Prevalence of metallo-β-lactamase genes among Pseudomonas aeruginosa isolated from various clinical samples in China

Background: Pseudomonas aeruginosa is an opportunistic pathogen which is associated with nosocomial infections and causes various diseases including urinary tract infection, pneumonia, soft-tissue infection and sepsis. The emergence of P. aeruginosa-acquired metallo-βlactamase (MBL) is most worrisome and poses a serious threat during treatment and infection control. The objective of this study was to identify antibiotic susceptibility, phenotypic detection of MBL production and to determine the prevalence of MBL genes in carbapenem-resistant P. aeruginosa isolated from different clinical samples. Methods: A total of 329 non-duplicate P. aeruginosa isolated from various clinical samples from two hospitals in China between September 2017 and March 2019  were included in this study. Phenotypic detection of MBL was performed by the combined detection method using imipenem and imipenem-ethylenediaminetetraacetic acid (EDTA) discs. MBL-encoding genes including blaVIM-1, blaVIM-2, blaIMP-1, blaIMP-2, blaSPM-1, blaSIM, blaNDM-1 and blaGIM were detected by polymerase chain reaction (PCR). Results: Of the 329 P. aeruginosa, majority of the isolates were resistant to imipenem (77.5%) followed by meropenem (64.7%). Of the 270 P. aeruginosa isolates tested, 149 (55.2%) isolates were found to be positive for MBL detection. Of the different samples, 57.8% (n = 26) of P. aeruginosa isolated from blood were found to be positive for MBL production. Of the various MBL genes, blaIMP-1 (28.2%) was the most predominant gene detected followed by blaVIM-2 (18.8%), blaVIM-1 (16.1%), blaNDM-1 (9.4%), blaIMP-2 (6.7%), blaSIM (6.0%), blaSPM-1 (4.0%) and blaGIM (1.3%) genes. Conclusions: The high resistance of P. aeruginosa toward imipenem and meropenem and the high prevalence of blaIMP-1 and blaVIM-2 set the alarm on the increasing, perhaps the increased, carbapenem resistance. In addition to routine antibiotic susceptibility testings, our results emphasize the importance of both the phenotypic and genotypic MBL detection methods in routine practice for early detection of carbapenem resistance and to prevent further dissemination of this resistant pathogen.


Introduction
Pseudomonas aeruginosa, an opportunistic pathogen, is associated with nosocomial infections and causes various diseases including pneumonia, urinary tract infection, soft-tissue infection and sepsis [1]. Infections caused by multidrug-resistant (MDR) P. aeruginosa are associated with significant morbidity and mortality. A high level of intrinsic and acquired resistance to multiple antibiotics exhibited by P. aeruginosa makes it challenging to treat and limits the treatment options [2]. Pseudomonas aeruginosa exhibits almost all known resistance mechanisms; however, enzyme production is the major mechanism of acquired resistance, especially β-lactamase production [3]. The increased prevalence of extended-spectrum β-lactamase (ESBL)-producing P. aeruginosa led to the use of carbapenems. Carbapenems are the last choice of drug for the treatment of P. aeruginosa infections. However, the alarming increase in carbapenem resistance is a cause of serious concern in the treatment of P. aeruginosa infections [4]. The World Health Organization (WHO) has identified 12 most common bacteria that pose a challenge to human health. Among these, carbapenem-resistant P. aeruginosa was designated as one of the highly critical and poses a serious threat to patients who require ventilators and blood catheters [5]. β-lactamases are classified into four different classes: classes A, C and D act through a serinebased mechanism and metallo-β-lactamase (MBL), while class B requires a bivalent metal ion for its activity [6]. Of all these mechanisms, the emergence of P. aeruginosaacquired MBL is the most worrisome and poses a serious threat during treatment and infection control [7]. Except for monobactams, MBL-producing strains can hydrolyze all other β-lactam antibiotics including penicillin, cephamycins, cephalosporin and carbapenems [8]. The MBLproducing P. aeruginosa strains carry co-resistance genes for other classes of antibiotics.
When patients with severe infections caused by MBLproducing P. aeruginosa are treated with antibiotics, it often leads to poor clinical outcome. Thus, it is highly essential to detect MBL-producing P. aeruginosa as early as possible for the effective treatment of critically ill patients within clinical settings. The present study aimed to identify antibiotic susceptibility, phenotypic detection of MBL and to determine the prevalence of MBL genes in carbapenem-resistant P. aeruginosa isolated from different clinical samples.

