Evaluation of bla GES-5 and bla veb-1 genes with multidrug-resistant extend, pandrug resistance patterns (MDR, XDR, PDR), and biofilm formation in Pseudomonas aeruginosa isolates

: Pseudomonas aeruginosa is a ubiquitous microorganism that is difficult to treat due to the increasing prevalence of multidrug resistance patterns. A total of 227 samples were taken from different clinical samples during the study period from January 2018 to December 2018. The isolates were identified with antibiotic sensitivity testing with ESBL by the Vitek-2 automated method. MDR, XDR, and PDR were determined. 40 (17.6%) isolates were positive for P. aeruginosa , maximum of ESBL and MBL. Positive isolates were detected in the burn, coexisting ESBL + MBL enzymes in 21 (52.5%) of our isolates. Imipenem followed by Meropenem were found to be effective against ESBL and MBL producers. Resistance was reached between 72-100% to 5 antibiotics. The frequency of PDR, MDR, and XDR were 5%, 50%, and 45%, respectively. The frequency of co-production between MDR, XDR, and PDR with MBL, ESBL, and Biofilm was 35%, 12.5% and 5%, respectively. Among the ESBLs, the frequency of distribution of bla VEB-1 gene and bla GES-5 gene was 50% and 40 %, respectively. Bacterial isolates simultaneously carrying bla VEB-1 gene with multiple β-lactamases of different classes of biofilm, MDR, PDR, and XDR as same as a coexisting bla GES-5 gene . One isolate was detected as new isolates registered in global gene bank as locally P. aeruginosa isolates in Erbil city (LOCUS MN900953). The phylogenetic trees of the bla VEB gene isolates were demonstrated a genotype closely related to others, deposited in GenBank similar to the P. aeruginosa gene; gene sequencing revealed a 99% similarity with other isolates deposited in GenBank.


Introduction
Pseudomonas aeruginosa is the major cause of nosocomial infection in medical settings, bringing about severe infection in patients with background diseases (1). For such infections, antimicrobial therapy may become a difficult task because P. aeruginosa is resistant to many antibiotics and even develops resistance to several antibiotics during treatment. In this context, infections of P. aeruginosa that demonstrate acquired resistance to beta-lactam drugs are one of the most challenging targets for antimicrobial therapy and are responsible for high rates of therapeutic failure, increased mortality, morbidity, and overall cost of treatment (2). Those strains of P. aeruginosa that are resistant to at least one or more of the three or more classes of anti-pseudomonal antibiotics are called multi-drug resistant (MDRs). The term extremely drug-resistant (XDR) refers to those isolates that are resistant to at least one antibiotic agent from six or more of the anti-pseudomonal antimicrobial groups. If a P. aeruginosa isolate is resistant to all anti-pseudomonal antimicrobials, it is called Pandrug resistant (PDR) (3). ESBLs are a group of enzymes produced by certain microorganisms with the ability to hydrolyze a broad range of β-lactams, including penicillins, cephalosporins, and aztreonam, whereas cannot hydrolyze cephamycins and carbapenems (i.e., Imipenem) professionally.In general, ESBLs are not codified by the bacterial chromosome but are usually codified by transferable plasmids. The transferable plasmids can harbour many different genes and have the ability to transmit a copy of themselves to other bacteria (4). These enzymes are a major concern regarding the future of antimicrobial chemotherapy because of their rapid transition among microorganisms. Microbial biofilm is a structure of the microbial community that is found attached to living or non-living surfaces. These three-dimensional structures are always enclosed within a complex matrix. Biofilm-producing microorganisms -compared to non-producing counterparts -are often more resistant to antimicrobial agents; so that in some cases, three to four times the concentration of antibiotic compounds is required to kill biofilm-producing microorganisms, depending on the antibiotic composition used (5). Various mechanisms including delayed penetration of the antimicrobial agents through the biofilm matrix, the altered growth rate of biofilm organisms, and other physiological changes endow resistance to biofilm-forming microorganisms (6). Thus, biofilm formation paves the road for pathogen's survival and persistence under unfa-vorable conditions like host invasion or following antibiotic treatment (7). The epidemic prevalence of MDR / XDR strains in the healthcare setting has always been a concern of pathologists. In recent years, evidence of MDR / XDR spread in various hospitals across the world has been reported, indicating the presence of high-risk clones (8). Early detection of gram-negative bacteria such as P. aeruginosa, which produces MBL and ESBL, is of particular importance to medical centers to prevent the spread of these strains and to provide appropriate medical services to hospitalized patients and patients with acute illness (9). In P. aeruginosa, five types of acquired class A ESBL were identified, VEB-, PER-, GES, TEM-and SHV. OXA type ESBLs belong to molecular class D. VEB, PER, and OXA are the most predominant ESBL types reported in P. aeruginosa. The molecular classification of β -lactamase bacteria relies on the sequence of nucleotides and amino acids in these enzymes (4). bla VEB (for Vietnamese extendedspectrum beta-lactamase), bla VEB-has the highest aminoacid similarity with Pseudomonas Extended Resistance (PER-1) and (PER-2) (38%), which confers remarkable resistance to Ceftazidime, Cefotaxime and Aztreonam (10). Genes encoding several enzymes that cause resistance to Beta-lactame antibiotics are classified in Class A which includes but is not limited to bla VEB, and GES enzymes encoding genes. ESBL are enzymes produced by Gram-negative bacteria that are encoded by plasmid gene sequences (11). The bla VEB-1 gene has been reported in several enterobacterial isolates from Southeast Asia (12) and P. aeruginosa clinical isolates (13), GES-type b-lactamase was originally identified as a cephamycinhydrolysing extended-spectrum b-lactamase family. Twenty-six variants of the GES group have been identified, some of which are classified as carbapenemases (14) bla  have been reported worldwide as a carbapenemase harbored Klebsiella pneumoniae, Escherichia coli, and P. aeruginosa (15). Prior to the outbreak described in this report, only GES-3 and GES-4 had been detected in Japan (in K. pneumoniae). The MDRP of the initial case was either acquired within the community or imported from abroad. As this is the first report of GES-5 detection in Japan, we will be able to monitor the possible spread of GES-5 in the surrounding area. Molecular detection and monitoring of resistance genes are of great importance to establish appropriate antimicrobial therapy and to evaluate and monitor the spread of drug-resistant P. aeruginosa. This research was carried out to determine the presence of blaveb-1 and blages-5 among ESBL and MBL production and biofilm formation among MDR, XDR, and PDR producing P. aeruginosa isolated from different clinical samples (16).

