254 J Contemp Med Sci | Vol. 8, No. 4, July-August 2022: 254–258 Original Molecular Analysis of Carbapenem Resistant Genes in Pseudomonas aeruginosa isolated from Baghdad Hospitals Ali H. Salih, Adnan H. Aubaid*, Ghada B. Ali Department of Medical Microbiology, Faculty of Medicine, University of Al-Qadisiyah, Al-Qadisiyah, Iraq. *Correspondence to: Adnan H. Aubaid (E-mail: adnan.uobeed@qu.edu.iq) (Submitted: 28 April 2022 – Revised version received: 12 May 2022 – Accepted: 26 May 2022 – Published Online: 26 August 2022) Abstract Objectives: This study aimed to molecular investigation of prevalence the carbapenem-resistant genes in P. aeruginosa in isolates collected from Baghdad hospitals. Methods: In a cross-sectional manner, P. aeruginosa were isolated and identified from patients who attended to Hospital in Baghdad city during the period of December 2021 to June 2022. Genotypic characterization of 16SrRNA gene, plasmid profile, exoA gene, carbapenem resistance gene were tested. Results: Diagnosis of P. aeruginosa isolates was confirmed genotypically via the amplification of 16SrRNA gene with 1504 bp by using PCR amplification of genes encoding Ambler class B MBL (blaIMP, blaVIM, IMI, blaNDM, blaSPM-1, and blaGIM), and Ambler class D carbapenemase (blaOXA-23, blaOXA-24, blaOXA-40, blaOXA-48, blaOXA-50, blaOXA-51 and blaOXA-58) were performed. The carbapenem resistant isolates were also evaluated for the presence of class D carbapenemase (blaOXA 50), genes by PCR. Conclusion: This study proved that P. aeruginosa isolated had carbapenem-resistant genes that strongly correlated with antibiotic resistance according to phenotypic and genotypic characterization. Keywords: Carbapenems, Pseudomonas aeruginosa, Hospitals, Iraq ISSN 2413-0516 Introduction Pseudomonas aeruginosa is a ubiquitous, Gram-negative bac- terium and versatile opportunistic pathogen, which is consid- ered a significant reason for an ever-widening array of various life-threatening infections.1 Over the past decades, the emer- gence and dissemination of P. aeruginosa and Enterobacte- riaceae, which are resistant to carbapenems, which are the broadest spectrum agents of the β-lactam group, has become apparent as an urgent threat to public health. The finding P. aeruginosa has intrinsic resistance to numerous antimicrobial agents and also easily acquires resist- ance to many antibiotics, including carbapenems resistance is an ominous development that challenges this last-resort anti- biotic. Unfortunately, carbapenems resistant P. aeruginosa has now emerged and is disseminating worldwide.2-3 Resistance to carbapenems in P. aeruginosa can be mediated by several mechanisms, including MexAB-OprM, AmpC, decreased outer membrane permeability, up-regulation of the efflux pumps, hyper production of a chromosomal AmpC-type cephalosporinases or the production of carbapenemases OprD.4 There is scarce information available on the distribution of MBLs producing P. aeruginosa isolates in Baghdad hospi- tals. Therefore, immediate determination of carbapene- mases and other mechanisms creating isolates is main to avoid the spread of P. aeruginosa inside and between hospi- tals and to correctly treat infections caused by this. The study aimed to identify the variations in phenotypic, geno- typic characteristics and antibiotic resistance profile of Pseudomonas aeruginosa isolated from inpatients in Baghdad hospitals. Additionally, the use of carbapenems has increased markedly during the past few years in Baghdad city. The current study suspected that increased use of these antibiotics could cause the selection of isolates resistance to carbapenems. Materials and Methods Isolation and Identification of P. aeruginosa A total of 2000 clinical samples were collected from inpatients of many hospitals in Baghdad city during the period from December 2020 to June 2021, which included: diabetic foot (50), otitis media (550), lower respiratory tract (150), urinary tract (725), wound (200), Blood and burns (250). The clinical samples were transported to the laboratory without delay. All samples were cultivated, by using the standard loop of urine and sterile swabs of other samples, on the blood agar, Mac- Conkeys agar Pseudomonas chromogenic agar, and cetrimide agar as selective