Phenotypic and molecular detection of metallo-β-lactamase-producing Pseudomonas aeruginosa isolates from patients with burns in Tehran , Iran

Introduction: Health care-associated infections caused by metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa are a significant growing concern in patients with burns worldwide. The aims of this study were to determine the antibiotic susceptibility of and detect the presence of MBLs among P. aeruginosa isolates and assess their clonal relationship using enterobacterial repetitive intergenic consensus (ERIC)-PCR. Methods: Non-duplicated clinical isolates (160) of P. aeruginosa were collected from patients with burns at the Motahari Hospital in Tehran, Iran. All isolates were identified using standard laboratory methods and further characterized for antimicrobial susceptibility. Any carbapenem-resistant isolates were then examined for MBL production by the E-test and MBL-encoding genes were detected by PCR. The clonal relatedness of MBLproducing isolates was assessed by ERIC-PCR. Results: For multidrug-resistant isolates, the highest rates of susceptibility were observed for colistin 160 (100%), polymyxin B 160 (100%), and ceftazidime 32 (20%). In total, 69 (43.7%) isolates were identified as MBL producers. Twenty-eight (17.5%) isolates were positive for the blaVIM-1 gene followed by the blaIMP-1 (15.6%) and blaSPM-1 (5.6%) genes. ERIC-PCR revealed three separate genotypes, where type A (76.8%) was the most prevalent, followed by B (20.3%), and then C (2.9%). Conclusions: Our present study found that the blaIMP-1 and blaVIM-1 genes were present at a significant frequency and also detected the blaSPM-1 gene in P. aeruginosa isolates for the first time, highlighting the need for establishing suitable infection control measures to successfully treat patients and prevent further spread of these resistant organisms among patients with burns.


INTRODUCTION
Burn infections are a major health concern due to increasing morbidity and mortality worldwide, especially within developing countries 1 .Pseudomonas aeruginosa is regarded as one of the most important opportunistic pathogens among aerobic non-fermentative bacterial species and causes nosocomial infections in patients with burns 2 .The appearance of resistance to routinely used antimicrobial agents among pathogens associated with burn wounds restricts the accessible therapeutic options available to effectively treat these infections, which is frequently associated with a high mortality rate 3 .Carbapenems have been effective in the treatment of severe infections caused by P. aeruginosa.However, resistance to carbapenems has been reported to now be on the rise throughout the world 4,5 and several carbapenem-hydrolyzing β-lactamases have been identified in P. aeruginosa.The most common mechanism of resistance to carbapenems involves the acquisition of carbapenem-hydrolyzing β-lactamases belonging to Ambler classes B (metalloenzymes) and D (oxacillinase) 6 .Metallo-β-lactamases (MBLs) can hydrolyze a wide variety of β-lactams, including penicillins, cephalosporins, and carbapenems 7 .Various types of MBL genes have been identified in P. aeruginosa, including the imipenemase (IMP), Verona integron-encoded metallo-β-lactamase (VIM), São Paulo metalo-β-lactamase (SPM), Germany imipenemase (GIM), Seoul imipenemase (SIM), and New Delhi metallo-β-lactamase (NDM) types.IMP and VIM are the most prevalent types of acquired MBLs 8,9 .IMP-type enzymes initially emerged in early 1991 in Japan, while the VIM type was initially reported in Italy.However, both types have been increasingly reported in other parts of the world 10 .SPM-1, i.e., Sao Paulo MBL, was initially identified in a clinical isolate of P. aeruginosa in Brazil in 1997, while the New Delhi MBL-1 (NDM-1) type of MBL was found in Klebsiella pneumoniae isolated from a Swedish patient with a previous history of hospitalization in India 8,11 .The genes encoding these enzymes are usually carried on integrins with some located on transferable plasmids that have the potential to horizontally spread among different bacterial species 12 .Several non-molecular techniques have been used to study these zinc-dependent enzymes with the application of chelating agents, such as ethylenediaminetetraacetic acid (EDTA), to inhibit the enzymatic activity 13 .There are no standard phenotypic confirmatory tests for the detection of MBLs among P. aeruginosa isolates 14 .Molecular methods are necessary to confirm the presence of MBL genes in clinical isolates and are also considered proper tools for screening.Polymerase chain reaction (PCR) detection has high sensitivity and reliability for detection of MBL genes among MBL-producing P. aeruginosa isolates 15 .
The Motahari Burn Hospital is located in Tehran and is the most frequently referred burn center, accepting the majority of referrals from a number of medical centers in Iran.Determining the prevalence of MBLs in P. aeruginosa isolates from patients with burns is of particular importance because the appearance of resistance in such clinical isolates is usually coupled with the emergence of higher rates of treatment failure and mortality.At present, little is known about the molecular epidemiology of MBL-producing P. aeruginosa isolates in the Motahari Burn Hospital.Therefore, this current study aimed to characterize the antibiotic susceptibility of and detect the presence of bla IMP-1 , bla IMP-2, bla VIM-1 , bla VIM-2 , bla SPM-1 , bla GIM , bla SIM , and bla NDM-1 genes among P. aeruginosa isolates and assess the clonal relationship of these isolates using enterobacterial repetitive intergenic consensus (ERIC)-PCR.

