Prevalence of Clinically Isolated Metallo-beta-lactamase-producing Pseudomonas aeruginosa, Coding Genes, and Possible Risk Factors in Iran.

BACKGROUND &
OBJECTIVE: The spread of carbapenem-resistant Pseudomonas aeruginosa is a global concern. Metallo-beta-lactamase (MBL) enzymes cause extensive drug resistance among Gram-negative bacteria. The current study aimed at determining the prevalence of MBL-producing P. aeruginosa in Iran. DATA EXTRACTION: A total of 43 studies were found out of which 36 were adopted. Data were collected from Google, Google Scholar, Science Direct, PubMed, Scopus, Embase, and Sciverse. The terms "Pseudomonasaeruginosa", "metallo-beta-lactamase", "prevalence", "carbapenems", and "Iran" were searched. Data from the isolates not producing MBLs were excluded from the study. Data were analyzed with Graph Pad Prism 6, meta-analysis section.
RESULTS: According to the results of the current study, 36 surveys indicated that 55% of the clinically isolated P. aeruginosa in Iran were resistant to imipenem and meropenem, among which 37.72% were the MBL producers. Among genes encoding MBLs, blaVIM and blaIMP were predominant with the prevalence of 12.91%±11.01% and 12.50%±23.56%, respectively. No report of harboring blaNDM1 and blaSPM1 by P. aeruginosa was found, similar to most of the other countries in Asia. The prevalence of blaVIM and blaIMP from burn settings were 11.50%±3.5% and 24.65%±23%, respectively. Furthermore, the prevalence of these genes was not significantly different among burn and non-burn isolates (P=0.942 and P=0.597, respectively). Moreover, no relationship was observed between the MBL production and patients' age range.
CONCLUSION: Approximately half of P. aeruginosa isolates were carbapenem-resistant in Iran, and approximately half were the MBL producers. The blaVIM and blaIMP were the predominant MBLs among P. aeruginosa strains, while other genes were not found in P. aeruginosa. Moreover, there was no significant difference between blaVIM and blaIMP among burn and non-burn isolates. Due to the multiple drug resistance conferred by MBLs, detection and control of their spread alongside proper therapeutic regimens in hospitals and community settings are essential to prevent infection acquisition.

Introduction

Carbapenem resistance among clinically isolated Pseudomonas aeruginosa (P. aeruginosa) is a great concern worldwide, as this class of antibiotics is among the last resorts to eradicate infections with Gram-negative species (1-3). The prevalence of multidrug-resistant P. aeruginosa (MDRP) non-susceptible to quinolones and aminoglycosides in addition to beta-lactams is reported worldwide (4-6). Pseudomonas aeruginosa isolates acquire resistance to carbapenems via several mechanisms including overexpression of efflux systems, change or lack of outer membrane proteins (such as OprD porin), chromosomal AmpC beta-lactamase, and production of carbapenemases, overall named heteroresistance (7). The most important carbapenemases produced by P. aeruginosa are zinc-dependent metallo-beta-lactamases (MBLs) capable of hydrolyzing imipenem, meropenem, ertapenem, and cephalosporins, but not monobactams and aztreonam (8, 9). Other types of carbapenemases include class D enzymes such as OXA-23, OXA-27, OXA-48, and class A including clavulanic acid inhibitory enzymes (SME, NMC, IMI, and KPC) (10). MBLs are determined in Enterobacteriaceae and other Gram-negative non-fermenters (8). Phenotypic detection of MBLs include (1 Combined disk using imipenem and imipenem + EDTA (ethylenediaminetetraacetic acid)/dipicolinic acid; 2) The Hodge test in which Escherichia coli ATCC is lawn on Mueller-Hinton agar, and then, test strains are cultured horizontally, and 3) The carbaNP test as the most sensitive protocol. Carbapenem and vancomycin exposure were shown as risk factors for carbapenem-resistant P. aeruginosa in Brazil (11). There are various MBL genes among carbapenem-resistant P. aeruginosa including Verona integron-encoded MBL (VIM), Germany imipenemase (GIM), imipenemase (IMP), Sao Paulo MBL (SPM), New Delhi MBL (NDM), and Florence imipenemase (FIM). Each MBL gene is encoded by specific genetic elements including transposons, integrons, plasmids, or on the chromosome, carrying genes encoding determinants of resistance to several antibiotics in addition to carbapenems, causing the advent of MDR P. aeruginosa. Moreover, these genetic determinants are transferable to other Gram-negative species, extending the antimicrobial resistance rate and complicating the treatment of infected patients (12). Therefore, it is necessary to understand the epidemiology, molecular characteristics, and resistance mechanism of MPPA to control infection and prevent a possible global health crisis. These beta-lactamases are inhibited by the EDTA chelator. The most important MBL types for epidemiological spread and clinical relevance are IMP, VIM, SPM, and currently NDM (13, 14). The predominant MBLs are VIM and IMP reportedly carried by the mobilizable elements such as integrons. The VIM-2 integrons are detected in MDR strains (15). It is reported that VIM-type MBLs are predominant in some areas (16, 17). The blaNDM1 was reported from Klebsiella pneumonia in Iran in 2013 (18).