Isolation and identification of bacteria
This present study was carried out in Rizgary, Teaching hospital, Laboratory center, Raparin, Nanakaly hospitals in Erbil province at a period from (September 2016 -March 2017). A total of 227 samples (sputum, urine, wound, burn, Bronchial wash) were collected and inoculated onto Blood Agar and MacConkey Agar plates. Plates were incubated at 37°C for 24-48 hours under aerobic conditions. Provisional identification of P. aeruginosa was performed based on microscopic properties, culture characteristics, biochemical tests, definitive identification by Vitek 2 Compact: colorimetric technology for bacterial identification; gram-negative (GN) card was used for P. aeruginosa identification. All the following steps were done according to the manufacturer's instructions (Biomerieux, France). The Ethics Committee approved the study of Hawler Medical University/College of Health Sciences.

Antimicrobial susceptibility test
In vitro susceptibility testing of all collected isolates was performed using the VITEK 2 compact system, an automated ID and susceptibility (AST) system for a wide range of antimicrobials. VITEK (17). Final results were represented using the Advanced Expert System (AES) that is the experiment was repeated wherever indicated by the AES (18). Multidrug-resistant (MDR, strain resistant to at least one antibiotic out of three or more antimicrobial classes), extensively drug-resistant (XDR, resistant to at least one antibiotic in all but two or fewer antimicrobial classes (i.e. bacterial strain shows susceptibility to only one or two categories), (PDR, resistant to all antibiotic classes) were determined based on the guideline of European Centre for Disease Prevention and Control (ECDC), and Centers for Disease Control and Prevention (CDC).

