Distribution of sul genes and their variants in uropathogenic Escherichia coli isolated from two hospitals of Sabah

Sulphonamides resistant strains are highly prevalent in uropathogenic Escherichia coli (UPEC) isolates. Sul genes encode sulphonamide resistance and are present on transferrable plasmids. Integrons (IGNs) are genetic elements containing integrase gene, attl site and gene cassettes which carry multiple antibiotic resistant genes. Class 1 integrons have been extensively studied because these were most prevalent among clinical isolates. In this study, UPEC isolates were determined for the antibiotic susceptibility patterns to four antibiotics commonly used for urinary tract infections, which include co-trimethoxazole (TMP-STX). Distribution of sul genes and integrase1 gene (intI1) was studied in TMP-STX resistant UPEC isolates by using multiplex polymerase chain reaction (mPCR). Sul genes variants were investigated by DNA sequencing of the whole open reading frame of sul1 and sul2 genes and PCR product of sul3 gene. Sul1, sul2 and sul3 genes were prevalent in 37 (24.7%) of 150 UPEC isolates. IntI1 is positive in 22 sul genes positive isolates. Of six isolates positive with sul2 genes, sul2(a) and sul2(b) variants, which were described in the previous study, in the four isolates and the two isolates respectively were observed. This is the first mPCR which investigates the prevalence of three sul genes and intI1 in the UPEC clinical isolates from two hospitals of Sabah.


Introduction
The major problem in treatment of UTI is the resistance of UPEC to antimicrobial agents.The previous studies indicated that there is increasing trend of UPEC resistance to TMP-STX.In Europe, TMP-STX resistance rates among uropathogens increased from 0-5% before 1990 to 9-26% in 1999 and 2000 (Blahna et al., 2006).After their clinical introduction in 1935, sulfonamides were used to treat bacterial and protozoal infections.Sulfonamides have generally been used in combination with diaminopyrimidines to overcome the rapid emergence of resistance since 1970s (Huovinen et al., 1995;Huovinen, 2001).The combination trimethoprimsulfamethoxazole is still commonly used in clinical practice and one of the drugs of choice for the treatment of urinary tract infections.Dihydropteroate synthase (DHPS) is an enzyme important in the folic acid biosynthesis pathway.Competition of sulfonamides with the structural analog p-aminobenzoic acid for binding to dihydropteroate synthase (DHPS), results in inhibition of the formation of dihydrofolic acid (Sko¨ld, 2000).Resistance to sulfonamides in E. coli can result from mutations in the chromosomal DHPS gene (folP) as well as the acquisition of an alternative DHPS gene (sul).The protein encoded by sul gene has a lower affinity for sulphonamides (SwedbergandSko¨ld, 1980;Swedberg et al., 1993;Ra°dstro¨m and Swedberg, 1998;Vedantam et al., 1998;Sundstro¨m et al., 1998).Three alternative sulfonamide resistance DHPS genes (sul1, sul2 and sul3) in gram-negative bacteria have been described.The DNA sequences of sul1 and sul2 from E. coli are different from all the known chromosomal DHPS genes from E. coli and other bacteria and their origin remains unknown (Ra°dstro¨m and Swedberg, 1998).Sul1 has been found on large conjugative plasmids carrying on class 1 integrons (Ra°dstro¨m et al., 1991;Antunes et al., 2005;Hammerum et al., 2006;Trobos et al., 2008).The sul2 gene has also been recently found on a wide range of large conjugative plasmids as well as small non-conjugative plasmids (Hammerum et al., 2006;Trobos et al., 2008;Bean et al., 2009).Perreten and Boerlin described a new alternative sulfonamide resistance determinant called sul3 which is probably acquired by E. coli from a distantly related organism.The presence of sul3 on different plasmids in different E. coli clones, this gene was flanked by two insertional elements and a distinct DNA sequence of it suggested that it was a new sul gene (Perreten and Boerlin, 2003) Rapid transmission of drug resistance in bacterial pathogens is the consequence of the widespread use of antibiotics as well as transfer of drug resistance determinants mediated by plasmids, transposons and gene cassettes in IGNs.The presence of variable no. of drug resistance genes in their gene cassettes makes the genetic organizations of IGNs diverse (Li et al., 2013).An IGN is defined as a genetic element that consists of three parts.The parts are the gene which encodes integrase that mediates site-specific recombination events, the site, attI, at which gene cassettes can be integrated by site-specific recombination and gene cassettes.Although IGNs themselves are not mobile, the integrase enzyme excises and integrates the gene cassettes from and into the IGN (Bennett, 1999;Fluit and Schmitz, 2004).There are five classes of IGN and the first three classes are resistant integrons while class 4 and 5 are superintegrons.The most prevalent IGN is class I and it is present in gram-negative bacteria including E. coli.Class 1 IGNs have been extensively studied because these were most prevalent among clinical isolates as well as commensals out of three classes (Bass et al., 1999;Chang et al., 2000;Mazel et al., 2000;Naas et al., 2000;Sundeand Sorum, 2000) Out of three sul genes, the sul2 gene has been reported to be the most prevalent gene in Escherichia coli.This gene was highly conserved, and is usually carried on large conjugative resistance plasmids.Sul2 genetic variation in E.coli isolates from animal, meat and human clinical samples and normal human was previously studied.Six point mutations were observed with four synonymous mutations and two non-synonymous mutations (Trobos et al., 2009).However, there were no reports of sul1 and sul3 genetic variants.In this study, UPEC isolates were determined for the antibiotic susceptibility patterns to four antibiotics.Multiplex PCR of three sul genes and intI1 gene was set up to screen TMP-STX resistant UPEC and application of this PCR method to detect three sul genes in UPEC isolates from two hospitals, Hospital Queen Elizabeth and Hospital Papar, of Sabah State, Malaysia.Most of the sul genes were present on IGN and detection of class 1 integrase was included in this study.In addition, DNA sequencing of three sul genes were undertaken to find out the variants of sul genes.

