This prospective cross-sectional multicenter study was started on July 2018 to the end of June 2019 at six hospitals centers across the country, Iran, including Sanandaj, Mashhad, Zanjan, Zahedan, Ahvaz, and Hamedan. Hospitalized and outpatients as well as healthcare-associated equipment (HAE) samples were considered for this study. One isolate per patient was included. Biochemical reactions including growth on MacConkey agar, oxidase reaction, sugar consumption in TSI medium, Oxidation-Fermentation (OF) activity, mobility, and growth at 42 °C were assessed to isolate Pseudomonas species.

PCR was used to species detection. DNA was extracted by boiling and 5΄-CCTGACCATCCGTCGCCACAAC-3΄ and 5΄-CGCAGCAGGATGCCGACGCC-3΄ primers were used [9]. The PCR process was performed, and PCR products were run in 1.5% agarose gel. The 222-bp band represented the target P. aeruginosa. P. aeruginosa ATCC 27,853 was used as a positive control in all experiments.

The antibiotic resistance profile of P. aeruginosa isolates was determined by Kirby-Bauer disk diffusion method on Müller-Hinton agar (Himedia, India) and the results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) criteria [10]. Colistin (10µgr), meropenem (10µgr), ceftazidime (30µgr), ciprofloxacin (5µgr), levofloxacin (5µgr), tobramycin (10µgr), amikacin (30µgr), gentamicin (10µgr), and piperacillin (100µgr) (BD, Sparks, MD, USA) were considered for this study. Colistin broth disk elution (CBDE) method was performed for colistin susceptibility [11]. P. aeruginosa ATCC 27,853 standard was used as quality control. Multidrug resistance (MDR), extensively drug resistance (XDR), and pan drug resistance (PDR) patterns were defined based on Magiorakos criteria [12]. According to the criteria, it is defined that MDR is acquired non-susceptibility to at least one agent belonging to three or more antimicrobial categories. As defined by XDR, it is non-susceptibility to all antimicrobial categories except two or fewer (i.e., bacterial isolates remain susceptible to only one or two antimicrobial categories), and as defined by PDR, it is non-susceptibility to all antimicrobial agents.

β-lactam/inhibitor (BLI) combined disks including ceftazidime/clavulanic acid and cefepime/clavulanic acid were considered for isolates that showed resistance phenotype to cefepime and ceftazidime. On the other hand, isolates that demonstrated carbapenem resistant or intermediate phenotype in susceptibility testing were subjected for modified carbapenem inactivation method (mCIM) [10, 13]. mCIM is only valid in Enterobacterials and P. aeruginosa. All the cephalosporin resistant isolates, regardless of positive combined disk result, as well as mCIM positive isolates, were further investigated to find β-lactamase genes by molecular PCR assay.

To detect β-lactamase genes, multiplex PCR was done, as previously described [14,15,16,17]. The list of primers including narrow-spectrum β-lactamases (NSBL), ESBLs, plasmid-borne ampC (PBampC), and carbapenemases are presented in Table 1. Ready-to-use MasterMix (Parstous, Iran) was used in 25 µl reaction. Strains with favorable genes in our repertoire that were collected from previous studies considered as positive control [18, 19]. Those without positive control in our collection, were sequenced through Sanger sequencing by ABI 3730XL sequencer. PCR products were run in 1.5% agarose and the gels were examined using ethidium bromide and visualized by UV.

To find the genomic relatedness between and amongst the isolates, REP-PCR was performed, as previously described [21]. Captured pictures were analyzed by GelJ (v. 1.3) software with dice tolerance 2.0 and UPGMA method to draw the dendrogram. To find clusters, 80% of similarity was considered [21].

MLST by seven housekeeping genes was performed for those isolates that were carbapenem-resistant [20]. PCR was performed on the extracted DNA of the isolates and PCR products were sequenced. The obtained sequences were embedded at www.pubmlst.org to identify the desired alleles and the sequence type of the strains was finally determined.