Molecular characterization of NDM-1-producing Pseudomonas aeruginosa isolates from hospitalized patients in Iran

The emergence of carbapenem-resistant Pseudomonas aeruginosa is one of the most important challenges in a healthcare setting. The aim of this study is double-locus sequence typing (DLST) typing of blaNDM-1 positive P. aeruginosa isolates. Twenty-nine blaNDM-1 positive isolates were collected during three years of study from different cities in Iran. Modified hodge test (MHT), double-disk synergy test (DDST) and double-disk potentiation test (DDPT) was performed for detection of carbapenemase and metallo-beta-lactamase (MBL) producing blaNDM-1 positive P. aeruginosa isolates. The antibiotic resistance genes were considered by PCR method. Clonal relationship of blaNDM-1 positive was also characterized using DLST method. Antibiotic susceptibility pattern showed that all isolates were resistant to imipenem and ertapenem. DDST and DDPT revealed that 15/29 (51.8%) and 26 (89.7%) of blaNDM-1 positive isolates were MBL producing isolates, respectively. The presence of blaOXA-10,blaVIM-2, blaIMP-1 and blaSPM genes were detected in 86.2%, 41.4%, 34.5% and 3.5% isolates, respectively. DLST typing results revealed the main cluster were DLST 25-11 with 13 infected or colonized patients. The presence of blaNDM-1 gene with other MBLs encoding genes in P. aeruginosa is a potential challenge in the treatment of microorganism infections. DLST showed partial diversity among 29 blaNDM-1 positive isolates.


Background
Pseudomonas aeruginosa is one of the most important hospital-acquired pathogens that causes miscellaneous opportunistic infections [1]. The emergence of multidrug-resistant (MDR: was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories) and extremely drug resistant (XDR: was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories) P. aeruginosa isolates has been considered as a major concern for the treatment of infections caused by these isolates [2]. Carbapenemases are a wide spectrum group of beta-lactamase which hydrolyzes carbapenems to other b-lactams including monobactams, penicillins, and cephalosporins. Although carbapenems are a commonly last resort treatment used for MDR P. aeruginosa infection, the emergence of carbapenem-resistant P. aeruginosa is becoming a main public health concern and is associated with high rates of mortality and morbidity among hospitalized patients [3,4].
Resistance to carbapenems can be related to producing carbapenemase enzymes such as serine carbapenemases and the MBLs encoding genes such as IMP, VIM, and

Annals of Clinical Microbiology and Antimicrobials
NDM enzymes [5]. The MBLs encoding genes such as bla VIM and bla IMP are one of the most clinically important classes of beta-lactamases; but, discovered transmissible New Delhi metallo-beta-lactamase-1 (NDM-1) is becoming the most threatening carbapenemase, recently [6][7][8].
The bla NDM-1 producing strains are resistant to a wideranging of other antibiotic groups and transport numerous additional resistance genes such as genes encodings resistance to fluoroquinolones, aminoglycosides, sulfonamides, and macrolides. Furthermore, the NDM-1 enzyme is surfacing, resulting in almost whole resistance to antibiotics [8][9][10]. Molecular typing of P. aeruginosa is important to understand the local epidemiology, but it remains a challenging issue. The epidemiology of P. aeruginosa has been analyzed by an array of different typing methods such as Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) that are costly, required specific technical abilities and time to consume [11]. The newly described double-locus sequence typing (DLST) methods based on the partial sequencing of two highly variable loci to typing P. aeruginosa isolates which allowed us to obtain an unambiguous and standardized definition of types [12]. DLST has remarkable discriminatory power, reproducibility and is able to recognize high-risk epidemic clones [12]. Although bla NDM-1 positive isolates are rare, knowledge of its occurrence is considered as a serious menace, however, this study is the first report of DLST typing of bla NDM-1 positive P. aeruginosa isolates obtained from different part of Iran.
A total of 29 non-duplicate bla NDM-1 positive P. aeruginosa were collected from different clinical samples. The identification of P. aeruginosa was done by the conventional microbiology tests and confirmed by PCR with specific primers for gyrB gene [14].

