Molecular Characterization of Carbapenemase- Producing Gram-negative Bacteria Isolated from Clinical Specimens in Baghdad, Iraq

The emergence and spread of carbapenem-resistant Gram-negative bacteria is a worldwide emerging public health threat responsible for large number of nosocomail infections. Metallo-β-lactamases including IMP, VIM, and NDM as well as carbapenem hydrolyzing class D β-lactamase (OXA-48 like) are the predominant types that confer resistance to Carbapenem group of antibiotics. The aim of this study was to identify the carbapenemase encoding genes among Gram negative bacteria isolates. 42 isolates were identified depending on routine morphological tests followed by species identification using the VITEK 2 system. The 16S rDNA gene sequence was used for confirmation of the detection of Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrobial susceptibility testing was performed using VITEK 2 system. For phenotypic detection of carbapenemase activity, modified carbapenem inactivation method (mCIM) was performed. The carbapenemases encoding genes (blaiMP, blasPM, blaVIM, blaNDM, blaKPC, blaBIC, blaOXA, blaAiM, blasiM, blaGiM, blaDIM) were amplified by PCR and the amplified products were sequenced. Forty two Gram-negative bacteria isolates including 25 of P. aeruginosa (59.5%) and 17 of Enterobacteriaceae family (40.4%) were identified. According to PCR-based method results, carbapenemase gene blaOXA-48 was detected in 31(73.8%) of isolates, blaVIM in 23 (54.7%) and blaNDM in 2(4.76%) of isolates. Twelve (28.5%) of isolates harbored a combination of blaOXA-48 and blaVIM, (2.4%) coexistence blaOXA-48 and blaNDM gene and (2.4%) of isolates harbored a blaOXA-48, blaVIM and blaNDM genes. No other carbapenemase genes were identified. Based on the present study, it was concluded that the high prevalence was in blaOXA-48 gene, followed by blaVIM gene among carbapenemase-producing Gram-negative bacteria isolates.


INTRODUCTION
Gram-negative bacteria that produce Carbapenemase have been related to increased mortality and critical nosocomial outbreaks that represent the main challenge in both therapeutic and infection control 1 .
The concern of carbapenemase-producing Gram-negative bacteria that emerged currently is due to it is often related to the occurrence of multiple drug resistant isolates for which few choices of antimicrobials stay available 2 .
Carbapenems are β-lactam antibiotics that used most frequently as last resource antibiotics for treating of multidrug-resistant Gram negative bacilli-causing infections, since they have the wide spectrum of bactericidal action and their stability against most of the β-lactamases, including ESBLs 3 .
The increase of carbapenem resistance in these microorganisms is a major concern globally. The most common mechanism of resistance is the production of carbapenem-hydrolysing enzymes, carbapenemases that hydrolyse most β-lactams 4 .
These enzymes encoding by multiple genes of resistance, which is associated with different mobile genetic determinants, thus conferring resistance to various classes of antimicrobials, such as aminoglycosides, fluoro-quinolones, tetracyclines, trimethoprim, sulphonamides, and phenicols 5 .
The major public health threat is with transmissible carbapenemases, which can increase the rate of mortality and limit the choice of appropriate antibiotic therapy 6 . The transmissible enzymes can be acquired unpredictably by important nosocomial pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii and members of the family Enterobacteriaceae 7 .
Because of the lack of implementing standardized protocols for detection of carbapenemase-producing isolates in many countries that probable to be the major reservoirs, the actual prevalence of these producers is still unknown. It is estimated that we are on the rim of a global epidemic with carbapenemase-producing isolates, which in the hospital environment is likely to be caused mostly by all types of nosocomial carbapenemase-producers (e.g. KPC, IMP, VIM, NDM and OXA-48) 9 .
In Iraq, we showed that a significant increase in carbapenem-resistant bacteria in the last two decades, especially after the 2003 War, maybe is due to the Iraq's openness to the world and the entry of foreign workers, especially from the endemic area from the Indian subcontinent like Bangladesh and other countries.
For all of the mentioned above, as well as, few report investigated the molecular basis of resistance to carbapenems among Gram-negative bacteria, this study aimed to identify the genes which is responsible for encoding carbapenemase enzymes in these organisms.

MATERIALS AND METHODS Bacterial isolates
A total of 42 different Gram-negative isolates were collected from various patient specimens from different hospitals in Baghdad city, Iraq during a period between October 2017 and February 2018. The isolates were identified initially depending on morphological characteristics as described previously 10 , followed by identification by using VITEK® 2 compact system (bioMeriux, France). Genotypic method was used to confirm the identification of isolates at species level using 16S rDNA gene sequences.