media. P. aeruginosa and incubated overnight at 37°C for 18–24 hours. Initial diagnosis of isolates was made on the basis of Gram’s staining of culture, colonial morphology on different media, hemolysis on blood agar, pigment produc- tion, odor in cultures, size, edge, and oxidase test. Suspected Pseudomonas colonies were further identified to species level using routine biochemical tests and selective culture media.5 In addition to these tests, the P. aeruginosa isolates were also confirmed biochemically with the Vitek-2 automated system and by 16SrRNA as a molecular method. Oligonucleotide Primer Sequences Used for PCR Amplification 1. Specific primer sequences of 16SrRNA gene were used to confirm the identification of P. aeruginosa by PCR according to Jiang et al., 20066 provided by Alpha DNA company (Canada) and prepared according to the instruc- tions of the supplied company, as shown in Table 1. 2. Oligonucleotide primer for detection of carbapenem resistance genes in P. aeruginosa. These primers were pro- vided by Macrogen company from South Korea are listed in Table 2. 255J Contemp Med Sci | Vol. 8, No. 4, July-August 2022:254–258 A.H. Salih et al. Original Molecular Analysis of Carbapenem Resistant Genes in Pseudomonas aeruginosa isolated from Baghdad Hospitals PCR Protocols for Detection of Carbapenem Resistance Genes PCR protocols for detection of carbapenem resistance genes. An oligonucleotide primer was prepared depending on the manufacturer’s instruction by dissolving the lyophilized sample with nuclease-free water after rotating down briefly. A working primer tube was prepared by diluting it with nuclease-free water. The final pico-moles depended on the procedure of each primer. The PCR tubes were placed into a thermocycler and the right PCR cycling program parameters conditions were installed as in Table 3. PCR was used for detecting P. aeruginosa, the mixture of 25 μl consisted of 12.5 μl of GoTaq Hot Star master mix (which contains Taq DNA poly- merase, dNTPs, mgcl2, and reaction buffer at the optimal con- centration for efficient amplification of DNA templates by PCR), 5 μl DNA template (20 ng.), 1 μl of each forward and reverse primer (10 pmol.) 5.5 μl of nuclease-free water to com- plete the amplification mixture volume. The PCR tubes con- taining the mixture were transferred to preheated Thermo cycler under sterile condition. All requests, technical and preparations of agarose gel electrophoresis that were used for the detection of PCR prod- ucts were done according to Sambrook and Russel.8 The PCR products separated in 1.5% agarose gels (after staining with 0.5 mg/ml Ethidium bromide) were visualized using a gel ultravi- olet transilluminator system. The positive results were distin- guished when the PCR product base pairs were equal to the base pairs of the DNA ladder. Results and Discussion According to the results of the present study, the overall count constitutes a total of 100/2000 samples, P. aeruginosa Table 1. Oligonucleotide primer sequences of 16SrRNA gene in P. aeruginosa used for confirmatory identification Product sizeReferencePrimer sequences (5’→3’)Name of primer bp1504(Jiang et al., 2006)6AGA GTT TGA TCM TGG CTC AG F16SrRNA CGG TTA CCT TGT TAC GAC TTR Table 2. Oligonucleotide primer for detection of carbapenem resistance genes in P. aeruginosa Primer Gene name Sequence (5’→3’) Product size Genbank reference NDM bla-NDM F CAGTCGCTTCCAACGGTTTG 529 bp MF379690.1 R ATCACGATCATGCTGGCCTT IMP bla-IMP F CTTTCAGGCAGCCAAACCAC 371 bp Design to this study R TGGGGCGTTGTTCCTAAACA VIM-1 bla-VIM-1 F TCCACGCACTTTCATGACGA 503 bp Design to this study R AAGTCCCGCTCCAACGATTT GIM blaGIM F AGAACCTTGACCGAACGCAG 909 bp Design to this study R GCACCAGTTTTCCCATACAG OXA-48 blaOXA-48 F TTG GTG GCA TCG ATT ATC GG 744 bp Design to this study R GAG CAC TTC TTT TGT GAT GGC OXA-40 blaOXA-40 F CACCTATGGTAATGCTCTTGC 491 bp (Woodford et al., 2006)7 R GTGGAGTAACACCCATTCC OXA-50 blaOXA-50 F AATCCGGCGCTCATCCATC 869 bp (Woodford et al., 2006)7 R GGTCGGCGACTGAGGCGG SPM blaSPM-1 F CCTACAATCTAACGGCGACC 650 (Woodford et al., 2006)7 R TCGCCGTGTCCAGGTATAAC Oxa58 bla Oxa58 F AAGTATTGGGGCTTGTGCTG 599 (Woodford et al., 2006)7 R CCCCTCTGCGCTCTACATAC Oxa23 bla Oxa23 F GATCGGATTGGAGAACCAGA 501 (Woodford et al., 2006)7 R