Samples and controls
One hundred and fifty UPEC isolates stocked in Microbiology Laboratory, Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah were investigated for antibiotic susceptibility tests in this study.These UPEC isolates were derived from urine samples sent to microbiology laboratory (for culture and sensitivity) of two hospitals, Hospital Queen Elizabeth and Hospital Papar which are located around Kota Kinabalu, Sabah and we have checked for significant bacteriuria to include in the criteria for UPEC.The study was done on the urine samples collected between January and June, 2013.Positive controls for sul1, sul2, sul3, intI1 were pKTN117, pKTN118 and pKTN 119, pKTN 120 respectively.These plasmids were constructed with the PCR products of these genes, which were cloned into TopoTA plasmids.The genes inside the plasmids were sequenced, aligned with the sequences downloaded from the NCBI website and the DNA sequences were observed to be homologous.Escherichia coli ATCC 25922 was used as the negative control in this study.

Disc diffusion method
The stocked UPEC isolates were cultured on MacConkey agar and disc diffusion method was performed on Mueller-Hinton agar plates, according to Clinical and Laboratory Standards Institute (CLSI) guidelines to perform antibiotic susceptibility test (AST) (CLSI, 2012).The antibiotic discs used in this study were cotrimethoxazole (TMP-STX), ciprofloxacin (CIP), gentamycin (GM) andcefotaxime (CTX).

Determination of minimum inhibitory concentration
This procedure was performed using the agar dilution method as described by the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2012).The minimal inhibitory concentration (MIC) is defined as the lowest antimicrobial concentration able to totally inhibit bacterial growth.E. coli ATCC 25922 was used as a negative control for both disc diffusion method and MIC study (CLSI, 2012).