Phenotypic detection of carbapenemase production
The modified carbapenem inactivation method (mCIM) was performed according to CLSI guidelines 11 . In briefly, using sterile inoculating loop, 1µl of test organism was suspended in 2ml of tryptic soy broth, the bacterial suspension was homogenized by vortex. Then, a 10-µg meropenem disk was immersed into the suspension. Subsequently, the culture was incubated for 4 hours at 35°C, prepared 0.5 McFarland suspension of E. coli ATCC® 25922(a carbapenem-susceptible strain) that was inoculated on Muellar-Hinton agar(MHA) plates streaked as cell lawn.
After the incubation, the disk was removed using a 10-µl inoculating loop; the loop was dragged along the edge of the tube during removal to remove excess liquid, and the disk was placed onto the inoculated MHA plate, which was then incubated in for 18-24 hours at 35°C. Following the incubation, diameter of the inhibition zone around the disc was measured, a zone diameter of 6-10 mm or presence of colonies within a 16-18 mm zone was considered to be a positive result, 16-18 mm an indeterminate result, and 19 mm a negative result.

Genotypic identification of carbapenemaseencoding genes DNA isolation of bacteria
Pure culture of bacterial isolates were grown overnight in liquid nutrient broth medium (NB) for the isolation of genomic DNA using the Genomic DNA purification kit (Promega, USA) according to the manufacturers protocol.
All isolates were subjected to molecular screening to detect carbapenemase-encoding genes by using PCR amplification technique. In this study, multiplex PCR was used to detect carbapenemase encoding genes from clinical isolates and the PCR products were sequenced.

Multiplex PCR
Eleven pairs of primers (Alpha DNA, Canada) were used in this method, that defined into 3 multiplex reaction. No.1 included detection of bla IMP , bla SPM and bla VIM genes, No.2 included detection of bla NDM , bla KPC , bla BIC and bla OXA genes and No. 3 included detection of bla AIM , bla SIM , bla GIM and bla DIM genes.
Different primers (Table 2) were used, the PCR reaction mixture contained: 2µl of template DNA, 12.5µl of Go Taq Green Master Mix (2x) (Promega (USA), 1µl from each of the following primers : IMP, SPM ,VIM, NDM, BIC, KPC or OXA and the volume was completed to 25µl with nuclease free water. While the reaction mixture of 25µl for each of the following primers: AIM, SIM, GIM or DIM composed from 2µl of template DNA,12.5µl of Go Taq Green Master Mix, 1µl of each primers and 1.5µl of dimethyl sulfoxide (DMSO) and the volume was completed to 25µl with nuclease free water 12 . Cycle conditions were as followed: 10 min at 94°C and 36 cycles of amplification consisting of 30 sec at 94°C, 40 sec at 52°C and 50 sec at 72°C, with 5 min at 72°C for the final extension.
T h e a m p l i f i e d p r o d u c t s w e r e electrophoresed in 2% agarose gel in 1x TBE buffer containing red safe dye at 100 V for 50 minutes. Then, the PCR products were visualized under UV light by UV transilluminator. The E.coli ATCC 25922 strain was used as negative control.

Sequencing of PCR products
The amplified PCR products were sequenced at Macrogen DNA sequencing Company (Seoul, Korea). DNA sequences were analyzed and compared with standard strain using BLAST (Basic Local Alignment Search Tool) in National Center for Biotechnology Information website (http://www. ncbi.nlm.nih.gov/ BLAST).

Genebank accession numbers
The 16S rDNA gene, bla VIM -2 gene, bla OXA -48 gene and bla NDM gene sequences from this study were deposited in Genbank database under accession numbers MK182251 to MK182258, MK156197 to MK156202 and MK159338 to MK159352.
For mCIM, all isolates showed positive results (25 isolates of Pseudomonas aeruginosa and 17 isolates of Enterobacteriaceae family)  indicating the production of carbapenemase by these strains. Molecular tests confirmed that at least one carbapenemase gene in all isolates that were phenotypically carbapenemase positive, with the exception of one Pseudomonas aeruginosa isolate was carbapenemase-positive by phenotypic test despite being negative for the detection of carbapenemase encoding genes. Multiplex PCR-based methods were conducted to detect the carbapenem-resistant

