P. aeruginosa clinical isolates, materials, and equipment

In this cross-sectional study conducted in Iran, a country located southwest of the Asian continent, a total of 200 P. aeruginosa clinical isolates were utilized. These strains were collected from various specimens, including urine (n = 90), sputum (n = 55), wound (n = 28), blood (n = 26), and cerebrospinal fluid (CSF) (n = 1), obtained from patients referred to hospitals in Ardabil city, northwest of Iran. The hospitals included Imam Khomeini (n = 105), Alavi (n = 55), Imam Reza (n = 25), Bu-Ali (n = 6), Sabalan (n = 6), Fatemi (n = 2), and Ghaem (n = 1). The data collection period spanned from June 2019 to May 2023.

The initial identification of P. aeruginosa clinical isolates was performed using phenotypic standard laboratory tests, which included assessments of pigment production, colony morphology, oxidase, catalase, IMViC pattern, and Gram staining. Confirmation of the species was subsequently achieved using the polymerase chain reaction (PCR) with a specific species primer [3].

The most important materials used in our study were the Master Mix for PCR/ERIC-PCR and real-time PCR (Ampliqon, Denmark), primers (Metabion, Germany), TRIzol™ Reagent (Bio Basic, Ontario, Canada), cDNA synthesis kit (Yekta Tajhiz Azma, Tehran, Iran), Mueller Hinton agar (Conda, Pronasida, Spain), colistin sulfate salt powder (Sigma-Aldrich co, St. Louis, MO, ≥ 15,000 U/mg), antibiotic disks (Padtan Teb, Iran), Cetrimide agar (Conda, Pronasida, Spain), and Trypticase Soy Broth (TSB) (QUELAB/UK). Additionally, the following equipment was used: Eppendorf thermal cycler (Germany), LightCycler® System (Roche Diagnostics), NanoDrop 2000c Spectrophotometer (Thermo Scientific, USA), Agarose Gel Electrophoresis (Padideh Nojen Pars, Iran), and ELISA microplate reader (BioTek, USA).

Colistin agar test

The susceptibility pattern of P. aeruginosa clinical isolates to colistin was determined based on the agar dilution MIC (minimum inhibitory concentration) method on Mueller–Hinton agar as suggested by the Clinical and Laboratory Standards Institute (CLSI) [13]. For this purpose, 3–5 fresh P. aeruginosa colonies from Mueller Hinton agar plates were picked and transferred to 4–5 mL sterile saline to prepare 0.5 McFarland turbidity standards. Bacterial standard suspensions were diluted in saline (1:10). A 10 μL of each diluted bacterial suspension was poured onto a colistin agar plate. Colistin agar plates were prepared in required dilutions, i.e., 0.5–16 μg/mL. Incubation condition and length were maintained at 37 °C for 16–18 h. P. aeruginosa clinical isolates with MIC values ≥ 4 μg/mL were considered as resistant strains. A colistin-resistant Acinetobacter baumannii clinical isolate was used as the positive control (MIC = 16 μg/mL) (Ethics ID: IR.ARUMS.REC.1400.071).

The disk diffusion method was used to determine multiple drug resistance patterns (multidrug-resistant (MDR), extremely drug-resistant (XDR), and pandrug-resistant (PDR) strains) among colistin-resistant P. aeruginosa clinical isolates [14]. Furthermore, this method was employed to assess the resistance rates of colistin-resistant P. aeruginosa isolates against various antibiotics, including piperacillin (100 μg), piperacillin-tazobactam (100/10 μg), ceftazidime (30 μg), cefepime (30 μg), aztreonam (30 μg), imipenem (10 μg), meropenem (10 μg), gentamicin (10 μg), tobramycin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), norfloxacin (10 μg), and ofloxacin (5 μg), as per our previous study [3]. P. aeruginosa ATCC 27853 was used as a reference strain.

Biofilm formation assay

Evaluation of biofilm production among colistin-resistant P. aeruginosa isolates was performed by a colorimetric assay [15]. For this aim, of the 1:100 diluted suspensions of P. aeruginosa isolates which had grown in TSB mediums, 150 μL were inoculated into a sterile 96-well flat bottom plate and incubated at 37 °C for 24 h. The plate was washed with 200 μL of phosphate-buffered saline (PBS) (pH ~ 7.4) three times. Biofilm fixation was done with 100 μL of methanol (99%) for 15 min and then the wells were air-dried. 150 μL of crystal violet stain (1%) was added to wells for 20 min, unbound stain washed with water, and then bound stain released through 150 μL of acetic acid (33%). ELISA microplate reader was used to measure the optical density (OD) of wells at 590 nm. Colistin-resistant P. aeruginosa isolates were divided into four categories including no biofilm producer if the OD of a strain (ODs) was less or equal to the OD negative control (ODc), weak biofilm producer if ODc < ODs < 2 × ODc, moderate biofilm producer if 2 × ODc < ODs < 4 × ODc, and strong biofilm producer if 4 × ODc < ODs. P. aeruginosa ATCC 27853 and sterile TSB medium were used as positive and negative controls, respectively. All experiments are performed in triplicate.

