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

BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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 NDM enzymes [5]. The MBLs encoding genes such as blaVIM and blaIMP 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 [6-8]. The blaNDM-1 producing strains are resistant to a wide-ranging 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 [8-10].

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 blaNDM-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 blaNDM-1 positive P. aeruginosa isolates obtained from different part of Iran.

Methods

Study design, sampling, and bacterial isolates

A cross-sectional study was conducted at three major teaching Hospitals (Ahvaz, Tehran, and Isfahan) in Iran during three-year period. In total, 369 non-duplicate P. aeruginosa isolates were collected from different clinical sources such as trachea (84/369), wound (51/369), urine (79/369), punch biopsy (62/369), blood (34/369), sputum (35/369) and other (24/369). These samples were obtained from patient hospitalized in intensive care (ICU) and neonatal ICU (174/369), internal (149/369), emergency (11/369), other (15/369) and 20 samples from outpatients referred to laboratory center [13].

A total of 29 non-duplicate blaNDM-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 blaNDM, blaIMP, blaVIM, blaKPC, blaGES, blaSPM and blaOXA-10 using a set of specific primers on a thermal cycler (Eppendorf AG, Germany) as described previously [15-17]. Sequencing of the amplicons was performed by the Bioneer Company (Bioneer, Daejeon, South Korea) and the nucleotide sequences were analyzed using GenBank nucleotide database at http://www.ncbi.nlm.nih.gov/blast/.

Antimicrobial susceptibility testing

Antibiotic susceptibility of the blaNDM-1 positive isolates was determined by the Kirby–Bauer method as recommended by the CLSI. The 11 standard antibiotic disks used include: imipenem (10 µg), meropenem (10 µg), ertapenem (10 µg), ceftazidime (30 µg), cefotaxime (30 µg), cefepime (30 µg), gentamicin (10 µg), piperacillin/tazobactam (100/10 µg), amikacin (30 µg), ciprofloxacin (5 µg) and aztreonam (30 µg) (Mast Group Ltd, UK). The ESBL phenotype was identified using combined disk method by disks of ceftazidime (30 mg) with (10 mg) and without clavulanic acid (Mast Group Ltd, UK), applied to all blaNDM−1 positive isolates (15). Moreover, the minimum inhibitory concentrations (MICs) of imipenem (10 µg/ml) [≤ 2 mg/L (susceptible), 4 mg/L (intermediate), and ≥ 8 mg/L (resistant)] (Liofilchem, Roseto degli Abruzzi, Italy) were applied by gradient test strips to blaNDM−1 positive P. aeruginosa isolates [18].

Carbapenemase screening

The double-disk potentiation tests (DDPT) and double disk synergy test (DDST) was performed phenotypically for all blaNDM−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).

Fig. 1

Dendrogram of DLST result analysis of 29 blaNDM-1 P. aeruginosa clinical strains isolated from hospitals in three different cities (Tehran, Ahvaz, Isfahan) of Iran. Results were clustered by UPGMA and compared by Dice Methods; Is; Isfahan, Ah; Ahvaz, Te; Tehran, G; General hospital, B; Burn hospital

Results

During the three years of study, twenty-nine of blaNDM-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 blaNDM-1 isolates was 3.22 (n= 20:9). The most blaNDM-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 blaNDM-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 blaNDM-1 positive isolates were defined as MDR. The full results of antibiotic resistance pattern of blaNDM-1 positive P. aeruginosa isolates showed in Table 2.

Table 1

The detailed results of P. aeruginosa with and without blaNDM-1

Variable	P. aeruginosa (without blaNDM-1) N=340 (%)	P. aeruginosa (with blaNDM-1) N=29 (%)	p-value
Sex
Male	199 (58.5)	20 (69)	0.2
Female	141 (41.5)	9 (31)
Type of sample
Trachea	80 (23.5)	4 (13.8)	0.2
Urine	68 (20)	11 (37.9)	197
Punch	60 (17.6)	2 (6.9)	0.1
Wound	42 (12.3)	9 (31)	0.005
Sputum	34 (10)	1 (3.5)	0.2
Blood	32 (9.4)	2 (6.9)	0.6
Other	24 (70.5)	–	–
Type of patients
Burn	45 (13.2295)	6 (20.7)
ICU	153 (45)	21 (72.4)	0.09
Internal	143 (42.1)	6 (20.7)	0.02
Emergency	11 (3.2)	–	–
Outpatients	18 (5.3)	2 (6.9)	0.7
Other	15 (4.4)	–	–

