Phenotypic and Molecular Detection of IMP and SPM Metallo-Beta-Lactamases in Clinical Isolates of Carbapenem Resistant Pseudomonas aeruginosa

Objectives: Metallo-beta-lactamases play a major role in the resistance of Pseudomonas aeruginosa to carbapenems. The aim of this study was the phenotypic and molecular detection of IMP and SPM carbapenemase genes in 100 carbapenem-resistant clinical isolates of P. aeruginosa. The isolates identied using standard microbiological tests, and their antibiotic susceptibility pattern determined by disk agar diffusion (Kirby Bauer) method. Phenotypic identication of Metallo-beta-lactamase-producing strains assessed by the combined disk test (CDT). Then, PCR was used to detect the presence of IMP and SPM genes. Results: The highest and lowest levels of antibiotic resistance were observed against gentamicin (40%) and piperacillin-tazobactam (13%), respectively. Besides, 40 isolates (40%) had the Multi-drug Resistant (MDR) phenotype, while 5 (12.5%) MDR isolates were resistant to all antibiotics tested. The results of the CDT showed that among 43 carbapenem non-susceptible clinical isolates of P. aeruginosa, 33 (76.74%) isolates were Metallo-beta-lactamase-producing strains. Also, the frequency of the IMP gene was determined to be 9%, while none of these isolates carried the SPM gene. Due to the high prevalence of carbapenem-resistant and MDR P. aeruginosa in this study, routine antibiotic susceptibility testing and phenotypic identication of carbapenemase production by this bacterium are necessary for proper selection of antibiotics.


Introduction
Pseudomonas aeruginosa is an important opportunistic gram-negative pathogen causing almost 10% of nosocomial infections worldwide such as urinary tract infections, bacteremia, sepsis, and pneumonia in immunocompromised patients, especially in intensive care units (ICUs), as well as cystic brosis and burned patients (1)(2)(3). Today, one of the most important complications related to this bacterium in developing countries is the emergence of multidrug resistant strains (4). Carbapenems are the last line treatment option for the infections caused by this bacterium, but recently resistance to carbapenems has been increased resulting to high mortality, especially in immunocompromised patients (5). Decreased expression of outer membrane proteins, increased expression of e ux systems, and secretion of beta-lactamases are the most important mechanisms of antibiotic resistance in clinical isolates of P. aeruginosa (6). One of the most important mechanisms of carbapenem resistance in P. aeruginosa is the production of Metallo-beta-lactamases (MBLs). These enzymes belonged to the class B beta-lactamases requiring the zinc for activity and can hydrolyze a wide range of beta-lactamases (6,7).
MBL-producing P. aeruginosa rst reported in 1991 in Japan (8). The MBLs can hydrolyze penicillins and cephalosporins, and their encoding genes are often propagated horizontally among Gram-negative bacteria due to the presence of mobile genetic elements such as transposons and integrons (6,9). The IMP (Imipenemase) and SPM (Sao Paulo Metallo-beta-lactamase) genes are the most clinically common MBLs (8). The SPM gene rst identi ed in P. aeruginosa isolated from a blood culture sample of a 4-year-old girl with leukemia in Brazil (10), and the IMP gene rst identi ed in Japan (6). Prompt diagnosis and accurate reporting of the presence of these genes in hospitals can lead to better and more effective control of carbapenem-resistant strains and eradication of nosocomial infections. Therefore, due to the importance of the presence of Metallo-beta-lactamases in P. aeruginosa, this study aimed to phenotypic identify MBLs and molecular assessment of the presence of IMP and SPM genes in the clinical isolates of carbapenem-resistant P. aeruginosa in teaching and treatment hospitals of Mazandaran province, north of Iran.

Patients and bacterial isolates
In this descriptive-analytical study, 100 non-repetitive Pseudomonas aeruginosa isolated from different clinical samples collected from hospitalized and outpatients from May 2018 to June 2019. The clinical isolates identi ed using standard microbiological methods and biochemical tests (3) and con rmed by the API (Analytical Pro le Index) kit (France, BioMérieux, Lyon).

