Upstream region of OprD mutations in imipenem-resistant and imipenem-sensitive Pseudomonas aeruginosa isolates

The current study was aimed at investigating the prevalence of the mutations upstream of the oprD coding region and its promoters among imipenem-resistant and sensitive Pseudomonas aeruginosa isolated from educational hospitals in Yazd City, Iran. All isolates were identied by the conventional biochemical tests. Then, the antibiotic resistance of these isolates was determined using the disk diffusion method according to the CLSI guidelines. Also, the E.test was performed to determine the minimum inhibitory concentrations (MIC) of imipenem. The mutations of this gene were recognized by the amplication of this region and subsequently sequenced. Sequencing of the genomic region upstream of oprD these regions were done in the 29 clinical strains. Statistical analysis was done by the statistical software SPSS-18. Seventy (77.7%) of isolates had MIC ≥ 16 and were resistant to imipenem. Mutations of the upstream of the oprD gene and its promoters were seen in 25 (86.2%) isolates and 4 isolates had no mutation. One isolate had a base substitution A → Cat nt 25 in the coding region and this isolate had a point mutation leading to an amino acid change at positions 9 (I → L). Our study results indicated that none of the strains had mutation in Shine-Dalgarno and the point mutations were the most common mutations upstream of the oprD coding region among P. aeruginosa isolates. Mutations were observed in imipenem-resistant isolates and it seems this mechanism is effective in resistance of isolates to imipenem and this conrmed that the indiscriminate use of antibiotic should be controlled.


Introduction
Pseudomonas aeruginosa is an opportunistic pathogen that causes a variety of infections in immunocompromised patients. In recent years, Antibiotic resistance of P. aeruginosa is increasing and the selection of suitable treatments has become di cult and is associated with increased morbidity and mortality (Riera et al., 2011;Yan et al., 2014).
Carbapenems, mainly imipenem and meropenem, are important and useful antibiotics for the treatment of infections due to multidrug-resistant Pseudomonas. Carbapenems are a class of β-lactam antibiotics with good antimicrobial activity against P. aeruginosa (Lister, Wolter, & Hanson, 2009;Ocampo-Sosa et al., 2012). Carbapenem resistance of P. aeruginosa is mainly due to a combination of different factors, including low permeability of outer membrane porin and mutations in the gene encoding OprD, the production of the AmpC bate-lactamases, overproduction of e ux systems, and producing Carbapenemase (Hancock & Brinkman, 2002;Pirnay et al., 2002;Rostami et al., 2018). However, Among these mechanisms, the Loss or mutation of outer membrane porin (OprD) and promoter of this gene appears to be the most common mechanisms of intrinsic resistance to imipenem and a lesser extent to meropenem. This mechanism causes blocking of the entrance of carbapenems particularly imipenem into a bacterium (Amin et al., 2005;Shen, Pan, & Fang, 2015).
OprD, an outer membrane porin is a semipermeable barrier and substrate-speci c a penetrable protein consisting of 443 amino acids that allows the diffusion of sugars, small peptides, basic amino acids, and carbapenems typically imipenem into the cell (Cowan et al., 1992;Pirnay et al., 2002).
OprD mediated resistance occurs as a result of decreased transcriptional expression of oprD and imipenem resistance has been associated with(i) mutations that inactivate or destroy at least one of the oprD promoters, (ii) premature termination of oprD transcription, (iii) co-regulation with trace metal resistance mechanisms such as Zinc and copper, (iv) salicylate-mediated reduction, and (v) decreased transcriptional expression via co-regulation with the multidrug e ux pump encoded by mexEF-oprN (Amin et al., 2005).
The typed of mutations in the oprD gene and upstream regions and promoters of this gene are various such as nucleotide deletions, insertions, and point mutations that have been recognized to be the major mechanisms leading to inactivation of the oprD gene and promoter in imipenem-resistant isolates of P.aeruginosa (Gutiérrez et al., 2007;Pirnay et al., 2002). Transcription of oprD in P. aeruginosa PAO1 initiates with equal frequencies from two start sites, located 23 bases (SS1) and 71 bases (SS2) upstream of the structural gene. In the previous investigation, two or three types of imipenem-resistance mutants in clinical isolates were observed. The major type involves deletion and point mutations (Lynch, Drusano, & Mobley, 1987). These well-known alterations are commonly reported, include point mutations or insertion sequences (ISs) inactivating in the resistance to imipenem, especially in Iran. Therefore, this study aimed to evaluate the prevalence of mutations upstream of the oprD coding region and its promoters in imipenem-resistant and -sensitive Pseudomonas aeruginosa isolated from educational hospitals.