Objectives

The current study aimed at investigating the MBL production and MBL types among clinically isolated P. aeruginosa strains in Iran.

Data extraction

The current meta-analysis review collected data from search engines such as Google, Google Scholar, Science Direct, PubMed, Scopus, Sciverse, etc. The terms “Pseudomonas aeruginosa”, “metallo-beta-lactamas “prevalence”, “carbapenems” and “Iran” were searched. A total of 43 studies were found out of which 36 were adopted. Studies on phenotypic results of carbapenem resistance were included. All studies on burn isolates were also included. Studies on other mechanisms of carbapenem resistance were excluded. Data from non-metallo-beta-lactamase-mediated carbapenem-resistant strains were also excluded from the study. The prevalence of phenotypic and molecular studies was transferred into the software and the results were analyzed. The bias among published studies were the risk factors, sample size, the outcome of infections, the genetic relationship between strains in hospital settings (if the infection is clonally spread), age, and the economic stats of patients. The current study inclusion criteria were the possible influence of these risk factors on the prevalence of MBL-producing P. aeruginosa alongside the genetic characteristics of the strains.

Data were analyzed using GraphPad Prism 6, meta-analysis section, and the standardized mean difference by the Cohen d method basically reached by the employment of difference score divided by standard deviation (SD) of the scores analyzed by the software itself.

Results

The previous studies demonstrated that 55% of P. aeruginosa isolates were resistant to carbapenems. Of them, 37.72% were MBL-positive. The mean prevalence of blaVIM was predominant, while only 1% of the strains were blaIMP producers and none were positive for blaNDM1 and blaSPM1 (Table1) (19-26).

Table1

Phenotypic and Genotypic Prevalence of MBL Types in Different Cities of Iran

MBL Type	Percentage (%)	City	Total Isolates	MR (%)	Resistance	Year	Reference
VIM	6.3419.5117.3012.362.6213a16.12.1a32.855.177.5a21.3, 24a0.46	TehranAhvazTabrizTehranTehranTehranTehranTehranZanjanUrmiaShirazArakKermanshah	126100B104186B610100B4834837058240B108225	---82.6---------	ImipenemImipenemImipenemImipenemImipenemImipenemImipenemimipenemImipenemImipenemImipenemImipenemImipenem, meropenem	2007200820102010201020102012201220132013201220142015	(27)(19)(24)(22)(28)(21)(29)(29)(30)(31)(32)(33)(34)
IMP	5.7657.96.63.3b14.283.417.5b1.315.11	TabrizTehranTehranTehranZanjanUrmiaShirazTehranKermanshah	104100483483705824075225B	-8.3-------	ImipenemImipenemImipenemImipenemImipenemImipenemImipenemImipenemImipenem, meropenem	201020132012201220132013201220122015	(26)(30)(35)(34)