DISCUSSION
Carbapenemase-producing bacteria have become a major problem worldwide, which has emerged due to the increased dependence on carbapenems as a last resource to treat bacteria with multidrug-resistant 16 .
Carbapenemases represent the stringent threat for global human health and stand as one of the most challenging issues facing infectious disease containment in the subsequent years 17 .
Notwithstanding the small number of isolates, the author found the dominant OXA-48 carbapenemases among studied isolates.
T h e m o st c u r re nt a n d co n c e r n development is the rapid rise in emerging and dissemination of OXA-48, particularly in K. pneumoniae. In 2001, the OXA-48-producing Enterobacteriaceae was first identified in Turkey, then later reported in various countries including the Middle East, North Africa, and Europe 18 . In a local study carried by Abdulla et al. (2016), they reported that the bla OXA-48 genes were detected in 25%, of the E.coli isolates and 21.4% in K. pneumoniae isolates 19 .
Several studies in regional countries reported the predominance of bla OXA -48 among Gram negative bacteria, as it was 49% in Arabian Gulf 20 , 53.3% in the UAE 21 , 88% in Lebanon 22 , 49.2% in Egypt 16 and 86% in Turkey 8 , on the other hand, a study by Mohamed et al., reported decrease in the rate (22.4%) of this gene in P.aeruginosa isolates 23 .
In regard to existence of the bla VIM gene, the results also showed increasing in prevalence rate, as it was 54.7% of carbapenem-resistant isolates have possessed this gene.
A local study done by Al-Jubori et al, (2016) showed that the prevalence rate of bla VIM gene was 25% in A. baumannii 27 , while another study done by Hammadi et al .(2015), reported that all E.coli isolates did not carry bla VIM gene 28 .
The VIM types are the most frequent among class B carbapenemases which have been detected in all continents 29 . VIM enzymes were firstly reported in isolates of P. aeruginosa, and then emerged in Enterobacteriaceae as well. Subsequently in a number of regional countries, a study carried out in Saudi Arabia describing that P.aeruginosa strain harboring the bla VIM -2 gene from a Saudi patient hospitalized in France 30 .
In Iran, Rajabnia et al. (2015), reported that the bla VIM -1 gene are presence in 30% of K. pneumoniae isolates 31 , while in Turkey, Haciseyitoglu et al. (2017) found that the percentage rate of this gene was low when it reached only 10% in E.cloacae 32  Since the detection of NDM-1 is firstly reported in India, there has been a global rise in the dissemination of NDM-1 carrying organisms. At first, the existence of NDM-1 was predominantly reported in Enterobacteriaceae, but reports occurring recently pointed out to its spread in Acinetobacter spp. and Pseudomonas spp. as well 37 .
The author found that bla NDM gene is presence in two isolates (4.7%) of P.aeruginosa among carbapenem resistant isolates.
In local studies, Al-shara et al. (2014) reported that out of 36 carbapenem resistant P.aeruginosa isolates, only 5.6% of isolates harbored bla NDM gene 38 , another study by AL-Harmoosh (2015) showed that the prevalence rate of bla NDM -1 gene was 20% 39 , while in recent study by Hussein (2017), revealed that bla NDM -1 gene was 40% in E.coli isolates 40 , however Hammoudi et al. showed that the percentage of the prevalence of bla NDM -1 gene was 100% in isolates 41 .
Bacterial isolates that produce NDM-1 enzyme may express numerous other unrelated resistance genes, such as OXA-48 type and VIM type that encode other carbapenemases, AmpC, extended-spectrum beta-lactamases, and other classes of antimicrobials 42 .
The prevalence of NDM-1 producing isolates were reported from different countries including the Gulf Corporation Council (GCC) which investigated in a total of 200 isolates collected from 16 hospitals in Saudi Arabia, Kuwait, Oman and the United Arab Emirates. Overall, NDM-1 was the most common encountered carbapenemase gene 46.5% 43 , 47.6% in Egypt 16 , 29.5% in Turkey 8 and 7.8% in Tunisia 44 .
In Kuwait, Jamal et al. reported that two of the bla NDM -1-producing isolates co-harbored bla OXA -48 carbapenemase 48  A K. pneumoniae co-producing NDM-1 and OXA-232 (an OXA-48 variant) was imported to the USA from India 49 , and another was found also in a French hospital 50 , where its cross-transmission was documented 47 .
The ratio of carbapenemase producing isolates differs by geographic region, type of infection, specimen source, and selective pressure due to antibiotics. This difference also associated with variation among the different patients studied and the different rates of antibiotic used in different hospitals 51 .
The diversity of carbapenemases depends on the country; may be affected by historical and cultural relationships 17 . In our country, the wars, medical tourism, and Cross border transfer of patients particularly incoming workers might play a significant role in emerging and dissemination of different variants of carbapenemase encoding genes. There is an urgent need to find guidelines and appropriate procedures of infection control in order to deny such infections among patients.