Introduction

Shigella, a member of the Enterobacteriaceae family, is considered as a health problem across the world. Shigellosis is a self-limiting infectious disease in humans that is caused by Shigella, however, morbidity and mortality rates are highest among young children, elderly and immunocompromised patients. Upon antibiotic therapy, duration of symptoms and the transmission rate is reduced by shortening the period of Shigella excretion. However, life-threatening complications can occur in severe cases with Multi-Drug Resistant (MDR) Shigella, which defines the ability of organisms to withstand the inhibitory effect of at least one agent in ≥3 antimicrobial categories (1)(2)(3).

Over the past few decades, research in Iran (2), Malaysia (4), and West Nepal (5) revealed that Shigella is resistant to used traditional antimicrobials, similar to other bacteria. The rapid emergence of antimicrobial resistance is the public health is a concern that must be controlled (6).

The main mechanisms of Antimicrobial Resistance (AMR) are target site changes, production of an enzyme, reduced drug accumulation by the efflux pump or diminishing the permeability that can be inherent or adventitious. Control of antibiotic resistance is achieved by acquisition and dissemination of genes, through mobile genetic elements or mutations (7).

Tetracyclines, which were discovered in the 1940s, are broad-spectrum antibiotics for human, animals and plants. The first tetracycline-resistant bacteria, Shigella, was isolated in 1953, however, nowadays the majority of Shigella spp. have become resistant to it (8)(9).

The key mechanism of resistance to tetracycline arises due to the efflux pump and ribosomal protection system, which are under the control of structural and regulatory genes. Efflux pump genes code membrane proteins which export tetracycline from the cell that render it ineffective. Twenty-nine different tetracycline resistance (tet) genes were identified in gram positive and gram negative bacteria; eighteen of the Tet genes encode efflux pump, and it has been identified that tetA, tetB, tetC, tetD, tet E, tetG, tetH, tetI, tetJ, tetY, tet (30) and tet (31) are found in gram negative bacteria. Some of studies discovered tet A, tet B, tetC and tetD genesin Shigella, while the Antibiotic Resistance Genes Database (ARDB) does not report tetC for Shigella (8)(9)(10).

Efflux pumps belong to the six different groups, coding tetracycline resistance, which extrude tetracycline-cation complexes out of the bacteria for protons. Group 1 including tetA, tetB tetC and tetD, are frequently found in Shigella (10).

This study aimed to investigate tetA, tetB, tetC and tetD genes and the effect of efflux pump inhibitors on Shigella tetracycline resistant isolates.

Materials and Methods

Isolates

This study was conducted on 50 Shigella isolates collected from children diarrheic stools in Tehran, Iran (from November 2012 to October 2013). All of the isolates were identified and confirmed using conventional biochemical tests, during year 2015 (11) .

Serogrouping of Shigella isolates was done by slide agglutination assay using A, B, C and D antisera (Baharafshan Institute of Research & Development, Tehran, Iran). Polymerase Chain Reaction (PCR) assay targeting the ipaH (for genus Shigella), rfc (for species Shigella flexneri), and wbgZ (for species Shigella sonnei)genes was performed, Table 1 (12) (13).

Antimicrobial Susceptibility Testing

The susceptibility of isolates was examined using the disk diffusion method, in accordance with Clinical and Laboratory Standards Institute (CLSI 2015 ) guidelines  (14). The antibiotic susceptibility of Shigella isolates was examined against the following antibiotics (MAST Company, Merseyside, and U.K): minocycline (30 µg), tetracycline (30 µg), doxycycline (30 µg), ampicillin (10 µg), streptomycin (10 µg), trimethoprim-sulfamethoxazole (25 µg), nalidixic acid (30 µg), norfloxacin (10 µg), ciprofloxacin (5 µg) and levofloxacin (5 µg) on Muller –Hinton agar. This study was conducted for all of isolates in duplicates and Escherichia coli ATCC 25922 was used as a quality control strain in susceptibility testing.

Table 1. Primers Used in This Study

Determination of Minimal Inhibitory Concentration (MIC) with and without efflux pump inhibitor

Tetracycline resistant isolates in Disk diffusion method were used for Minimum Inhibitory Concentration (MIC) determination.