ATTTCTGACCGCATTTCCAT Oxa24 bla Oxa24 F GGTTAGTTGGCCCCCTTAAA 246 (Woodford et al., 2006)7 R AGTTGAGCGAAAAGGGGATT Oxa 51 blaOxa 51 F TAATGCTTTGATCGGCCTTG 353 (Woodford et al., 2006)7 R TGGATTGCACTTCATCTTGG IMI blaIMI F CCA TTC ACC CAT CAC AAC 440 (Woodford et al., 2006)7 R CTA CCG CAT AAT CAT TTG C 256 J Contemp Med Sci | Vol. 8, No. 4, July-August 2022: 254–258 Molecular Analysis of Carbapenem Resistant Genes in Pseudomonas aeruginosa isolated from Baghdad Hospitals Original A.H. Salih et al. Table 3. Programs of PCR thermocycling conditions for detection of carbapenem resistance genes Genes Temperature (C˚)/Time Cycle number Initial denaturation Cycling condition Final extension Denaturation Annealing Extension NDM 95°C/5 min 95°C/20 sec 56°C/30 sec 72°C/40 sec 72°C/5 min 35 IMP 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 VIM-1 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 GIM 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 SPM 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 OXA-48 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 OXA-40 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 OXA-50 95°C/5 min 95°C/20 sec 58°C/30 sec 72°C/40 sec 72°C/5 min 35 OXA-51 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 OXA-23 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 OXA-24 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 SPM 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 IMI 95°C/5 min 95°C/20 sec 50–60°C/30 sec 72–40°C/40 sec 72°C/5 min 35 representing (5%) of all the collected samples in this study. It’s well known that P. aeruginosa considers an important nosoco- mial pathogen in many medical centers throughout the world and a source of infections in any part of the body. Also, this bacterium is able to cause infection in healthy individuals at a low rate and creates a serious public health disaster resulting in an enormous burden of morbidity, and mortality in both developing and developed countries.9 One of the reasons for the high pathogenicity of P. aeruginosa is the intrinsic high resistance to several antibiotics, as well as the development of multiple drug resistance.10 The results of amplification of 16SrRNA gene with DNA extracted from one hundred P. aeruginosa isolates showed positive PCR product with amplicon size 1504 base pair (Figure 1). These results agreed with Alornaaouti11 who used the 16SrRNA gene which is considered one of the important gene to confirm the identification of P. aeruginosa and other bacterial species because have hyper constant sequencing and play a basic role in molecular identification and classification, also that can provide species-specific signature sequences useful for bacterial identification all type of bacteria.12 Detection of Class B (MBL) and Class D Carbapenemase The production of carbapenemases is of the utmost concern and became the mechanism of greater relevance towards car- bapenem resistance due to the growing enzyme diversity. These enzymes have high versatility, as they are characterized by a very wide hydrolytic spectrum and affect almost all β-lactams, with the exception of monobactams.13 P. aeruginosa isolates producing carbapenemases are also associated with XDR phenotype. Accordingly, the detection of carbapene- mases production in P. aeruginosa is important not only for the adequate selection of antibiotic therapy but also for hos- pital epidemiology surveillance and infection control. In recent reports in Najaf, the most common carbapene- mases are the MBL and OXA variant enzymes of Ambler class D.14-16 Class B acquired MBL is one of the most important enzymes including IMP, VIM, SPM, NDM, SIM and GIM, which expose huge-level of resistance against carbapenem.17–19 In this study, the PCR technique was used to identify the bla-IMP, bla-VIM, bla SPM, bla-NDM, bla-SIM and bla-GIM genes only (Figure 2), while the bla-AIM, bla-KHM, bla-DIM and bla-FIM genes was not included due to very few occur- rences in adjacent nations to Iraq. Furthermore, as the present results revealed, two isolates carry the NDM encoding gene in addition to coexisting with the OXA-50 encoding gene. The co-harboring of two carbap- enemase genes in P. aeruginosa isolates has been reported in several studies worldwide.19–21 This observation led to the emergence of a new drug- resistant model for P. aeruginosa. However, this result is of great concern and has shed light on the fact that NDM- producing XDR P. aeruginosa is now alarmingly on the increase in Baghdad hospitals. Although only three carbapenem-resistant P. aeruginosa isolates were identified to carry this gene in the present study, it is of concern as blaNDM producers may be disseminated rapidly in Baghdad hospitals and this finding implies that Fig. 1 Agarose gel electrophoresis for 16SrRNA gene amplicons. 