agent susceptibility patterns of bacteria in hospital from 1971-82. Clin Microbiol 1984;20:791-5.

6 Barret F, Casy JI, Wicox C. Bacteriophage types and antibiotic susceptibility of Staphylococcus aureus in Boston city hospitals. Arch Intern Med 1970;125:867-70.

7 Damon AH, Soussey CJ, Curvalin P. Characterization of mutation in the rpoB gene that confer in rifampin resistance in Staphylococcus aureus. Antimicrob Agent Chemother 1998;42(10):2950-4.

8 Archer G, Coughter JP, Johnston JL. Plasmid encoded trimethoprime resistance in Staphylococcus aureus. Antimicrob Agent Chemother 1986;29:733-40.

9 Lyon BR, Skurray R. Antibiotic resistance of Staphylococcus aureus. Genetic bais. Microbiol Rev 1987;51:88-92.

10 Lacey RW. Antibiotic resistance plasmid of Staphylococcus aureus, their clinical importance. Bacteriol Rev 1975;39:1-45.

11 Catlin BW. Iodometric detection of Hemophilus influenzae ß-lactamase. Rapid presumptive test for ampicillin and cephalosporin resistance. Antimicrob Agen Chemother 1975;7:265-70.

12 Birnboim HC. A rapid alkaline extraction method for the isolation of plasmid DNA. Method Enzymol 1983;100: 243-55.

13 Bonfiglio G, Livermore DM. ß-lactamase types amongst Staphylococcus aureus isolates in relation to susceptibility to ß-lactam inhibitor combinations. Antimicrob Chemother 1994;33:465-81.

14 Mansouri S, Khaleghi M. Antibacterial resistance pattern and frequency of methicillin resistant Staphylococcus aureus isolated from different sources in southeastern Iran. Irn J Med Sc 1997;22(3&4):89-93.

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