Bacterial strains
A total of 329 non-duplicate P. aeruginosa isolated from various clinical samples from the Beijing Friendship Hospital, Capital Medical University, Beijing, China and The Second Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan, China between September 2017 and March 2019 were included in this study. The isolates were identified as P. aeruginosa using the VITEK 2 system (bioMérieux, Craponne, France). Demographic data and other relevant details of patients from whom P. aeruginosa was isolated were collected from the hospital medical record department. The Institutional Ethical Board approved the study (IRB: 037-2017).

Phenotypic detection of MBL production
All isolates that were resistant and intermediately resistant to imipenem by disc diffusion method were subjected to MBL production test by the combined disk method [14]. Briefly, overnight culture adjusted to 0.5 MacFarland standard was inoculated on to MHA plates. Two disks of 10 μg imipenem (10 μg) and imipenem (10 μg) with 0.5 M ethylenediaminetetraacetic acid (EDTA) were placed 25 mm apart on the MHA plates. The plates were incubated at 37 °C for 16-18 h and observed for the zone of inhibition. A zone of ≥7 mm in the imipenem plus EDTA disc compared to imipenem alone disc was considered as positive for the presence of carbapenem resistance (MBL resistance).

Detection of MBL genes
Carbapenem-resistant isolates were subjected to polymerase chain reaction (PCR) for the detection of MBL genes including bla VIM-1 , bla VIM-2 , bla IMP-1 , bla IMP-2 , bla SPM-1 , bla SIM , bla NDM-1 and bla GIM as described by Azimi et al. [15]. Inhouse P. aeruginosa isolates positive for all tested genes and confirmed earlier through sequencing were used as controls for PCR. The gene-specific primer sequence and their annealing temperature are presented in Table 1 [ [16][17][18]. DNA was extracted using a commercial genomic DNA extraction kit (Thermo Fisher Scientific, Waltham, MA, USA). The PCR reaction mixture contained 12.5 μL of ReadyMix™ Taq PCR Reaction Mix (Sigma-Aldrich, St. Louis, MO, USA), 2.5 μL of the DNA (20 pg), and 0.5 μM of each primer and nuclease-free water made up to 25 μL. The PCR cycling conditions were as follows: initial denaturation at 96 °C for 10 min, followed by 30 cycles of 96 °C for 1 min, the specific annealing temperatures of the respective primers as presented in Table 1 for 1 min, and 72 °C for 1 min, and a final extension step at 72 °C for 10 min. After PCR, the amplicons were resolved in 1.2% (w/v) agarose gel electrophoresis and visualized under a ultraviolet (UV) transilluminator (BioRad, Hercules, CA, USA).

Statistical analysis
Descriptive statistics were performed to determine the frequencies. The chi-square (χ 2 ) test, Student's t-test and Pearson's correlation coefficient tests were performed to analyze the data using SPSS statistical software (IBM SPSS Inc., Chicago, IL, USA, Ver. 2015). A p-value of <0.05 was considered statistically significant.
Wang and Wang: Metallo-β-lactamase genes among Pseudomonas aeruginosa coefficient showed that there was a significant association between the P. aeruginosa isolated from patients with respiratory diseases and imipenem resistance (r = 0.98, p = 0.029). Thirty-two (9.7%) isolates were found to be susceptible to all the antibiotics tested. A total of 207 (62.9%) isolates were found to be MDR strains as they were intermediately or fully resistant to at least three different classes of antibiotics. None of the P. aeruginosa isolated from blood samples was resistant to colistin (Table 3).