Detection of colistin resistance genes

Molecular identification of the genes encoding efflux pumps (i.e., mexA, mexC, mexE, and mexY genes), TCSs (i.e., phoP, phoQ, pmrA, and pmrB genes), outer membrane porin (oprD gene), and mcr-1 gene, as well as genes involved in biofilm formation of P. aeruginosa (i.e., algD, pslD, pelF, Ppgl, and PAPI-1 genes) were performed by the PCR method. Used primers along with the PCR program for the detection of each gene were listed in Table 1. In brief, genomic DNA was extracted from the 200 P. aeruginosa clinical isolates by the boiling method [3] and confirmed by a spectrophotometer. Amplification of the genes was performed in a final volume of 15 μL (10 μL of Master Mix, with 3 μL of template DNA (50 ng/µL), and 2 μL of primers (10 μmol/L)) and then their presence was confirmed using the agarose gel electrophoresis and sequencing (Sanger method, Pishgam, Iran) techniques. It is worth mentioning that for some genes with non-specific bands on agarose gel, the values mentioned above along with the PCR conditions were changed. Finally, we employed the enterobacterial repetitive intergenic consensus (ERIC)-PCR method to assess the genetic relatedness among colistin-resistant P. aeruginosa isolates. In pursuit of this, amplification reactions were conducted in a final volume of 50 μL using the primers and ERIC-PCR program outlined in Table 1. Subsequently, the ERIC-PCR products were electrophoresed on a 2% agarose gel, and the resulting band patterns were analyzed using the Dice distance method for similarity and the UPGMA analysis method for clustering (GelQuest software version 3.3.5.0). ERIC-PCR band patterns exhibiting > 80% similarity were categorized as belonging to the same cluster [5].

Table 1 Used primers along with PCR/ERIC-PCR and qRT-PCR programsMutational analysis of the PhoPQ and PmrAB

Detection of colistin resistance-associated mutations among P. aeruginosa clinical isolates with multiple drug resistance was performed using the sequencing method. The PCR products of the phoQ and pmrB genes from P. aeruginosa clinical isolates MDR, XDR, and resistant to colistin were sent for sequencing. The nucleotide sequences were compared with colistin-susceptible P. aeruginosa reference strain ATCC 27853 using the BioEdit software (version 7.2.5). Additionally, an online data analysis service (available at https://web.expasy.org/translate/) was utilized to assess amino acid alterations.

Expression of the genes encoding efflux pumps, TCSs, and outer membrane porin

Expression levels of the mexA, mexC, mexE, mexY, phoP, pmrA, and oprD genes were determined among resistant isolates using the quantitative reverse transcription PCR (qRT-PCR) and specific sets of primers (Table 1). In brief, the total RNA of colistin-resistant P. aeruginosa isolates was extracted using the TRIzol™ Reagent. After confirming the quality and quantity of extracted RNAs (1 μg), cDNA synthesis was done according to the manufacturer’s instructions. The qRT-PCR of the genes was carried out under conditions presented in Table 1 and in a final volume of 15 μL (SYBR Green PCR Master Mix (7 μL), primers (2 μL, 10 μmol/L), cDNA (1 μg/μL), and DEPC-treated water (5 μL)). The 30S ribosomal rpsL gene was used as the normalizing gene. The expression levels of genes in colistin-resistant P. aeruginosa isolates were determined relative to their expression levels in P. aeruginosa ATCC 27853 using the 2−ΔΔCt method. Expression for each gene was assessed in duplicate.

Interpretation of the results of qRT-PCR was performed as follows: for the mexA and mexC genes; twofold, for the mexE gene; tenfold, and for the mexY gene; fourfold expression rates compared with the reference strain of P. aeruginosa ATCC 27853 were considered as overexpression [16]. For the oprD gene, the expression rate ≤ 30% relative to the reference strain was considered as down-regulation [17]. In addition, for the phoP and pmrA genes, expression levels higher than those of P. aeruginosa ATCC 27853 were considered as increased gene expression [12].