Study design and bacterial isolates
This was a cross-sectional study in which a total of 160 nonduplicated P. aeruginosa isolates were collected from patients with burns referred to the Motahari Hospital in Tehran, Iran during the 15 months from March 2015 to June 2016.Bacterial species were identified using standard laboratory methods, including Gram staining, oxidase, motility, catalase, citrate, and oxidation-fermentation tests, and assessments of growth at 37 and 44°C 16 .Species identity was confirmed by detection of oprI and oprL genes as described previously 17 .The isolates were stored at -70°C in trypticase soy broth containing 20% glycerol and subcultured twice prior to testing.

Ethical considerations
This study was approved by the Ethics Committee of Qazvin University of Medical Sciences (code IR.QUMS.REC.1394.726).Written informed consent was obtained from all individuals enrolled in this study.

MBL E-test
The isolates that were fully or intermediately resistant to imipenem and/or meropenem were screened for MBL production using the MBL E-test (AB Biodisk, Solna, Sweden).MBL E-test strips were double-sided and contained a sevendilution range of imipenem (4-256 µg/ml on one side and 1-64 µg/ml on the other) in the presence of a constant concentration of EDTA.A reduction in the imipenem maximum inhibitory concentration by ≥3 log 2 dilutions in the presence of EDTA or a maximum inhibitory concentration ratio of imipenem/imipenem plus EDTA of ≥8 was considered MBL positive 19 .

Detection of MBL-encoding genes
Carbapenem-resistant isolates were tested by PCR for MBL-encoding genes, including bla IMP-1 , bla IMP-2 , bla VIM-1 , bla VIM-2 , bla SPM-1 , bla GIM , bla SIM , and bla NDM-1 , using the specific primers listed in Table 1 [20][21][22] .Total genomic deoxyribonucleic acid (DNA) was extracted using a commercial extraction kit (Bioneer Company, Korea).PCR amplification was performed in a thermocycler (Applied Biosystems, USA) as follows: initial denaturation at 96ºC for 10 min, followed by 30 cycles of 96ºC for 1 min, the annealing temperature for each primer set indicated in Table 1 for 1 min, and 72ºC for 1 min, and a final extension step at 72ºC for 10 min.The amplification reaction was prepared in a total volume of 25μl containing 1U of Taq DNA polymerase 2X Master Mix, 1.5mM MgCl 2 (Ampliqon, Denmark), 0.5μM forward primer, 0.5μM reverse primer, 9μl nuclease free water, and 2.5μl DNA template (50pg concentration).PCR products were separated by agarose gel electrophoresis at 100 volts for 40 min, stained with ethidium bromide, and visualized on an ultraviolet (UV) transilluminator (UVtec, UK).The PCR products were then purified and both strands directly sequenced by Macrogen Company (Seoul, South Korea) to confirm the presence of the detected genes.Finally, online sequence alignment and analysis were conducted using the National Center for Biotechnology Information BLAST program (http://blast.ncbi.nlm.nih.gov/Blast) to compare the data found in our study with that available in the database library.