Bacterial Isolates
In a descriptive study, 90 isolates of P. aeruginosa were collected from June 2018 to April 2019 at the Teaching Hospitals of Shahid Sadoghi University of Medical Science, Yazd, Iran. These isolates were originated from different clinical specimens of hospitalized patients, including blood, burn wounds, urine, lungs, etc.
After transferring the plate containing Gram-negative rod colonies to the Laboratory of Microbiology, suspected colonies were identi ed by Gram staining and conventional biochemical tests such as catalase, oxidase, growth in 42°C, Oxidative/fermentative test, and Differential media such as TSI (Merck, Germany). Isolate identi ed as P. aeruginosa were stored at 70°C in trypticase soy broth (Merck) supplemented with a 20°C glycerol unit.
Minimum Inhibitory Concentration and Phenotypic Con rmatory Tests.
Antibiotic susceptibility testing of the isolates was performed using the disk diffusion method (Kirby-Bauer) according to Clinical and Laboratory Standard Institute guideline (CLSI, 2019) using Mueller-Hinton agar (Merck, Germany) and Imipenem, meropenem, ertapenem, cipro oxacin, ceftazidime, Cefepime, ceftriaxone, gentamicin, and tobramycin (MAST, UK). P. aeruginosa ATCC27853 was used as quality control. The Minimum Inhibitory Concentration (MIC) of imipenem was performed by E. test strips (Lio lchem, Italy) as described in the manufacturer's instructions. MIC breakpoint was de ned according to CLSI guidelines (CLSI, 2019).

DNA extraction
DNA extraction was performed using by salting out method and was stored at -20°C until further use (18).
PCR for detection of oprD gene.
PCR technique was performed. Primers were developed for each gene using Primer 3. The primers used for DNA ampli cation, as follows: 5΄-AGACATGCCGTGGATACAAA − 3΄ for the forward and 5΄-AGTGCTACCTGCGGAAACC − 3΄for the reverse primers. The nal optimized PCR reaction consisted of 0.5 µl MgCl2 (100 mM), 0.5 µl dNTP (10 mM), 0.2 µl (1 unit) Taq DNA polymerase (Cinnagen, Iran), 1 µl of each primer (10 pmol) (Alpha DNA, Canada), 2.5 µl PCR buffer (10 X), and 0.5 µl of DNA template (100 µg/ml) in a total volume of 25 µl with double distilled water. DNA ampli cation was carried out with a thermocycler (Quanta Biotech, England), PCR ampli cation was performed as follows: one cycle at 95°C for 300 seconds, then 30 cycles at 95°C for 45 Sec, 56°C for 45 Sec, and 72°C for 60 Sec and a nal extension at 72°C for 10 min using an initial denaturation step for 5 min at 94°C (one cycle), followed by 35 cycles of 1 min at 94°C, 1 min at 50°C, and 1min at 72°C. The ampli ed products were analyzed by 1.5% (w/w) agarose gel electrophoresis and were visualized on an ultraviolet illumination after staining with ethidium bromide.
DNA sequencing and analyses of sequence data According to imipenem MIC results, 29 isolates were selected randomly (due to Financial Limitations) for evaluation of the mutations. We ampli ed and sequenced the genomic region upstream of oprD genes in the imipenem-resistant (n = 25) and imipenem-sensitive (n = 4) bacteria. For DNA sequencing, upstream regions and fty-four (54) primary nucleotides of the oprD gene were sequenced. The sequence results were aligned and analyzed using MEGA 6 software and CLUSTAL W2, Vector NTI Advance version9.0.0 software (InforMax; Invitrogen). Protein alignments were carried out using. ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). A mutation in the promoter and the upstream coding region of the OprD gene (Table 3) was identi ed by DNA sequencing.