Phenotypic detection of MBL production
All the isolates that were resistant (255, 77.5%) and intermediately resistant (

Detection of MBL genes
All the 149 P. aeruginosa that were positive for carbapenem resistance were subjected to MBL gene detection by PCR.

Discussion
Pseudomonas aeruginosa infections are effectively treated by carbapenems; however, resistance toward these antibiotics spread across hospitals due to the extensive use of antibiotics [19]. Intrinsic resistance among P. aeruginosa  leads to MDR and is associated with high mortality. This study reported a high level of carbapenem resistance among P. aeruginosa isolated from various clinical samples. Although not significant, P. aeruginosa (77.2%) isolated from sputum samples were found to be resistant to imipenem (p > 0.05). The majority of our isolates were from sputum samples (28.0%) and the majority of our sputum samples (72.4%) were from patients with respiratory diseases. A significant association between the P. aeruginosa isolated from patients with respiratory diseases and imipenem resistance (r = 0.98, p = 0.029) was found in this study. In our hospital, imipenem is one of the most commonly used antibiotics as first-line therapy for most of the cases with pseudomonas infection, especially for respiratory diseases and patients who are on ventilator support. An overexposure of this drug could possibly be the reason for this higher rate of imipenem resistance among our isolates.
A wide range of resistant mechanisms make P. aeruginosa swiftly change to the selective environmental pressure and makes it resistant to several classes of antibiotics. In this study, 77.5% of the isolates were found to be resistant to imipenem, which corroborates to that reported from Egypt (78.3%) [20] and China (73.3%) [21]. Compared to other studies from the Asia-Pacific region including Iran (25.2%) [22], China (17.1%, 43.3%) [23,24], Japan (28.5%) [25] and Taiwan (16%) [26], this study reported a much higher resistance toward imipenem. Another study from Iran reported a resistance of 98.8% [15]. In a review article, Hong et al. presented an overview of the epidemiology and molecular characteristics of MBL-producing P. aeruginosa.
The study which analyzed several publications from Asia, Europe, America and Africa reported imipenem resistance among P. aeruginosa ranging from 8% to 66% [9]. In this study, meropenem resistance was the second most common resistance reported (64.7%) among our isolates. In addition to imipenem resistance, Hong et al. reported meropenem resistance ranging from 8% to 57% [9]. In the aforementioned study, which covers multiple countries of all regions, the reported imipenem and meropenem resistance ranges among P. aeruginosa do not exceed the rate reported in this study [9]. This implies that imipenem and meropenem resistance among our isolates was higher than that reported worldwide and needs special attention. In addition to the overuse of imipenem and meropenem in our hospital settings, the presence of resistant genes on mobile genetic elements may have also contributed to the spread of resistance within our isolates [8]. In our study, 62.9% of the isolates were found to be MDR strains, which was higher than that reported from Brazil (37%) and Asia (42.8%) [27,28].
In this study, 55.2% of the isolates were found to be MBL producers. A study from Iran, which included P. aeruginosa isolated from burnt wound, reported a lower rate (43.7%) of MBL producers compared to this study [15]. Similarly, another study from Iran also reported a lower rate (37.7%) of MBL producers among their clinical isolates of P. aeruginosa [29]. Hong et al. in a review article analyzed reports with varied carbapenem resistance rates. Compared to this study, the review article reported lower carbapenem-resistant isolates from South and Southeast Asia including Philippines (31.1%), Singapore (23.3%), Thailand (28.7%), Vietnam (46.7%) and India (32%); Oceanic including Australia (16%) and New Zealand (10.3%); East Africa including Kenya (13.7%); and North America including Canada (3.3%) and the United States (20%). A much higher rate was reported from Russia (75.3%) and Costa Rica (63.1%) [9]. A meta-analysis from Iran, which included 14 publications on P. aeruginosa isolated from burn patients, reported a pooled prevalence rate of 76.8% carbapenemresistant P. aeruginosa, which is much higher than that reported in this study [30]. However, a study from China, which included P. aeruginosa isolated from cystic