MBL, metallo-beta-lactamase;

VIM-2;

IMP-2;

isolates from burn injuries; MR, mortality rate

Furthermore, no significant difference was observed among burn and non-burn isolates regarding the prevalence of MBL genes. There was no significant relationship between patients’ age ranges and the presence of MBLs. Furthermore, the mortality rate due to infection with MBL-producing bacteria was not fully elucidated, according to the data from some studies (47.25±3795, N=2).

As already mentioned, the bias among the published studies were seldom the detection of risk factors, sample size differences, lack of data on the outcome of infections, the obscure genetic relationship of strains within hospital settings (if the infection is clonally spread), weak uncovering ages, and the economic status of the patients. Hence, the possible risk factors for the acquisition of MBL-producing P. aeruginosa strains were not potentially achieved.

Worldwide incidence of MBL-producing

Middle-East and North African countries

A study by Bahar from Turkey showed the presence of VIM-5 beta-lactamase for the first time in Klebsiella pneumonia (36). Iraz reported blaGES-5 and a novel blaVIM variant, named VIM-38 (37).

Other studies from Saudi Arabia demonstrated the presence of VIM-2 in a patients with HIV (38), and 41% (16/39) blaVIM among the extended-spectrum beta-lactamases (ESBL)-producing P. aeruginosa (39). Another study revealed that 20.57% (72/350) of the isolates produced MBL and all of them

carried blaVIM-2 (40). In Egypt, the first report of blaNDM-1 associated with blaVIM-2 was published in 2014 (41). Another study from Egypt revealed that 39.34% of P. aeruginosa species isolated from patients with cancer were imipenem-resistant among which 27% were the MBL-positive strains. The blaVIM-2, blaNDM-1, and blaIMP-1 were detected among 58.3%, 4.2%, and 2.1% of MBL-positive isolates (42). The VIM-2 was reported from Tunisia and Algeria in North Africa (43-45). In a study in Tunisia, of 30 MBL-positive isolates, 17.5% were blaVIM-2 positive (46).

Asia Pacific

In a study by Dong in China, among 59 carbapenem-resistant P. aeruginosa strains, 39 (66.1%) were positive for the MBL genotype; 35 (89.7%) and 4 (10.3%) of which carried blaIMP-1 and blaVIM-2, respectively (47). Qu showed that among 24 MBL-producing strains, 10 were positive for the blaVIM-2, while 13 were positive for blaIMP-9, and 1 for blaIMP-1 (48). Yu showed that 14/140 of P. aeruginosa species were positive for blaVIM-2; in addition 12 of which carried class 1 integrons (49). The blaIM-9 was first reported from China in 7 P. aeruginosa isolates in 2005 (50). In Southern China, only 1 of 61 imipenem-resistant isolates harbored blaIMP-9 (51). Of 368 isolates from several cities in China, 25 were positive for the blaIMP-6 and 3 positive for blaVIM-2 with the predominance of ST244 and ST235 sequence types (52). The blaKPC-2 was detected among 38 carbapenemase-producing P. aeruginosa isolates exhibiting ST463 (53). In Thailand, MBL was clearly positive in 24 (18.46%) and weakly positive in 12 (9.23%) isolates; IMP-1, IMP-14, and VIM-2 were detected among both of these sets of isolates (54). In Japan, 11 of 23 carbapenem-resistant isolates carried GES-5 (55). Another study showed that the prevalence of blaIMP/(AAC-6)-producing P. aeruginosa increased in Japan from 170/300 (56.7%) in 2011 to 230/300 (76.7%) in 2012 (56). In Korea, 20 (15.6%) and 11 (8.6%) imipenem non-susceptible isolates were positive for blaIMP-1 and blaVIM-2, respectively (57).