Statistical analysis
The data were analyzed using the Statistical Program for Social Sciences version 18. (SPSS Version. 18 IBM, Chicago, IL, USA). For the analysis of data, chi-square tests were employed to calculate the P-value. Statistical signi cance and levels were set at P < 0.05.

Antibiotic resistance patterns
The frequency of resistance to carbapenems was as follows: imipenem 48.9%, meropenem 56.6%, and Ertapenem 52.5%. The results of antimicrobial susceptibility testing using the disk diffusion method are shown in Table 1

PCR and sequencing
The oprD gene and genomic region upstream of oprD were ampli ed by PCR. The electrophoresis agarose gel was performing on PCR products that were shown in Fig. 1. The size of the ampli ed fragment is 570 base pairs. As shown in Fig. 1.
The frequency of mutations based on specimens was as follows: Burn 57.69%, Urine19.23%, and other specimens 23.07%. Most mutations were seen in P. aeruginosa isolated from burn specimens and burn ward. The Statistical analysis found a signi cant correlation between the type of specimens and MIC (P ≤ 0.05). The Statistical analysis found a signi cant correlation between MIC and resistance to imipenem(P ≤ 0.05).
Based on the observed mutations, none of the strains had no mutation in Shine-Dalgarno (GGAG; nucleotides − 12 to -9), -10 (TAAGTT; nucleotides − 84 to -79), and − 35 (TCGCCA; nucleotides − 107 to -102) sequences. Six isolates have point mutations in the promoter, Five isolates had T→C base substitution at nt -90 and One isolate had a base substitution G→ Cat nt -120. Also, One isolate had a base substitution A→ Cat n t 25 in the coding region, and this isolate had a point mutation leading to an amino acid change at positions 9(I→L). The insertion of one base was seen in ve isolates and the insertion of tree nucleotide was observed in one isolate. The rest of the results sequencing of upstream regions and promoter regions are shown in Table 3.
According to studies of antibiotic resistance in different parts of the world and the result of the present study; it can be concluded that resistance rates in P. aeruginosa isolates were higher than previous reports, which can be due to a combination of different factors such as the inconsiderate use or the previous use of antibiotics in prophylaxis, differences in the type of sample, and the geographical region and care of patients in hospitals and difference in the mechanism of resistance . Since the carbapenems are commonly used in the treatment and mutations in the genomic region upstream of oprD and promoter are the most current reason against resistance to these antibiotics, so identifying and assessing the prevalence of these mutations in the bacteria population can be very effective in controlling the resistance pattern. The mutational inactivation of the oprD gene and disruption in promoter represents the major cause of OprD loss in P. aeruginosa strains. In our study, alterations were observed in resistant isolates. Mutations of the upstream region oprD gene were seen in all (25) the imipenem-resistant isolates. Mutations in SS1 and SS2 were point mutations. One isolate had a base substitution A→ Cat n t 25 in the coding region and this isolate had a point mutation leading to an amino acid change at positions 9 (I→L). Also, the insertion of one base was seen in ve isolates and the insertion of tree nucleotide were observed in one isolate.
A similar study was performed by Damien Fournier et al (Fournier et al., 2013). Mutations of the oprD gene were seen in 86.2% of imipenem-resistant isolates and Reported the lack of OprD was due to tot the disruption of the oprD promoter by ISPsy2 in one strain and the other strains had a mutation or gene disruption by different insertion sequences ISPa1635, ISPa1328, IS911, ISPs1, IS51, IS222, and ISPa41). In a study conducted by Alain A et al (Ocampo-Sosa et al., 2012). seventy-seven (77%) isolates had mutations and mutations were observed in both sensitive and resistant isolates. Most isolates showed point mutations and deletion mutations. In a study performed by Aki Hirabayashi et al (Hirabayashi et al., 2017). Sequencing of oprD gene and the promoter and downstream regions were done and the results revealed that most of the resistant-isolates had insertion mutations in the oprD gene, also there was a direct