Antibiotic susceptibility testing
The antibiotic susceptibility pattern of the isolates was determined by the disk agar diffusion method (Kirby-Bauer) according to the instructions of the Clinical and Laboratory Standards Institute (CLSI) (11). In this test, we used the Pseudomonas aeruginosa ATCC 27853 as the control strain. The antibiotics studied included imipenem (10 µg), meropenem (10 µg), doripenem (10 µg), ceftazidime (30 µg), Aztreonam (30 µg), piperacillin-tazobactam (100 − 10 µg), cipro oxacin (5 µg), and gentamicin (10 µg) (MAST Diagnostic Co., UK). Also, the agar dilution test used to investigate the susceptibility pattern of the clinical isolates against colistin according to the instructions of the Clinical and Laboratory Standards Institute (CLSI) (11). Pseudomonas aeruginosa ATCC 27853 chosen as the control strain in this test.
Phenotypic identi cation of Metallo-beta-lactamase producing strains Combined Disk Test (CDT), using imipenem (10 µg) alone and imipenem-EDTA (10-750 µg), was used to phenotypic detection of the presence of MBLs in clinical isolates of P. aeruginosa. An increase in the diameter of the growth inhibition zone greater than or equal to 7 mm around the imipenem-EDTA combined disk compared to the imipenem disk alone indicates the production of MBLs in this test (12).
Extraction of the bacterial genome and ampli cation of the IMP and SPM genes by PCR The genomic DNAs of P. aeruginosa isolates extracted by DNA extraction kit (Bioneer, South Korea). Ampli cation of the IMP and SPM genes using speci c primers as IMP-F:GAAGGCGTTTATGTTCATAC, IMP-R:GTATGTTTCAAGAGTGATGC (13), and SPM-F:AAAATCTGGGTACGCAAACG, SPM-R:ACATTATCCGCTGGAACAGG (14) done by thermocycler (Bio-Rad, USA). The condition of the PCR tests was as follows: initial denaturation at 94 °C for 2 minutes, and 30 cycles including denaturation at 94 °C for 30 seconds, annealing of the primers at 57 °C for 30 seconds, and extension of the DNA fragments at 72 °C for 45 seconds, and a nal extension of the fragments at 72 °C for 10 minutes.

Patients and bacterial isolates
Among 100 Pseudomonas aeruginosa clinical isolates in this study, 61 of them collected from men. The age of the patients varied from a few days old to 91 years old, and the mean age range of the patients was 48.65 ± 13.13. Also, the ICU with 40 cases had the highest frequency of the isolates (Table 1), while 29, 26, 20, and 11 isolates collected from urine, sputum, wound, and other clinical samples, respectively. Besides, 14 isolates were related to outpatients, while the highest positive culture (71.4%) among outpatients belonged to the urine samples (Table 1).  isolates observed against gentamicin, while 38 (95%) isolates were resistant to this antibiotic. On the other hand, the highest antibiotic resistance observed in wound and catheter isolates, while P. aeruginosa isolated from feces and plantar secretions were resistant to all tested antibiotics. Also, only resistance to carbapenems was observed among the tracheal isolates of P. aeruginosa (Table 5).    and BICU had a positive combined disk test. However, none of the PICU, CCU, and oncology isolates were CDT positive, while 2/4 (50%), 2/6 (33.3%), 1/2 (50%), 3/7 (42.8%), 2/4 (50%), 1/4 (25%), and 3/6 (50%) of the P. aeruginosa isolated from Internal Medicine, Pediatrics, Women, Surgery, Neurology, Men, and Burn wards detected as CDT positive isolates, respectively.
On the other hand, the results of the PCR test showed that among 43 carbapenem non-susceptible isolates, only 3 (6.97%) isolates contained the IMP gene ( Figure S1), while none of the isolates carried SPM gene ( Figure S2).
All three isolates carrying the IMP gene were resistant to meropenem and doripenem, while 2 (66.6%) isolates carrying the gene were resistant towards imipenem. On the other hand, 1 (33.3%) IMP positive isolate detected as resistant against aztreonam as well as piperacillin/tazobactam. Interestingly, all three isolates carrying this gene showed resistance to ceftazidime and gentamicin, while resistance against cipro oxacin was observed in 2 (66.6%) IMP containing isolates.