Determination of MIC for tetracycline was achieved by micro broth dilution procedure in the range of 0.5 to 1024 µg/mL with and without efflux pump inhibitor. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as an efflux pump inhibitor at final concentration of 50 µg/mL (15)(16).

Then, effect of CCCP on growth of Shigella isolates and Escherichia coli (as a control) was examined (15). The isolates with tetracycline MIC of ≤4 µg/mL and ≥ 16 µg/mL were considered as susceptible and resistant, respectively, according to CLSI guidelines.

DNA Extraction

All of Shigella isolates were grown at 37 °C in Luria-Bertani (LB) broth media. Subsequently, 2 mL of overnight cultures were centrifuged and DNA extracted from the sediment sample using a DNA extraction kit (Exgene Cell SV kit Gene All Biotechnology Co. Ltd Korea).

Purified genomic DNA was stored in sterile DNase free tubes for PCR screening.

Oligonucleotides used in Polymerase Chain Reaction

Polymerase Chain Reaction amplification of ipaH, wbgZ, rfc, tetC and tetD genes was performed with primers used in previous studies (12)(13)(17). Other primers (tetA and tetB) were selected based on the sequences available on GenBank for each gene and then Primer3Plus-Bioinformatics was used to generate the candidate primer pairs. All of primers used in this study were synthesized by the TAGC Company (Tag Copenhagen A/S Kong Georgs Vej 12 DK-2000 Frederiksberg Denmark); primer sequences are listed in Table 1. Stock suspension (100 Pmol) was made in molecular grade water and stored at -70°C; all of primers were diluted to 10 Pmol before use.

Conventional Polymerase Chain Reaction

Originally, PCR was performed to detect the ipaH gene for confirming isolates as genus of Shigella, and then presence of wbgZ , rfc, tetA, tetB , tetC and tetD genes was assessed. The PCR conditions were as follows: initial denaturation step at 94°C for 5 minutes, followed by 30 cycles consisting of denaturation (at 94°C for 1 minute), annealing (1 minute, separately adjusted for each set of primer pair, according Table1), and extension (at 72 °C for 1 minute), followed by a final extension step at 72 °C for 5 minutes. Shigella flexneri ATCC 9290 and S. sonnei ATCC 1202 were used as controls in each assay.

Finally, an agarose gel was used to assess the PCR results.

RNA Extraction and cDNA Synthesis

All of isolates were cultured in LB agar media at 37 °C. ^.One colony from each isolate was suspended in 1 mL of LB broth media and incubated at 37 °C for 18 to 24 hours.

Next, concentration of bacterial suspension was adjusted to 10⁷   cfu /mL by OD in 625nm, and 400 microliter of bacterial suspension was centrifuged (3000 rpm/10 minutes). After centrifugation the supernatant was removed, the bottom sediment was resuspended in sterile normal saline, followed by another centrifugation. Subsequent to two washes, lysis buffer (340 µL)_, SDS (30 µL), carrier RNA (10 µL₎ and proteinase K (20 µL) were added to bacterial cell tubes. RNA was extracted by a kit and automated Mag Core Nucleic Acid Extractor.

Immediately after, 2µL ofRNA extracts was treated with mixture of DNase (2µL) and buffer (2µL)at 37°C/ 30 minutes.Subsequently, EDTA (2 µL)was added and incubated at 65°C ^/10 minutes.

Purified genomic RNA that was collected in sterile DNase free tubes was used to synthesize PCR cDNA.

In order to control quality of RNA extraction, 5 µL treated RNA and 5 µL untreated were used for agarose gel electrophoresis. Also, concentration and purity of RNA were assessed by measuring the ratio of UV absorbance at 260 nm and 280 nm, using a Nano Drop 2000C spectrophotometer.

Extracted RNA was used to synthesize cDNA by Revert Aid First Strand cDNA Synthesis kit (Thermo, Dreieich) and Step One plus (ABI). Also, concentration and purity of cDNA was assessed, and the cDNA was stored in sterile DNase free tubes at -20°C.