European countries

The blaGIM-1 was first reported from Germany among 28% of the isolates in 2004 (58). In the study by Valenza in Germany, among 489 isolates, 11.7% of MBL-producing isolates were blaVIM-positive, but no other encoding gene was detected (59). Another study showed the outbreak of blaVIM-2 among 11 specimens from urinary tract infection in Germany (60). There was a case report of NDM-1-producing P. aeruginosa in France (61). There was another report of blaNDM-1 in Balkan region, Serbia (62) as well as a report of IMP-29 in France (63). A novel MBL named VIM-14 carried in a class 1 integron with a new organization was detected in Italy. The integron harbored the genes aac7, blaVIM-14, blaOXA-20, and aac4 (64). A novel blaIMP, blaIMP-58, was recently reported from Denmark (65).

North America

The first report of MBL-producing blaVIM-2 in the United States was in 2005 (66). The blaNDM was reported in some Enterobactericeae isolates in USA (67).

Latin America and Africa

In a study in Brazil, MBLs included SPM-1 (55.6%), VIM-2 (30.6%), and IMP-1 (8.3%) enzymes (68). The NDM-1 was detected in K. pneumonia in South Africa (69).

Discussion

In the current review, the range of MBLs was from 16.68% in the study by Shahcheragi to 100% in the studies by Bahar and Saderi (21-23). The variation between phenotypic and genotypic detection of MBLs were not highlighted among previous studies from Iran, although it was reported by other studies (70). The studies showed that the presence of other resistance mechanisms may interfere in the MBL phenotypic detection, or EDTA can affect cell membrane (71). The NDM1, SPM1, and GIM genes are not present in Iran, and are reportedly detected in distinct geographic areas (5, 72, 73). Rojo Bezares demonstrated that 49.4% of carbapenem-resistant P. aeruginosa from Spain were MBL-positive, all were blaVIM2-positive, and 75% were integrin-class1-positive (74). In the study by NagKumar, 18.85% of P. aeruginosa isolates were MBL-positive. In Colombia, 60% of carbapenem-resistant strains were VIM-positive, but all were IMP- and NDM-negative, and also class 1 and 2 were detected among them(75). The GIM1, SPM1, and, FIM1 were reported from Germany, Switzerland, Brazil, and Italy, respectively (58, 76-78). The blaVIM is the main MBL encoding gene among Middle-East and most of other Asian countries. The blaNDM-1 emerged in some countries other than India, and thus, its spread is of great concern.

The current study determined no significant difference regarding the prevalence of blaIMP and blaVIM between the species isolated from burn and non-burn injuries.

Furthermore, it was not revealed if there was a relationship between the prevalence of MBLs and patients’ age ranges. In addition, the mortality rate was not fully elucidated; however, results of 2 studies showed a mean rate of 47.25% It was depicted that previous antibiotic use, catheterization, intravenous (IV) lines, >8 days hospital stay, mechanical ventilation, and endotracheal intubation were the risk factors for MBL-producing P. aeruginosa , but significant risk factors for MBL-positives species were graft application and surgical intervention (79). As already mentioned, the bias among published studies seldom detected risk factors, sample size differences, lack of the outcome of infections, the obscure genetic relationship of strains within hospital settings (if the infection was clonally spread), and weak uncovering ages and the economic status of the patients. For these reasons, the possible risk factors for the acquisition of MBL-producing P. aeruginosa infections were not potentially achieved.

Conclusion

Approximately half of P. aeruginosa isolates were carbapenem-resistant in Iran, among which nearly half were MBL-positive. The blaVIM and blaIMP were the predominant MBLs in P. aeruginosa strains, and the emergence of other genes is a concern. Moreover, there was no significant difference between blaVIM and blaIMP prevalence among burn and non-burn injuries. Due to multiple drug resistance conferred by MBLs, detection and control of their spread alongside proper therapeutic regimens in hospital and community settings is essential.