Abstract: Eighty five isolates of Salmonella spp. collected from different provinces of Iran during the years 2008 to 2010, were screened for the qnrA, qnrB and qnrS genes by PCR. PCR assays detected 22 of 85 (25.8%) Salmonella spp. isolates carried the qnrA gene, 1 (1.17%) of 85 isolates harbored the qnrB and 1 (1.17%) of 85 isolates contained the qnrS gene and 1 isolate carried all the three qnrA, qnrB and qnrS genes. Resistance to quinolones and fluoroquinolones by these genes has been confirmed. Antimicrobial susceptibility patterns of isolates were as follow: 49 (57.6%) isolates exhibited resistance to nalidixic acid and none to ciprofloxacin. As expected, MIC assay confirmed these results. Having detected the qnr genes and consequently resistance to quinolones and fluoroquinolones in Salmonella isolates indicated that qnr genes are disseminating among Salmonella serovars.
INTRODUCTION
Salmonella resistance to antimicrobial agents such as quinolones and fluoroquinolones has emerged worldwide in recent years (Shahina et al., 2011; Mansouri et al., 2011; Farivar et al., 2006). The molecular epidemiology of resistance plasmids indicates the importance and role of plasmids in the spread of antimicrobial drug resistance (Roy et al., 2002; Belaluddin and Shahjahan, 2003; Yekhan et al., 2000). The first plasmid-mediated with low-level quinolone resistance (PMQR), qnr (later termed qnrA), was described in a Klebsiella pneumoniae strain from the USA by Soto et al. (2003), Cheung et al. (2005), Jacoby et al. (2003), Jiang et al. (2008) and Wu et al. (2007). Recently the qnrA gene has been found in Enterobacteriaceae worldwide and it is more detected in Asian isolates. New qnr genes, the qnrB and the qnrS genes have been identified in klebsiella pneumonia strains isolated in USA and India as well as in Shigella flexneri isolated in Japan (Hopkins et al., 2007). Quinolone resistance mostly caused by chromosomal mutations. In recent years, plasmid-mediated quinolone resistance, qnrA, qnrB and qnrS, has been reported in several parts of the world and differing from each other 40% or more in nucleotide sequence (Vasilaki et al., 2008). This gene, encoding a pentapeptide repeat protein which protects DNA gyrase and topoisomerase IV from quinolone inhibition (Hopkins et al., 2005; Cheung et al., 2005; Park et al., 2006; Jacoby et al., 2006; Jiang et al., 2008). In Gram-negative bacteria, topoisomerase IV is a secondary target for quinolone agents (Hopkins et al., 2005; Kilmartin et al., 2007). The serious problem is that pentapeptide has been shown to block the action of ciprofloxacin, a member of the large and widely group of antimicrobial drug. The qnrB and qnrS genes appear to be more widespread; The qnrB gene having been found in Senegal, USA and Korea while the qnrS genes have been found in Enterobacteriacea in Germany, USA, Taiwan, Vietnam and France. Pentapeptide repeat family amino acid in the qnrB gene indicates 39.5% identity with the qnrA gene and 37.4% amino acid identity with the qnrS gene (Jacoby et al., 2006; Hopkins et al., 2007). The plasmid increased resistance to both nalidixic acid and ciprofloxacin had a widespread range and that are not belong to plasmid group IncC (Jacoby et al., 2003). The qnr genes corroborate resistance to nalidixic acid as well as reducing susceptibility to ciprofloxacin but the main level of quinolone resistance by the qnr genes is low (Crump et al., 2003). Some research detected that ESBL gene and integron have been often associated with qnr gene, sequencing was performed to identify qnr and association ESBL gene (Hopkins et al., 2007; Sharma et al., 2009). In this study, Molecular epidemiology of the qnrA, qnrB, qnrS genes and decreased susceptibility to quinolones and fluoroquinolones in eighty five isolates of Salmonella spp. were studied.
MATERIALS AND METHODS
Bacterial isolates: A total of 85 isolates of Salmonella spp. were collected from several hospitals in different provinces of Iran, during the years 2008 to 2010. Each isolate was obtain from stool, blood, Synovial fluid, abscess, urine and bone marrow specimen and each strain came from a unique patient in different hospital. Bacterial isolates were mainly recovered from stool. All isolates were identified by biochemical and serological methods as previously described (Ahmed et al., 2009).
Antimicrobial susceptibility test: Antimicrobial susceptibility of clinical isolates were studied by disk diffusion method. All isolates tested and inoculated by clinical and laboratory standard institute (CLSI) (CLSI, 2009; Sharma et al., 2009). Disks prepared by MAST company (Mast Co, Merseyside, UK) were used to determine the susceptibility of isolates to nalidixic acid 30 μg mL-1 and ciprofloxacin 30 μg mL-1.
Minimum Inhibitory Concentration (MIC) of selected antimicrobial agents were determined by broth microdilution method (Sensitite, West lake, OH, USA) and then were confirmed by E test (AB Biodisk, Piscataway NJ, USA). Klebsiella pneumoniae ATCC 700603 was used as quality control strain (Table 1).