fibrosis patients, reported that 56.25% of their isolates were found to be MBL producers, which is comparable to that reported in this study [21]. Although not significant, a relatively high number (41, 27.52%) of P. aeruginosa isolated from sputum samples were found to be MBL producers. However, in terms of percentage, P. aeruginosa isolated from blood samples yielded a higher rate (57.8%) of MBL producers. Pseudomonas aeruginosa isolated from male Most of the MBL genes are located in the integrons within mobile genetic elements and are responsible for the dissemination of antibiotic resistance through horizontal gene transfer. In the past decade, the emergence and dissemination of the newly identified bla NDM and bla AIM and the most prevalent bla IMP , bla VIM , bla SIM and bla GIM MBL genes have been widely reported around the world [20]. In this study, bla IMP-1 (28.2%) was the most common gene detected; bla VIM-2 (18.8%) was the second most common gene detected, followed by bla VIM-1 (16.1%). Similar to this study, a study from Iran which included MBL-producing P. aeruginosa reported that bla IMP (24.65%) was the predominant gene detected followed by bla VIM (11.5%) [29]. Similarly, a study from Japan which included 180 P. aeruginosa reported that bla IMP-1 was the predominant gene detected (116/180 isolates) followed by bla VIM-2 (63/180 isolates) [16]. While a meta-analysis from Iran which included P. aeruginosa from burn patients reported that the pooled prevalence of bla VIM was 21.4% and of bla IMP was 13.1%, the reported rates were higher than in this study [30]. A study from Iran also reported that bla VIM (17.5%) was the most common gene followed by bla IMP (15.6%) [15]. A study from China that included 63 MBL-producing P. aeruginosa reported that bla VIM was the most common gene but with a higher rate (84.1%) and bla IMP was the second most common gene (76.1%), however, with higher rates than those reported in this study [21]. A study from Iran that included 19 MBL-producing P. aeruginosa reported that 31.5% of their isolates carried the bla VIM gene and 10.5% carried bla IMP , which was higher than that reported in this study [22]. In contrast to our study, a study from Egypt reported that none of their P. aeruginosa was positive for the bla VIM gene [20]. In the present study, 9.4% of our isolates were positive for the bla NDM-1 gene, which is lower than that reported from India (27%) [31]. A study from Bahrain reported a lower rate of bla NDM-1 (2.5%) than that reported in this study [32]. In the present study, the bla IMP-1 gene was significantly higher among the isolates tested (p = 0.023). The bla IMP-1 gene along with bla IMP-2 was the most common combination found in this study. There was no significant difference in the presence of MBL genes with that of hospital settings, including ICU/in-ward patients, patients under ventilator and burnt wound patients (p > 0.05). Fourteen (9.4%) isolates did not amplify any of the genes tested, and these isolates could possibly carry other resistant gene variations that were not tested in this study. A study to test more resistant gene variations could possibly reveal the resistant genes associated with these isolates. The present study suggests that with the predominance of the bla IMP-1 gene, bla VIM is also increasing in our region. In addition, the high rate of the bla NDM-1 gene signifies its spread and emphasizes the need for continuous monitoring for better patient management and infection control.

Conclusions
In conclusion, the relatively high resistance of P. aeruginosa toward imipenem and meropenem and the high prevalence of bla IMP-1 and bla VIM-2 set an alarm on the increasing, perhaps the increased, carbapenem resistance. In addition to routine antibiotic susceptibility testings, our results emphasize the importance of both the phenotypic and genotypic MBL detection methods in routine practice for early detection of carbapenem resistance and to prevent further dissemination of this resistant pathogen.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: Authors state no conflict of interest. Informed consent: Informed consent was obtained from all individuals included in this study. Ethical approval: Research involving human subjects complied with all relevant national regulations, institutional policies and is in accordance with the tenets of the Helsinki Declaration (as revised in 2013), and has been approved by the authors' institutional human Ethics Committee (037-2017).