Clonal analysis by ERIC-PCR
ERIC-PCR was performed to detect any clonal relationships between the isolates.The final PCR reaction volume was 25μl and contained 2.5µl 10X PCR buffer, 0.5µl deoxynucleotide triphosphate (dNTP) Mix (10mM), 5µl MgCl 2 , 25pM primer F, 25pM primer R, 2U Taq DNA polymerase, 3µl (50-100 ng) extracted template DNA, and 16.1µl distilled water.The amplification conditions were as follows: initial denaturation at 95ºC for 5 min, followed by 30 cycles of 94ºC for 1 min, 45ºC for 1 min, and then 72ºC for 2 min, and a final extension at 72ºC for 16 min.The PCR products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide.Visual analysis of the ERIC-PCR profile was performed and profiles that had identical or very similar banding patterns (up to two bands different) were regarded as originating from the same DNA groups 23 .

Statistical analysis
Descriptive statistics was conducted to determine frequencies.Microbiological, clinical, and demographic characteristics were assessed using the Statistical Package for the Social Sciences (SPSS)-based computer program version 16.

DISCUSSION
Patients with burn injuries face a higher risk of contracting nosocomial infection due to having partial or complete loss of the skin barrier and a weakened immune system that makes delivery of immediate specialized care a necessity to reduce the rates of mortality and morbidity 24 .P. aeruginosa is a leading cause of nosocomial infections that frequently occur in patients undergoing long-term hospitalization and, particularly, those admitted to burn units.Antimicrobial treatment of infections caused by this organism in a patient with burns is often complicated by the appearance of resistance to highly effective antibiotics, such as carbapenems 1 .During the last decade, acquired MBL-carrying P. aeruginosa isolates with the potential to efficiently hydrolyze carbapenems and most β-lactam drugs have emerged 8 .
The present study uncovered a significantly high level of resistance to routinely prescribed antibacterial agents in our target hospital.All isolates were MDR and, among the antimicrobials tested, were fully resistant to amoxicillin clavulanate, ciprofloxacin, and cefoxitin.Moreover, more than 90% of the isolates were resistant to gentamicin, amikacin, gatifloxacin, cefepime, aztreonam, imipenem, and meropenem, whereas colistin and polymyxin B were the most active antimicrobial agents against our P. aeruginosa isolates.Overall, the frequency of MDR isolates in the present study was higher than in recent studies carried out by Asadpour et al. on P. aeruginosa isolates from patients with burns in Northern Iran (72.2%) 25 , and Rostami et al. in southwest of Iran in 2018 (72%) 26 .In total, these results indicate that the available choices for appropriate treatment of infection caused by these resistant isolates are currently limited.However, the data on colistin and polymyxin B used in our study were comparable to those in other studies [27][28][29] and, therefore, these are considered the antibiotics of choice against P. aeruginosa strains isolated from the patients with burn infections in this hospital.The presence of higher rates of resistance against the antimicrobial agents tested in the current study reflects the irrational and extensive administration of these antibiotics in our medical settings, which eventually resulted in the emergence of this resistance among bacterial isolates.Furthermore, our results highlight the necessity for launching local and nationwide surveillance guidelines on monitoring antibiotic administration to prevent antimicrobial resistance and the occurrence of resistance against antibiotics among bacterial agents in this major referral burn center in Tehran.
In the current study, 158 (98.8%) isolates were nonsusceptible to the carbapenems used.Among those, 69 (43.7%) isolates were MBL producers based on the MBL E-test.This frequency was higher than in other studies, such as in one report from India 29 and two other recent studies from Iran 30,31 .