Multiplex PCR for detection of sul1, sul2, sul3 and intI1 genes
Those isolates having resistance to TMP-STX were studied for detection of sul1, sul2, sul3 genes and intI1by multiplex PCR.The bacterial isolates were inoculated in 3 ml of Luria-Bertani broth and incubated at 37˚C for 18 hrs.The bacterial DNA was extracted by boiling method (Abdallah et al., 2011;Ifeanyi et al., 2015).The primer sets for sul1, sul2, sul3 genes, intI1 gene and 16srRNA gene were mentioned in Table 1 (Kerrn et al., 2002;Gundogdu et al., 2011).The 16s rRNA gene was amplified in each reaction as an internal positive control for confirmation of E. coli.The ingredients for multiplex PCR were 6 μL of DNA template, 1x PCR buffer, 3mM MgCl 2 , 0.4mM dNTPs, 1.5U of Taq polymerase, 0.4 mM each of 16srRNA gene, sul1, sul2 and intI1 sets of primers, 0.8mM of sul3 forward and reverse primers and sterile distilled water was added to make total 25 μl of PCR mixture.The PCR conditions were 94°Cx5min, 30 cycles of 94°C x 30s, 58°C x 30s, 72°C x 1min and a final extension of 72°C x 7min.Amplification was performed using Applied Biosystems Thermocycler and First base Taq DNA polymerase.The sizes of PCR amplicons were checked by 1.5% TAE agarose gel electrophoresis and Alpha Imager ® HP System gel documentation apparatus after the gel was stained with ethidium bromide.

PCR for sul1 and sul2 whole gene sequence for detection of sul gene variants
For sul1 gene and sul2 gene the whole sequence amplification, the primer setssul1ws and sul2ws primer sets shown in Table 1 were used.The primers sets were taken from NCBI website taking care of the precautions in primer design.The PCR ingredients were 6 μL of DNA template, 1x PCR buffer, 3mM MgCl 2 , 0.4mM dNTPs, 1.5U of Taq polymerase, 0.4 mM of each forward and reverse primers of sul1ws for sul1.The PCR conditions were 94°C x 5min, 30 cycles of 94°C x 30s, 56°C x 30s, 72°C x 1min and a final extension of 72°C x 5min.The PCR ingredients were the same for sul2 gene with the exception that forward and reverse primers of sul2ws were used.The PCR conditions were 94°C x 5min, 35 cycles of 94°C x 45s, 54°C x 45s, 72°C x 1min and a final extension of 72°C x 5min.Amplification and gel documentation were the same as mPCR method.For the sul3 gene DNA sequence, PCR products of mPCR method were used.

DNA sequencing of PCR products
DNA sequencing of PCR products was performed with Applied Biosystems highest capacity-based genetic analyzer using Big Dye® Terminator v3.1 cycle sequencing kit.

Alignment of DNA sequences to find single nucleotide polymorphisms (SNPs)
The resulting DNA sequences were analyzed with A plasmid Editor (ApE) software and nucleotide sequences were aligned with wild type sul gene sequences downloaded from NCBI website to detect sul genes variants and nucleotide sequence numbers were counted according to wild type sequences.

Results of AST and MIC value
One hundred and fifty UPEC isolates were tested for antibiotic susceptibility patterns with four antibiotics commonly used for UTI, which were mentioned in the method.Out of 150 isolates, forty, five, four and three isolates were resistant to TMP-STX, gentamycin, ciprofloxacin and cefotaxime, respectively.In case of MIC determination for the TMP-STX resistant isolates, it was observed to be 128/2432 µg/mL

Results of mPCR setting up and screening of sul genes and intI1 gene in TMP-STX resistant UPEC isolates
After setting up of multiplex PCR (Figure 1), all the TMP-STX resistant isolates were investigated for sul genes and intI1 gene.The distribution of sul genes were mentioned in Table 2. Presence of at least one of the sul genes was observed in 37 (92.5%) of 40TMP-STX resistant isolates.Sul2 gene was most prevalent (68%), sul1 gene was 52% and sul3 gene was the least (16%).Of total 22 isolates positive for intI1, 11 were sul1 positive, 13 were sul2 positive and 5 were sul3 positive (Table 2).There was absence of isolate having sul1, sul2 and sul3 genes together.An interesting finding was no coexistence of sul1 and sul3 in the studied UPEC isolates.