PCR amplification of resistance genes
PCR amplification was performed for detection of bla NDM , bla IMP , bla VIM , bla KPC , bla GES , bla SPM and bla OXA-10 using a set of specific primers on a thermal cycler (Eppendorf AG, Germany) as described previously [15][16][17]. Sequencing of the amplicons was performed by the Bioneer Company (Bioneer, Daejeon, South Korea) and the nucleotide sequences were analyzed using Gen-Bank nucleotide database at http:// www. ncbi. nlm. nih. gov/ blast/.

Carbapenemase screening
The double-disk potentiation tests (DDPT) and double disk synergy test (DDST) was performed phenotypically for all bla NDM−1 positive described by Yong et al. [19].

Double-locus sequence typing method
DLST typing was carried out using amplification of ms172 and ms217 loci using specific primers as previously described (Basset and Blanc, 2014) and according to DLST website (http:// www. dlst. org/). PCR products were purified and were sequenced by Bioneer Corporation (Bioneer, Daejeon, South Korea).
In the cases of any results of allele assignment, the allele was considered as a null allele. The allele profiles were compared and clustered by the UPGMA and Dice methods ( Fig. 1), using an online data analysis service (nslico.ehu.es).

Results
During the three years of study, twenty-nine of bla NDM-1 positive isolates were collected, of them, 6 (20.7%) and 23 (79.3%) isolates were from burn patients and non-burn patients, respectively. The male to the female proportion in bla NDM-1 isolates was 3.22 (n= 20:9). The most bla NDM-1 positive strains were isolated from wound/ punch (n = 11; 37.9%) followed by urine (n = 11; 37.9%) samples, whereas, the majority of the bla NDM-1 isolates were obtained from ICU ward (n = 21: 72.4%), followed by internal ward (n = 6; 20.7%) and burn ward (n = 6; 20.7%) ( Table 1). Antibiotic susceptibility pattern showed that all isolates were resistance to imipenem and ertapenem, moreover, the resistance rate of meropenem was 96.5%. In contrast, the highest sensitivity was against to piperacillin/tazobactam and amikacin (41.4%). All bla NDM-1 positive isolates were defined as MDR. The full results of antibiotic resistance pattern of bla NDM-1 positive P. aeruginosa isolates showed in Table 2.
The distribution of carbapenemase genes and other antibiotic resistance genes among bla NDM-1 positive P. aeruginosa isolates are presented in Table 2. However, PCR analysis showed none of the bla NDM-1 positive P. aeruginosa isolates contained bla KPC and bla GES genes. The presence of bla VIM-2 , bla IMP-1 and bla SPM genes were detected in 41.4%, 34.5% and 3.5% isolates, respectively. Among bla NDM-1 positive P. aeruginosa isolates, the bla OXA-10 beta-lactamase was the most frequently gene recognized in 86.2% (25/29).
In the current study, the DLST method was tested for all bla NDM-1 isolates recovered over a period of four years from various hospital wards. DLST results revealed partial diversity among 29 bla NDM-1 positive isolates. Totally,  Table 2). The most common type including 13 isolates (45%) from different hospitals (in Ahvaz and Isfahan). A total of 29, 27 sequences were ms172 and ms217 whereas, 2 strains carried null alleles for these loci. The DLST profile 5-91 were detected in 3 (50%) burn isolates. The details of information about DL type of P. aeruginosa isolates are presented in Table 2.