Discussion
The production of carbapenem hydrolyzing enzymes as well as producing a polysaccharide matrix in respiratory infections is one of the most signi cant mechanisms for resistance to carbapenems in P. aeruginosa strains (14,15). However, not only the sputum isolates of this study were resistant to all antibiotics tested, but also 11 (42.3%) of them were detected as MDR. On the other hand, our clinical isolates had a relatively low resistance rate (< 40%) against all antibiotics tested, except gentamicin, as well as other Iranian studies (3,16). However, the studies conducted by Radan et al. in 2016, and Mirsalehian et al. in 2017, reported the higher antibiotic resistance rate (5,13), may be due to the type of samples, as all isolates collected from burn sections. Since patients with burns may be exposed to broad-spectrum antibiotics pressure, high antibiotic resistance rate observes in these wards (13). In another study conducted in Hamedan, as in our study, the lowest antibiotic resistance of the P. aeruginosa clinical isolates was reported against piperacillin-tazobactam (17). It seems that this antibiotic is still one of the effective drugs against this bacterium in Iran.
In the present study, the resistance rate against imipenem (28%) was similar to other studies in Iran (6,18). On the other hand, the resistance towards imipenem and doripenem in the present study was less than meropenem.
According to our assessment, the use of meropenem for the treatment of infections caused by this bacterium in our investigated hospitals was more than imipenem, while the doripenem is not widely used in the treatment of these infections. In another Iranian study conducted in 2017, the resistance to imipenem (8.4%) and meropenem (9.5%) was higher than that of penicillin (7.4%) (3). Also, in some studies, colistin has reported as an effective drug against multidrug-resistant as well as carbapenem-resistant P. aeruginosa (13), while 26% of our isolates detected as colistin-resistant. However, 8 (20%) MDR isolates in our study were resistant to colistin. The rate of colistinresistance in clinical isolates of P. aeruginosa in other studies conducted in Iran and Iraq have reported as 11% and 18.2%, respectively (17,19).
In the present study, out of 43 carbapenem-resistant isolates, 33 (76.74%) isolates were CDT positive consistent with some studies conducted in Iran and other countries (12,20). According to the recent studies, we are witnessing an increasing prevalence of MBLs in clinical isolates of P. aeruginosa. The rate of MBLs in Kerman was negative in 2008 (21), while in 2015, it was 48% in Isfahan (7), however, in another study conducted in India, 37.5% of the P. aeruginosa clinical isolates have reported as MBL-producer (22). IMP is one of the most important Metallo-betalactamases causing resistance to beta-lactams and carbapenems (23), whereas in this study, all three isolates carrying the IMP gene were resistant to the carbapenems. In an Iranian study conducted in 2019, 4.7% of the isolates contained the IMP gene (6), while in another study conducted in India, out of all CDT positive isolates, 3% of them were carrying the IMP gene (20). Studies in Asian, African, and North American countries such as South Korea, India, Egypt, and Canada (9,20,24,25) have reported an IMP gene prevalence of between 2 and 8%, indicating that the prevalence of this gene is low as well as our results.
On the other hand, some studies in Iran and other countries reported no SPM positive P. aeruginosa (7,8,26), however, Azimi et al. in 2018 reported a prevalence of 5.6% and 15.6% of the SPM and IMP gene, respectively, while all of their isolates collected from children admitted to the burn ward (27). The absence of the SPM gene and the low prevalence of the IMP gene in this study indicate that other mechanisms such as increased expression of e ux pumps, decreased expression of outer membrane proteins, and production of other carbapenemases may have involved in the development of carbapenem-resistant strains in this region. Considering the importance of carbapenem-resistant, colistin-resistant, and MDR isolates of Pseudomonas aeruginosa, routine antibiotic susceptibility testing and using more modern phenotypic tests such as Modi ed Hodge test and CarbaNP test are essential for the initial identi cation of carbapenemase-producing P. aeruginosa.

Limitations
The present study had some limitations. Lack of information about other carbapenemase encoding genes belonging to MBL group and genetic relationship between the resistant strains are not determined.