Molecular Detection of qnrA, qnrB and qnrS Resistance Genes among Salmonella spp. in Iran
N. Sepehri Rad,
S. Gerayesh Nejad
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.
to cite this article:
R. Saboohi, S.D. Siadat, M.R. Aghasadeghi, M.R. Razavi, B. Rajaei, N. Sepehri Rad, A. Moshiri, S.F. Mousavi, S. Javadian, K. Kave, A. Dashtbani-Roozbehani, S.M. Sadat, S. Gerayesh Nejad and N. Kashanizadeh, 2012. Molecular Detection of qnrA, qnrB and qnrS Resistance Genes among Salmonella spp. in Iran. Current Research in Bacteriology, 5: 24-30.
Received: November 06, 2011;
Accepted: February 20, 2012;
Published: May 21, 2012
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
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®,
|| The information of blaCMY-2 positive Salmonella
|CP: Ciprofloxacin (=.0.64 μg mL-1), +: Present,
-: Absent, MIC: Minimum inhibitory concentration
|| 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).
|| PCR products of qnrA and 1 kb DNA ladder
|| 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
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).
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).
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.
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