1% agarose (100 min at 100 volt/50 mAmp). Lane:1 (M 100 bp ladder) Lane 1-10 local isolates, PCR product 1504 bp size. 257J Contemp Med Sci | Vol. 8, No. 4, July-August 2022:254–258 A.H. Salih et al. Original Molecular Analysis of Carbapenem Resistant Genes in Pseudomonas aeruginosa isolated from Baghdad Hospitals several new blaNDM cases will be found in the near future. Consequently, the detection of five NDM-positive isolates in this study suggested possibilities of spread via its high rate of genetic transfer among pathogenic bacteria in Baghdad hospi- tals, or possibilities to human factors such as hygiene and international tourists. It is believed that the emergence of blaNDM carried isolates in Baghdad may have been a result of the introduction of NDM isolates via increasing medical tourism of Iraqi patients to the Indian subcontinent. At present, the prevalence of NDM-1 has increased signif- icantly throughout the world and has been identified mostly in Asia,19 Europe, Africa.20 Therefore, the detection of NDM harboring P. aeruginosa isolates in this study indicates the immediate importance of the establishment of surveillance to prevent nosocomial infections and dissemination of NDM in Baghdad hospitals.21-24 In the study of these genes blaSPM, IMP, VIM1, GIM, IMI, OXA-48, OXA-40, OXA-58, OXA-23, OXA-24, and OXA51 were negative results (Figure 3). In further studies conducted in Najaf, 14 showed that two (5.9%) out of 34 carbapenem-resistant P. aeruginosa isolates were SPM-type positive. Interestingly, the present study showed that OXA-50 was the most frequent carbapenemase identified in XDR P. aeruginosa isolates (98.%). However, the occurring Class-D oxacillinase OXA-50 was shown to be expressed constitutively in P. aeruginosa (Figure 4). A similar enhancement in the prevalence of blaOXA-50 carrying P. aeruginosa isolates has been noticed earlier in a recent local study reported by Rasool et al.,15 who found that the majority of the carbapenems resistant P. aeruginosa iso- lates carried the blaOXA-50 gene (53.8%). While Al-Janahi14 reported that only 8.8% of the carbapenem resistant P. aeruginosa isolated from Najaf hospitals were harbored blaOXA-50. Ethics Consideration This study is in accordance with the ethics committee of Al-Diwaniaya teaching hospital, Iraq. A verbal agreement was Fig. 2 Agarose gel electrophoresis for NDM gene amplicons. (1.5% agarose,100 min at 100 volt/50 mAmp). Lane:1 (M 1500 bp ladder) Lane 4,11 and 28 local isolates, Positive PCR product 528 base pair. Fig. 3 Agarose gel electrophoresis for OXA-50 gene amplicons. (1.5% agarose (100 min at 100 volt/50 mAmp). Lane:1 (M 1500 bp ladder) Lane 1,2,3,4,5,6,7,8,10,11,12,13,14,15,17,18. Positive PCR product 869 base pair, and 2 local isolates (9,16) Negative OXA-50 gene. Fig. 4 Agarose gel electrophoresis for OXA-50 gene amplicons. (1.5% agarose, 100 min at 100 volt/50 mAmp). Lane:1 (M 1500 bp ladder) Lane 4,17,4 and 2 local isolates, Positive PCR product 869 base pair. obtained from participants in the study of the relative’s pre- taking samples. Conflict of Interest No known conflict of interest correlated with this publication. Funding This research did not receive any grant from agencies in the public, commercial, or not-for-profit sectors. Availability of Data and Materials The data used and/ or analyzed throughout this study are available from the corresponding author on reasonable request.  References 1. Rashid, A., Akram, M., Kayode, O.T. and Kayode, A.A. (2020). Clinical features and epidemiological patterns of infections by multidrug resistance Staphylococcus aureus and Pseudomonas aeruginosa in patients with burns. Biomed. J. Sci. Tech. 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