Evaluation of gene expression by real time Polymerase Chain Reaction

Gene expression analysis was performed by Evergreen kit (Maxima® SYBR Green/Fluorescein qPCR Master Mix) in Step One plus (ABI) and ECO (Illumina USA) instruments, using tetA and tetB specific primers for efflux pump. The rpsL gene was chosen as a reference primer housekeeping gene (Table 1). Normalization of cDNA and primers concentration was done for each reaction, the volume of cDNA for each isolate was adjusted to 20 ng per reaction. The real time PCR conditions of initial denaturation of 10 minutes at 95^◦C, 40 cycles of 15 seconds at 95^◦C for denaturation, 45 seconds annealing and 1-minute at 72^◦C for extension, and at 72^◦C for 5 minutes, followed by a melting curve analysis. Calculation of gene expression was done using comparative quantification of 2^(∆∆Ct)-based on Pfaffl method (18). Amplification efficiency was determined from analysis of a serial dilution of a control strain cDNA (at certain concentrations). Analysis of data was performed with Step One Software v2.3, and qPCR products were used for electrophoresis on agarose gel for controlling the results. Mean values of duplicated runs of each isolate in real time PCR was used for analysis.

Results

Isolates

A total of 50 Shigella spp. were isolated during the period of the study between November 2012 and October 2013. The isolates of Shigella were identified by conventional biochemical tests and sero-grouping method. Among the 50 Shigella isolates confirmed by PCR, 16% (n=8) was identified as Shigella flexneri (S. flexneri) and 84% (n=42) as Shigella sonnei (S. sonnei).Species-specific PCR analyses of ipaH gene confirmed the identity of all isolates as Shigella spp.  Moreover, amplification of the genes wbgZ and rfc confirmed serogrouping results (Table1).

Antimicrobial susceptibility testing

The results of antimicrobial susceptibility of Shigella spp. demonstrated that all of the isolates were MDR and resistant to three or more classes of antibiotics.

According to the results of antimicrobial susceptibility testing listed in Table 2, 96% of all isolates were resistant to tetracycline, 94% to doxycycline, and 2% to minocycline

Table 2. Results ofAntimicrobial Susceptibility

Table 3. Results of Minimum Inhibitory Concentration With and Without Efflux Pump Inhibitor

CCCP: Inhibitor n: Number of isolates

Minimum Inhibitory Concentration Test

All tetracycline resistant isolates were subjected to MIC determination with or without efflux pump inhibitor (CCCP). All of the isolates showed resistance by the MIC method (Table 3).

Extraction of nucleic acid

Majority of extracted nucleic acids (RNA, cDNA) indicated the acceptable ratio of UV absorbance at 260 nm and 280 nm for assessing the purity of DNA (about 2) and electrophoresis. A few extractions of RNA with poor results of electrophoresis were repeated.

Conventional PCR

The IpaH gene amplified with PCR, visualized a 619-bp band on agarose gel for all isolates and confirmed them as the genus of Shigella. In addition, amplification of wbgZ and rfc genes revealed 430 bp and 537 bp bands, confirming Shigella isolates as S. sonnei and S. flexneri, respectively.

Polymerase Chain Reaction amplification of Shigella isolates showed that 66% of isolates were positive for tetA, while only 12% of them were positive for tetB.  Neither of the isolates was positive for tetC and tetD (Table 1).

Real Time Polymerase Chain Reaction

As Table 4 shows, PCR analysis of isolates demonstrated 78% against 12% for presence of tetA and tetB (Table 4).

Expression of tetB genewas seen in S. flexneri isolates, which were resistant to tetracycline.

Shigella sonnei isolates did not express tetA, and their MIC did not change after using CCCP (Table 4).

Table 4. Results of Expression of tetA and tetB Genes by Real Time Polymerase Chain Reaction

¹Presence of tetA by conventional PCR ²Presence of tetA by conventional PCR ³0: No expression ⁴D= S. sonnei ⁵B=S. flexneri ⁶R: Resistance ⁷S: Sensitive ⁸P; Positive ⁹N: Negative MIC ≥ 16 μg/ml indicate resistance to tetracycline. MIC≤4 μg/mL indicates susceptibility to tetracycline. T=Tetracycline D= Doxycycline MIN=Minocycline S=Streptomycin SXT= trimethoprim-sulfamethoxazole AM=Ampicillin NA=Nalidixic Acid CIP=Ciprofloxacin