Investigation of sul genes variants
Altogether, six of each sul genes were undertaken for DNA sequence analysis and all the sul1 and sul3 genes were totally identical to the sequence of NCBI downloaded respective sequences.However, two isolates positive with sul2 genes have one synonymous mutation at nucleotide position (NP) no.159 (T to C variant) and another non-synonymous mutation at NP no.197 (C to A variant), which changed from amino acid alanine to glutamic acid.Trobos et al. (2009) have named sul2(a) for T at NP 159 and C at NP 197 (TC variant) and sul2(b) for C at NP 159 and A at NP 197 (CA variant), which were observed in their study.As we followed the system described in that study, sul2(a) and sul2(b) variants in the four isolates and the two isolates respectively were observed among six sul2 genes positive isolates sequenced (Figure 2

Discussion
This is the first study to use multiplex PCR for detection of three sul genes in human UPEC isolates obtained from hospitals of Sabah.There has been a study on three sul genes in E. coli isolates from healthy humans, pork and pigs in Denmark and clinical samples were not included and the study did not mention that multiplex PCR was used.However, the literature has shown one PCR method for the four genes sul1, sul2, sul3 and intI1 indicating that their PCR was multiplex one (Hammerum et al., 2006).
In addition, Gundogdu et al. (2011) studied all three sul genes in UPEC.However, in their PCR sul1 and sul2 were detected in one PCR reaction while sul3 was detected in another PCR reaction.Wu et al. (2010) studied on 109 TMP-STX resistant E. coli strains isolated from pig feces, pig carcasses and human stools.In their study, 26 isolates from human stool were resistant to TMP-STX.Of these isolates, sul2 gene was the most prevalent gene followed by sul1 gene.There was no coexistence of sul1 and sul3 in these human isolates.Sul1 and sul2 coexistence was higher while sul2 and sul3 genes were together in lesser no. of isolates.These above mentioned findings were consistent with the findings in our study.Teichmann et al. (2014) indicated that sul2 gene was highly prevalent in TMP-STX resistant UPEC isolates up to 67% in their study.In this study, sul2 genes were present in 68% of TMP-STX resistant isolates showing that sul2 genes was more prevalent than two other sul genes.
IGNs are capable of collecting of promoter-less gene cassettes through the actions of specialized site-specific recombination enzymes, intl.By mean of these mobile elements such as plasmids and transposons, IGNs can be horizontally transferred drug resistant genes between bacterial species commonly within the Enterobacteriaceae family (Hall et al., 2003).Therefore, in this study screening of intI1 gene was undertaken together with sul genes in mPCR assay.
In the Danish study on E. coli isolates from UTI cases and bacteremia cases, sul1, sul2 and intI1 gene were included in the multiplex PCR and sul2 gene was the most common gene among these.The study did not include the sul3 gene.In that study, 97% of TMP-STX resistant isolates had carried sul genes and 96% of sul1 gene positive strains had intI1gene (Kernn et al., 2002).In case of Class 1 integron, sul1 gene was commonly located at the 3' conserved region which is downstream of gene cassettes (Carattoli, 2001).In our study, 92.5% of TMP-STX resistant isolates had positive for one of sul genes and 53% of sul1 gene positive strains were positive for intI1 gene.(Sorum H and L'Abee-Lund, 2002).As an alternative hypothesis, it can be assumed that DHPS enzyme cannot tolerate many changes to its amino acid sequence (Trobos et al., 2009).
In this study, TMP-STX resistant UPEC were studied for distribution of sul1, sul2 and sul3 genes and their variants because we have observed that sul genes positive isolates had consistent AST pattern and MIC values.These data support the information there is no much genetic variation of sul genes at the DNA level.However, the previous work in Denmark indicated that there were sul2 variants.Therefore it will be an interesting information if we find sul gene variants in our study.
Regarding two sul2 gene variants, the two variants observed in this study were included and the most common variants found in the other study (Trobos et al., 2009).The observed variants we observed in this study were common in human clinical isolates in their study and sul2(a) variant is more prevalent compared with variant sul2(b) in both studies.Although geographical regions were far away between Denmark and Sabah, Malaysia, it is surprising that the common two variants existing in sul2 gene in the two countries were consistent.We need further information on sul2 gene variants to draw a firm conclusion.

