Investigation of the Genetic Basis of Tetracycline Resistance in Staphylococcus aureus from Pakistan

Purpose: To determine the prevalence and genetic basis of tetracycline resistance in Staphylococcus aureus. Methods: One hundred and thirty (130) clinical isolates of S. aureus were collected from Khyber Teaching Hospital, Peshawar, Pakistan. Susceptibility to antibiotics (doxycycline, tetracycline and minocycline) was determined by Kirby-Bauer disc diffusion method with minimum inhibitory concentration (MIC) evaluated on Muller-Hinton agar as described by the Clinical and Laboratory Standards Institute (CLSI). The tetracycline-resistant strains (TET-R) were screened by polymerase chain reaction (PCR) for the presence of four common tetracycline resistance determinants, viz, tet(K), tet(L), tet(M) and tet(O). Results: Sixty (46.0 %) of these isolates were methicillin-resistant S. aureus (MRSA) while 70 (54.0 %) were methicillin-susceptible S. aureus (MSSA). Seventy four (56.9 %) strains were resistant to tetracycline (TET-R), 30 (23.1 %) to minocycline and 23 (17.7 %) to doxycycline. A majority of the MRSA were resistant to tetracyclines and all the MSSA were sensitive to doxycycline and minocycline. The tet(K) gene was found in 58 isolates and tet(L) in one isolate. No tet(M) and tet(O) were detected. Conclusion: This study indicates that resistance to tetracyclines is mainly by efflux pumps mediated by tet(K) in S. aureus in northwestern Pakistan.


INTRODUCTION
Tetracyclines are broad spectrum antibiotics used against a wide variety of bacterial infections, including both Gram-positive and Gram-negative. After their discovery in the 1940's, tetracyclines have continuously been used in both humans and animals with no major adverse effects. Tetracyclines inhibit bacterial ribosome from associating with the aminoacyl-tRNA and as a result, protein synthesis is inhibited. The discovery of tetracyclines sparked the development of many chemically altered antibiotics: including semisynthetic drugs such as minocycline and doxycycline [1].
A large number of tetracycline resistance genes have been identified. There are 38 acquired tetracycline resistance genes that are known and all use one of three strategies to render the bacteria resistant [2]. These include (1) efflux proteins, (2) ribosomal protection proteins and (3) enzymatic inactivation of tetracycline.
The majority of these genes (60 %) code for energy-dependent efflux pumps, and different bacterial genera tend to have the same efflux or ribosomal protection genes [3]. This indicates that tetracycline resistance genes can be transferred amongst the bacterial population. In fact, resistance to tetracycline in most bacteria is due to the acquisition of new genes; these genes tend to be associated with mobile elements such as transposons and plasmids [1]. Acne patients often require sequential treatment with tetracycline with several courses, which can last from three months to several years [4]. This long-term antibiotic treatment exerts a long selective pressure on both the targeted propionibacteria and the other microflora of the skin.
Tetacycline resistance genes: tetK, tetM, tetO and tetL are four major genes associated with tetracycline resistance amongst Grampositive bacteria. The tetK and tetL genes code for efflux proteins; these are energy-dependent membrane-associated proteins which prevent tetracycline from accumulating within the cell [3]. The other two genes, tetM and tetO, code for ribosomal protection proteins, which reduce the affinity of tetracycline to the ribosome [5].
Surveillance for detection of various tet determinants has been carried out worldwide. Tetracyclines are extensively used in Pakistan for skin and throat infections. But little surveillance work has been done to assess the extent of resistance. The aim of this study was to determine the genetic basis of tetracycline resistance in S. aureus. This is the first report disclosing genetic basis of tetracycline resistance in S. aureus from Pakistan.

EXPERIMENTAL Bacterial strains
Between April 2005 and May 2006, 130 nonduplicate, consecutive S. aureus isolates were collected from in-and out-patients at Khyber Teaching Hospital, a tertiary care hospital in Peshawar. In this study, 130 S. aureus were obtained from various sources. There were 69 male and 61 female patients. A majority of the isolates were obtained from wound and burn/skin infections. Identification was carried out by Gram stain, catalase, coagulase and DNAse tests.