relationship between the alteration or loss of oprD and the increase in MIC, for imipenem but not meropenem and other carbapenems (Cowan et al., 1992;Ocampo-Sosa et al., 2012;Shen et al., 2015;Zarei-Yazdeli et al., 2014). In a study conducted by Yumiko Sanbongi et al (Sanbongi et al., 2009). Most mutations were frame-shift mutations or deletion mutations. Gutiérrez et al (Gutiérrez et al., 2007). Have reported different mutations in the oprD gene, the most frequent mutations were frameshift mutations produced by one nucleotide insertions or deletions and point mutations leading to the creation of a premature stop. In a study performed by EL Amin et al (Amin et al., 2005). Sequence analysis revealed mutation of inactivation, including the insertion or deletion of one and two or more nucleotides and insertion sequences (IS). In investigating Performed by Wolter DJ et al (Wolter, Hanson, & Lister, 2004). PCR and sequence analysis revealed an interpolation of a large fragment in the oprD gene was known as IS elements that are not observed in this study. Jill Shen et al (Shen et al., 2015). Reported 96.5% (136/141) of the resistant isolates had mutations. Ninety-six strains had a small deletion in the OprD gene or multi-site mutations and 34 strains had a large deletion in the OprD gene, 6 strains had IS, and 4 strains had no mutation and showed a normal OprD2 gene. In this study, the insertion of one base was seen in ve strains. Twenty-ve strains had point mutations and 4 strains had no mutation.
Yoneyama et al (Yoneyama & Nakae, 1993). Reported a large deletion encompassing a region from upstream to downstream across the promoter region (from nucleotides 519-685) that prevented transcription of oprD and also deletion mutations were observed, including deletion an11 bp. Qinghui Sun et al (Sun et al., 2016). Have reported an insertion sequence element (ISRP10) that causes disrupt of the oprD gene and is seen in 96% of imipenem-resistant P. aeruginosa isolates. In a study performed by Yingjun Yan et al (Yan et al., 2014). The result of the analysis, indicatied that the 4-bp insertion in the oprD gene resulted in a frameshift in the OprD gene and imipenem resistance.
A different study conducted by Hussein Chalhoub (Chalhoub et al., 2016). DNA sequencing showed several mutations in the coding region oprD, but no mutation was observed in the promoter region of the gene. Reports had shown that mutation and inactivation or loss of an oprD gene, disruption in promoter and upstream region of oprD gene in P. aeruginosa strains are the major mechanisms that cause resistance to imipenem . This result was in accordance with the previous investigation of the clinical isolates of P. aeruginosa.
The results of this study show, increase in the resistance of P. aeruginosa to imipenem. Sequencing of the genomic region upstream of oprD in clinical strains revealed the point mutations in resistant strains. One isolate had a base substitution in the coding region oprD gene and this isolate had a point mutation leading to an amino acid change. All the imipenem-resistant isolates had mutations and Sensitive strains had no mutation. Judicious use of antimicrobials and controlled usage of imipenem may prevent P. aeruginosa from acquiring resistance to IPM. Neutralization of the mutation or the presence of a substance that can inactivate the mutation could lead to bacterial susceptibility to imipenem antibiotics.In our country, there is little information about the contribution of different mechanisms to imipenem resistance in these isolates, especially about oprD mutations in the upstream region of gene and promoter in imipenem-resistance isolates. Awareness of resistant mechanisms in P.aeruginosa isolates could help to regulate infection control strategies and to enhance the e cacy of imipenem for the treatment of infections due to these bacteria. Thus, there is a need to focus on intrinsic resistance mechanisms, especially Porin alteration which also confers signi cant imipenem resistance, it also suggests in the future other mechanisms such as gene expression and its relationship with the oprD mutations are evaluated and investigated in other isolates and other places. Availability of data and material The data are available. All data generated or analyzed during this study are included in this study.