Discussion
P. aeruginosa, one of the most common opportunistic pathogen associated with nosocomial infections, including pneumonia, urinary tract infections, and wound infections [1,20]. Although carbapenems are often used as a therapeutic agent for treating infections caused by P. aeruginosa, the high emergence of carbapenem resistance significantly decreases their usefulness [21,22]. The presence of bla NDM−1 producing isolates which may increase resistance to carbapenems is increasing among patients in healthcare systems [23]. In the current study, we described molecular characterization of bla NDM-1 producing of P.aeruginosa isolates with phenotypic and genotypic methods.
The overall data show that the frequency of bla NDM-1 producing P. aeruginosa isolates was 7.8% (29/369). In addition, there are variable reports of bla NDM-1 from different countries in Europe and Asian. The bla NDM-1 producing P. aeruginosa isolates has been also detected in Iran, recently. Shokri et al. from Isfahan (Center of Iran) in 2017 reported 6% of P. aeruginosa isolates were bla NDM-1 positive which is slightly lower than the result obtained in the present study [24]. In another study, Dogonchi et al. reported one isolate of P. aeruginosa harboring bla NDM-1 in the north of Iran [25]. Recently, Azimi et al. described a lower frequency rate for bla NDM-1 (7%) among carbapenem-resistant P. aeruginosa [26]. Moreover, the bla NDM−1 gene was also revealed by Riahi Rad et al. in Iran (21.4%) [27] and Takahashi et al. in Nepal (7%) [28].
With regard to the fact that our isolates were collected from hospitals of different cities in Iran and also regarding the results of previous studies, an increasing trend of bla NDM-1 producing P. aeruginosa strains can be observed in Iranian hospitals where it could be an endemic and serious concern in future. One of the important reason of possible increase of this phenomenon among Gram-negative isolates is inappropriate and excessive prescription and use of carbapenems in our hospitals, which leads to selective pressure.
According to our results, the most bla NDM-1 positive isolates (72.4%) were collected from the ICU ward that these findings are broadly consistent with the previous studies conducted in Iran [24,25]. This finding suggests that the ICU ward is can be a risk factor and major source for the dissemination of resistant genes in the Iranian hospitals. Our results presented that bla NDM-1 positive isolates had highly resistant to all antibiotics commonly used in the clinic which is in agreement with to the results of other studies [24,29,30].
In spite of the fact that the bla NDM-1 gene demonstrating the sensitivity of bacteria to aztreonam, 62% of bla NDM-1 positive isolates were resistant to this agent that could be related to the presence of other beta-lactamase genes. Based on screening of other carbapenemase and metallo β-lactamase genes, bla OXA-10 was the most frequently detected beta-lactamase among bla NDM-1 positive strain and bla IMP-1 was second, which is in contrast to other reports where bla VIM was significantly associated with bla NDM-1 [31,32].
The previous studies revealed that the acquisition of MBL determinants such as bla NDM-1 , bla VIM , bla IMP and bla SPM led to the emergence of MDR or XDR P. aeruginosa [36,37].
To the extent of our knowledge, this study is the first to report of Molecular typing of bla NDM-1 positive isolates in P. aeruginosa by DLST method.
Among various genotyping technique, DLST as a reliable genotyping method, provide a new, rapid, typability, stability and low-cost of epidemiological surveillance of P. aeruginosa isolates [11,38]. Analysis of DLST types revealed that the majority (19/29) of the isolates belonged to DLST type 25-11 and 5-91.
In addition, 8 bla NDM-1 positive isolates were clustered into two DLST types and 3 singletons. Accordingly, bla NDM-1 positive isolates were relatively heterogeneous, however, the route of transmission is not clear. These results highlight the importance of investigating carbapenem-resistant P. aeruginosa isolates in health care settings in our region.

Conclusions
The occurrence of bla NDM-1 isolates in P. aeruginosa is a large challenge in the treatment and worrying for global health. DLST type 25-11 is a significant cluster because a large number of bla NDM-1 isolates showed this genotype and also DLST type 5-91 known as an alarming type in burn patients. This work suggests that the DLST as an appreciated method in typing of bla NDM−1 strains; this technique reducing considerably the time and the cost of the molecular analysis and providing a reliable phylogenetic study. This information can help to generate the proper strategies for accurate and specific use of this antibacterial which can help to control of bla NDM-1 isolates.