Conclusions
Up to date, there were no mPCR technique which can detect three sul genes and intl1 in isolates from UTI cases of hospitals in Malaysia.This study will be beneficial for the microbiologists to detect sulphonamide or TMP-STX resistant strains because PCR technique provides rapid and reliable results.UPEC isolates in this study were not highly resistant to gentamycin, ciprofloxacin and third generation cephalosporin indicating that antibiotics restriction policy was well controlled in the hospitals of Sabah.Furthermore, sul2(a) and sul2(b) variants could be investigated by PCR-RFLP analysis using Taq I restriction enzyme.

Figure. 1 .
Figure.1.Gel electrophoresis picture of multiplex PCR for three sul genes, intl 1gene and 16s rRNA of E. coli.All the sample lanes are 16s rRNA and intI1 gene positive.Lane 2,8 aresul1 positive isolate.Lane 3 is sul2 positive isolate.Lane 4 and 5 have no sul genes.Lane 6 is sul1 and sul2 positive isolate.Lane 7 is sul2 andsul3 positive isolate.Lane 9 is sul3 positive isolate.Lane 1 & 10 are100bp ladder molecular marker.The sizes of PCR products are 433bp for sul1 gene, 293bp for sul2 gene and 790bp for sul3 gene while 200bp for 16s rRNA gene and 160bp for intI1 gene.

Figure 2 .
Figure 2. Six UPEC isolates with two sul2 variants and differentiation of these variants by PCR-RFLP analysis (a) Nucleotide sequence alignment among six isolates with two sul2 variants, sul2(a) and sul2(b)is shown.Isolates no.038,Q88,096,109 have sul2(a) variants and isolates no.023 and 225 havesul2(b) variants with change of amino acid from A to E (non-synonymous nucleotide change at 197 NP) (b) Restriction map of sul2(a) variant and sul2(b) variant are shown for PCR-RFLP analysis by TaqI restriction enzyme to differentiate sul2 variants.Sul2(b) variant has extra-TaqI restriction site.
(a)).Mutation at nucleotide sequence no.197 could be investigated by PCR-RFLP analysis with Taq I restriction enzyme giving rise to 194 bp DNA fragment in case of sul2(b) variant gene while 251 bp DNA fragment was observed in sul2(a) variant (data not shown) (Figure 2 (b)).

Table 2 . Distribution of sul genes in TMP-STX resistant isolates and intI1 positive isolates which were TMP-STX resistant.
Footnote -*Percentage of sul genes in total UPEC isolates Trobos et al. (2009)had studied on sul2 genetic variation in E. coli isolates from animal, meat and human clinical samples and normal human.Six point mutations were observed in 68 samples.The most common variant is sul2(a) and the second common variant was sul2(b) in their study.TC variants rarely contained an extra non-synonymous mutation at position 427 (A instead of G) and this mutation was named sul2(c) in that study and this variant was observed in two isolates.Other three synonymous mutations occurred at 288, 489 and 672 nucleotide positions and each mutation was present in only one isolate.Presence of the sul2 gene without much genetic variation in both human and animal isolates suggests horizontal transfer of the gene.Because only two to three point mutations, it did not indicate genetic drift and has been concluded by researchers that this had occurred within a short time