ICU Intensive care unit

Table 2

The detailed results of blaNDM−1 isolates

Imipenem (IPM), meropenem (MEM), ertapenem (ETP), piperacillin-tazobactam (TZP), cefepime (CFP), amikacin (AN), ciprofloxacin (CIP), gentamicin (GEN), ceftazidime (CAZ), cefexime (CTX), azteronam (AZT).َ A, Ahvaz; I, Isfahan; T, Tehran; OP, Outpatient; ICU

The MICs of imipenem against blaNDM−1 positive P. aeruginosa isolates are presented in Table 1. Overall, the results of MICs of imipenem showed 82.7% (24/29) isolates were high-level imipenem-resistant isolates (MIC ≥32). DDST and DDPT revealed that 15/29 (51.8%) and 26 (89.7%) of blaNDM-1 positive isolates were MBL producing isolates, respectively. In addition, the results of MHT showed that 27/29 (89.6%) of blaNDM−1 positive P. aeruginosa isolates were carbapenemase-producing isolates.

The distribution of carbapenemase genes and other antibiotic resistance genes among blaNDM-1 positive P. aeruginosa isolates are presented in Table 2. However, PCR analysis showed none of the blaNDM-1 positive P. aeruginosa isolates contained blaKPC and blaGES genes. The presence of blaVIM-2, blaIMP-1 and blaSPM genes were detected in 41.4%, 34.5% and 3.5% isolates, respectively. Among blaNDM-1 positive P. aeruginosa isolates, the blaOXA-10 beta-lactamase was the most frequently gene recognized in 86.2% (25/29).

The combination of blaVIM-2/ blaOXA-10 and blaIMP-1/blaOXA-10 beta-lactamase was found in 8 (27.6%) and 10 (34.5%) isolates, respectively. Moreover, among blaNDM-1 positive P. aeruginosa isolates the co-harboring of three genes, blaOXA-10, blaIMP-1 and blaVIM-2 was found in two isolates and only one isolate contained blaSPM that in combination with blaVIM-2 and blaOXA-10.

In the current study, the DLST method was tested for all blaNDM-1 isolates recovered over a period of four years from various hospital wards. DLST results revealed partial diversity among 29 blaNDM-1 positive isolates. Totally, 8 different DLST profile (DL type) (four different common types and three single type) were detected (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 blaNDM−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 blaNDM-1 producing of P.aeruginosa isolates with phenotypic and genotypic methods.

The overall data show that the frequency of blaNDM-1 producing P. aeruginosa isolates was 7.8% (29/369). In addition, there are variable reports of blaNDM-1 from different countries in Europe and Asian. The blaNDM-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 blaNDM-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 blaNDM-1 in the north of Iran [25]. Recently, Azimi et al. described a lower frequency rate for blaNDM-1 (7%) among carbapenem-resistant P. aeruginosa [26]. Moreover, the blaNDM−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 blaNDM-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 blaNDM-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 blaNDM-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 blaNDM-1 gene demonstrating the sensitivity of bacteria to aztreonam, 62% of blaNDM-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, blaOXA-10 was the most frequently detected beta-lactamase among blaNDM-1 positive strain and blaIMP-1 was second, which is in contrast to other reports where blaVIM was significantly associated with blaNDM-1 [31, 32].

One of the important findings in this study was the emergence of the co-harboring of blaNDM-1 positive P. aeruginosa isolates with more than one carbapenemase gene and metallo β-lactamase determinants, simultaneously. Accordingly, we report the first isolate of P. aeruginosa producing four carbapenemases co-existence blaNDM-1, blaVIM--2, blaIMP-1 and blaOXA-10 from Iran. One of them that was obtained from urine sample was resistant to all antibiotics used except to TZP that were intermediate. Furthermore, we demonstrated the co-harboring of blaNDM-1 with metallo-β-lactamases genes such as blaOXA-10, blaIMP-1 and blaVIM-2 in P. aeruginosa. The coexistence of carbapenemases encoding genes with blaNDM-1 positive P. aeruginosa isolates has been reported in several Asian and European countries including in India (blaNDM-1 + blaIMP+ blaVIM+ blaSPM), Denmark (blaNDM-1 + bla VIM-5+ blaVIM-6) [33], Bangladesh (blaNDM-1, blaVIM-1, blaVIM-2, bla IMP-1) [34] and Turkey (blaVIM-1+ blaVIM-2+ blaGES-5 [35].

The previous studies revealed that the acquisition of MBL determinants such as blaNDM-1, bla VIM, bla IMP and blaSPM 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 blaNDM-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 blaNDM-1 positive isolates were clustered into two DLST types and 3 singletons. Accordingly, blaNDM-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 blaNDM-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 blaNDM-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 blaNDM−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 blaNDM-1 isolates.