Antimicrobial agents susceptibility testing
Susceptibility to antimicrobial agents was determined by Kirby-Bauer disc diffusion and minimum inhibitory concentration (MIC) method on Muller-Hinton agar (Oxoid, England) as per Clinical and Laboratory Standards Institute (CLSI) guidelines [8]. The tetracycline antibiotics used against the collected strains were: doxycycline (DOX), tetracycline (TET), and minocycline (MN). E. coli NCTC 10418 was used as control for susceptibility testing.

Bacterial DNA extraction
Achromopeptidase method was used for DNA extraction. In this method, 50 ul of NaCl-EDTA Tris (NET) buffer (1x TE buffer in PCR water plus 20ul 5M NaCl) was added to 10 ml achromopeptidase solution in a small tube. Thereafter, 2 -3 fresh colonies (18 -20 h) of S. aureus were added and incubated for 15 -20 min at 50 o C. After incubation, the extracted DNA was diluted 10 times in NET buffer for molecular work. The DNA was stored at -4 o C and used for up to one month.

Confirmation of MRSA
The phenotypically identified MRSA, using 30 µg cefoxitn disc, were genotypically confirmed by a duplex PCR targeting mecA gene responsible for methicillin resistance and a specific region of 16s rDNA region of the S. aureus (nuc gene). The primers used are stated in Table 1.

Determination of tetracycline resistance genes using PCR
The presence of tetK, tetM, tetL and tetO genes responsible for tetracycline resistance was detected by PCR. The primer sequence, amplicon size and reference for the detection of each gene are given in Table 1.

Agarose gel electrophoresis
The amplified products were run on 1.5 % agarose gel in Tris-Acetate EDTA (TAE) buffer for 45 min. The ethidium bromide stained bands were examined under an ultraviolet transilluminator and photographed with a Kodak camera.

Statistical analysis
Statistical analysis was performed by Chisquare test (Minitab 15 software) and pvalues of ≤ 0.05 were considered significant.  (Table 2).
On screening the 74 isolates resistant to tetracycline for the presence of tetK, tetO, tetM and tetL genes, tetK gene was found in 58 isolates, made up of 34 among MRSA and 24 among MSSA. The electrophoretic gel photo for tetK is given in Figure 1. Only one tetL gene was found in MRSA. No tetM and tetO genes were found in these isolates.
All the tetK positive strains were highly resistant to tetracycline, MIC 50 being 64ml/ml. The MIC distribution curve is shown in Figure  2. Among tetK positive S. aureus, 40 (67.8 %) were susceptible to doxycycline and 19 (32.2 %) to minocycline.

DISCUSSION
This study was conducted to identify the molecular mechanisms of resistance against tetracycline by S. aureus in Pakistan. The results of tetracyclines susceptibility in this study are in agreement with those of others who observed 83 and > 50 % resistance to tetracycline, respectively. [14,15].  On screening the isolates for the presence of four commonly found tetracycline resistance genes, 58 (78 %) of the isolates were positive for tet(K). The tet(K) gene protects bacteria from tetracycline by a resistance mechanism known as tetracycline efflux. This mechanism prevents the accumulation of tetracycline within bacterial cells [1] by the synthesis of a cytoplasmic membrane protein which pumps tetracycline out of the cell at a quicker rate than it enters [3]. This may explain why tet(K) gene does not impart resistance to minocycline.
Twenty four of the isolates had raised levels of resistance to minocycline, which was reflected in the MIC 90 value of 64 ug/ml. The raised level of minocycline resistance may be due to other tetracycline resistance genes, besides tet(K) being present within the isolates that were not screened for.  [16]. The level of tet(K) genes present in the three studies provides evidence to suggest that the distribution of tet(K) genes in S. aureus are wide spread. In addition to this, the frequency at which tet(K) occurs signifies that in S. aureus, the main mechanism of resistance is through tetracycline efflux. This also suggests that when screening S. aureus for tetracycline resistance genes, tet(K) must always be screened for first.
No traces of tet (O) were found in the current study and these findings correlate with those of Bismuth et al. This suggests that this gene is rare in S. aureus, unlike tet(K). In addition, there is very little evidence to indicate the occurrence of tet(L) in Staphylococci species, as indicated by both Bismuth et al and Trzcinski et al. As a result, this study as qell as previous studies indicate that tet(K) is widely distributed, and therefore, is more likely to be found in S. aureus.

CONCLUSION
The findings of this study indicate the mechanism of resistance to tetracyclines in S. aureus isolates from Pakistan could be through the production of efflux pumps, encoded mostly by tet(K).