This study found 28 (17.5%)isolates carried bla IMP-1, which was the most common MBL gene, followed by the genes bla VIM-1 (15.6%) and bla SPM-1 (5.6%), either alone or in combination.In two similar studies from Iran, Lari et al. 31 and Salimi et al. 32 reported the presence of bla VIM-1 and bla IMP-1 genes among P. aeruginosa isolates obtained from patients with burns.In another study, Saffari et al. found that 27 (18%) and 8 (5.5%) of isolates were positive for the bla VIM-1 and bla VIM-2 genes, respectively 33 .Radan et al. showed 74.3% of the MBL isolates in their study carried the bla IMP gene 27 .In eastern Algeria, Meradji et al. stated that 46.7% of P. aeruginosa isolates from burn patients were MBL producers and contained the bla VIM-4 and bla VIM-2 genes 34 .We previously assessed the presence of the bla IMP-1 and bla VIM-1 genes within P. aeruginosa and Acinetobacter baumannii clinical isolates collected from medical centers in Iran 35,36 .Overall, our new findings highlight the widespread presence of the bla IMP-1 and bla VIM-1 genes in nosocomial P. aeruginosa, which contributes to numerous unpleasant health care-associated infections.To our knowledge, this is the first report on the existence of bla SPM-1 -related MBL genes among P. aeruginosa clinical isolates collected from patients with burns in Iran.In our present study, the bla IMP-1 and bla VIM-1 genes coexisted in 2 (1.3%) isolates.The co-existence of similar resistance genes in P. aeruginosa clinical isolates has been reported in a previous study in Iran 32 .The genes encoding these enzymes are often located on transferable genetic platforms and can potentially spread to other bacterial species through horizontal gene transfer.Therefore, appropriate infection control guidelines and treatment protocols are needed to stop the further dissemination of these resistance genes to other bacterial agents in medical centers.In our study, phenotypic tests yielded different positive results when screening for MBL-producing P. aeruginosa isolates compared to molecular methods.In total, 87% of isolates were confirmed to carry MBL genes by the PCR method.This discrepancy between the results obtained by PCR and MBL E-test could be due to the membrane permeabilizing effect of EDTA, which could increase the susceptibility of P. aeruginosa and Acinetobacter spp. to antibacterial agents 37 .
The ERIC-PCR results revealed that MBL-producing P. aeruginosa belonged to three separate clones, indicating clonal dissemination of these resistant isolates occurred in this referral burn center.The A genotype was the most common (76.8%) found in this study and was strongly associated with the clonal spread of resistant isolates and patient-to-patient transmission.
In conclusion, the results of this study revealed a noticeably high prevalence of MBL-producing P. aeruginosa isolates in our hospital.MBL-encoding genes are often carried by mobile genetic elements that can rapidly spread between different strains through horizontal gene transfer.However, early recognition of MBL-producing isolates, establishing comprehensive guidelines and infection control measures, and employing an all-inclusive protocol for antimicrobial therapy based on laboratory data are necessary to significantly decrease further dissemination of these resistant pathogens in our medical settings.

TABLE 1 :
Primers used for detecting metallo-β-lactamase-encoding genes in this study.
ERIC-PCR: FIGURE 1:Metallo-β-lactamase E-test results for a metallo-β-lactamase-positive Pseudomonas aeruginosa isolate.This figure presents a decreased imipenem (IP) maximum inhibitory concentration from 32 to 1.5µg/ml upon constant exposure to EDTA (IPI).This value is less than the cut-off value of 8.0, resulting in this isolate being classified as a metallo-β-lactamase positive P. aeruginosa isolate.IP: Imipenem; IPI: imipenem-inhibitor; EDTA: ethylenediaminetetraacetic acid.