PCR detection: Plasmid DNA was extracted according to the previous study (Sambrook et al., 1989). The qnrA, qnrB and qnrS genes were detected by PCR (Ependrof Master Cycler®, MA).
| Table 1: | The information of blaCMY-2 positive Salmonella sp. |
| CP: Ciprofloxacin (=.0.64 μg mL-1), +: Present, -: Absent, MIC: Minimum inhibitory concentration | |
| Fig. 1: | PCR products of qnrB and 50 bp DNA ladder |
The primer used for amplification of the qnr genes was described by Whichard et al. (2007). Programs used for these genes are as follow: The qnrA gene, 35 cycles of 1 at 94°C, 40 sec at 50°C, 40 at 72°C, 10 at 72°C, qnrB, 35 cycles of 1 at 94°C 30 at 40°C, 20 at 72°C, 10 at 72°C, The qnrS gene, 35 cycles of 1 at 94°C 30 at 40°C, 30 at 72°C, 10 at 72°C. The amplicon size of qnrA, qnrB, qnrS were 700, 120 and 280 bp, respectively (Fig. 1-3). K. Pneumoniae UAB1 Strain containing PMG252 plasmid (qnrA1) or PMG298 (qnrB1) and S. enterica serotype Bovis morbificans strain AM12888 (qnrS) were used as a positive control for presence of the qnr gene (Jacoby et al., 2003).
| Fig. 2: | PCR products of qnrA and 1 kb DNA ladder |
| Fig. 3: | PCR products of qnrS and 100 bp DNA ladder |
DNA sequencing: The qnrA, qnrB and qnrS genes were amplified and sequenced by using specific primers. Analysis was performed by using Bio Edit (www.Mbio.ncsu.edu/Bioedit/bioedit.html). DNA sequences were determined and used to screen the GenBank database using the BLAST algorithm available at the National Center of Biotechnology information web site (http://www.ncbi.nlm.nih.gov/BLAST; Veldman et al., 2008; Aschbacher et al., 2008).
RESULTS
Antimicrobial susceptibility test: Antimicrobial susceptibility patterns according to the disk diffusion method were as follow: 49 (57.6%) isolates exhibited resistance to nalidixic acid and none isolates to ciprofloxacin. Isolates indicated ciprofloxacin MIC range of 0.064-0.125 μg mL-1 (Table 1).
PCR detection of the qnr genes: Detection of the qnr genes in 85 Salmonella isolates was carried out by PCR amplification. The 700 bp amplicons of the qnrA gene were identified in 22 (25.8%) of 85 clinical isolates. The qnrB and the qnrS genes with the amplicon size of 120 and 280 bp, respectively were each found in 1 (1/17%) isolate of 85 clinical isolates.
Some isolates with the qnrA and only isolate with the qnrA, qnrB and qnrS were selected for sequencing. The comparison between sequence results and GenBank revealed identity to the sequence of the qnrA, qnrB and qnrS with the accession numbers No.AY906856.1 and No.DQ30391901 and No.FJ4181530.1, respectively (Fig. 1-3).
DISCUSSION
Quinolone resistance has been described previously by chromosomal mutation in bacterial topoisomerase gene and genes regulating expression of efflux pump or both. In recent years, plasmid-mediated quinolone resistance has been reported in several parts of the world. The qnr genes responsible for plasmid-born quinolone resistance have been found in clinical isolates of Enterobacteriaceae such as Salmonella and also resistance to different classes of antimicrobial agents is associated with the qnr gene (Oktem et al., 2008). This gene has been reported in different countries all over the world such as Senegal, USA, Korea, Germany, Taiwan, Vietnam and France (Hopkins et al., 2007).
A plasmid-mediated quinolone resistance conferring low resistance to quinolone, have been widely associated with clinical isolates of Enterobacteriaceae such as Salmonella (Cheung et al., 2005; Jiang et al., 2008; Jacoby et al., 2003; Wu et al., 2007). In fact, some reports suggested that the qnr gene confers only low-level resistance and its main contribution is to facilitate the development of QRDR mutations.
The present study demonstrates decreased quinolone and fluoroquinolone susceptibility as well as resistance to nalidixic acid and ciprofloxacin. It is very important to know that ciprofloxacin, a member of the large and widely used fluoroquinolone group of antimicrobial drugs, is considered the empirical choice treatment of infections in adults (Casin et al., 2003).
In this study, the isolates were of different serovars. Some of the isolates carried the qnrA and one isolate contained the qnrB and qnrS genes on their plasmids. All isolates were examined for the presence of the qnr resistance genes by PCR. Of 85 isolates one isolate harbored the three qnrA, qnrB, qnrS genes. The qnrA gene was more prevalent than the qnrB and qnrS gene among 85 Salmonella clinical isolates. Almost similar results are obtained in the United States in 2006 (Jacoby et al., 2006). Thus; the qnrA gene was more common among Salmonella clinical isolates in Iran. Despite possessing the gene encoding resistance to quinolone and fluoroquinolone in Salmonella, most isolates were susceptible to ciprofloxacin. Perhaps absence of an efficient promoter region is a compelling reason for this contradiction or perhaps these genes confer resistance or decreased susceptibility to nalidixic acid more than ciprofloxacin. Antibiotic resistance results to nalidixic acid and ciprofloxacin were almost the same for some countries, for example other parts of Iran (Tabriz) (Ghorashi et al., 2010; Manikandan et al., 2011).
The presence of qnr genes has been associated with an increase in the quinolone MIC values and therefore leads to incomplete treatment when quinolones are used as selective therapeutic drugs. Since plasmids carrying qnr resistance determinants have high prevalence and a worldwide distribution, care should be taken when quinolone are used to treat infections.