Phenotypic screening for ESBL
VITEK 2 system equipped with the antimicrobial susceptibility test extends AST-EXN8 card was used to test all isolates of P. aeruginosa. This system was designed to perform both screening and confirmatory tests for phenotypic detection of ESBL on the same plate. The test comprises a panel of six wells containing ceftazidime 0.5 mg⁄ L, cefotaxime 0.5 mg⁄ L, and cefepime 1.0 mg⁄ L; the same antibiotics in combination with clavulanic acid (4, 4, and 10 mg⁄ L, respectively) were poured in the other three wells. An optical reader was used for the quantitative measurement of bacterial growth. The reduced bacterial growth in wells containing Cephalosporin/clavulanate compared to those only containing the cephalosporin was regarded as the indicator of ESBL production. All phenotypic interpretations of ESBLs were reported as positive ESBL screening results. Strains were reported as ESBL-negative whenever phenotypic interpretations other than ESBLs were proposed by the AES (18) .

Metallo beta lactamase
The double disk approach was used to detect MBL production in P. aeruginosa isolates. Bacterial colonies were suspended in Mueller-Hinton broth; the suspension turbidity was calibrated to equal 0.5 McFarland stan-USA) through BLAST searches. Sequence alignment was performed with closely related reference sequences from other P. aeruginosa isolates, available in Gen-Bank, using the BLAST (http://www.ncbi.nlm.nih.gov). A neighbor-joining tree with combined bla VEB data was constructed, according to the maximum likelihood method, using Molecular Evolutionary Genetics Analysis (MEGA) 7 software (ver. 7, Pennsylvania State University, State College, PA, USA) (21).

Statistical analysis
Statistical Package for Social Sciences (SPSS) software, version 22 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Fisher exact test or chisquared test was performed in order to analyze categorical data. A P value of <0.05 was considered statistically significant.

Results and discussion
P. aeruginosa represents a major cause of acute nosocomial infections, particularly in critically ill and immune-compromised patients. Indeed, P. aeruginosa is the top pathogen causing ventilator-associated pneumonia and burn wound infections and is a major cause of nosocomial bacteremia, with a very high (>30%) associated mortality rate (22). Out of 227 samples investigated in this research, 40 (17.6%) isolates were positive for P. aeruginosa, as shown in Figure 1. It was observed that the most incriminating in burn infection account for (2.6%) of the total number of isolates, the results obtained agreed somewhat with the findings of (23), who found P. aeruginosa the most commonly cultured organism (54.2%) isolated from mild to severe form of external otitis and chronic suppurative otitis media. In contrast, 16% and 46.3% prevalence of P. aeruginosa obtained in wound swabs investigated was not similar to the findings of the present study. In contrast, Fifty-four clinical isolates of P. aeruginosa were collected from Selayang Hospital, Selangor, Malaysia most isolates thirty-six percent were identified from pus followed by dards and then the suspension was spread over Mueller-Hinton solid medium (Hi Media, Mumbai, India). Disks containing either Imipenem alone (10μg) or Imipenem (10μg)/ EDTA (750 μg/disc) were placed at the distance of 20 mm (center to center). After overnight incubation at 35 °C, a ≥ 7 mm increase in the inhibition zone of diameter around Imipenem-EDTA discs, as compared to imipenem discs alone, was interpreted as indicative of MBL production (19).

Phenotypic screening of biofilm formation
Congo red agar method P. aeruginosa suspension was inoculated on a specially formulated solid medium (BHI) with 5% sucrose and Congo red. The medium consisted of BHI (37 gms / L), sucrose (50 gms / L), agar no.1 (10gms / L) and red stain (0.8 gms / L) from the Congo. Red Congo was prepared as a condensed water solution and autoclaved for 15 minutes at 121 °C. The agar was then applied separately from the other medium constituent when it had cooled to 55 °C. Plates were inoculated and aerobically incubated at 37 C for  hours. Black colonies with solid, crystalline consistency showed a good outcome. Weak slime producers generally remained pink, while occasional darkening was observed in the center of the colonies. An indeterminate result was an obscuring of the colonies with the lack of a dry crystalline colonial morphology. The triplicate experiment was conducted and replicated three times (20).

Detection of bla VEB , and bla GES genes
Polymerase chain reaction (PCR) using specific primers (Table 1) was performed to detect bla VEB-1 , and bla Ges-5 genes. Total DNA was extracted from the isolates using a DNA extraction kit (Bioneer, Daejeon, Korea). PCR procedure was conducted in a thermocycler (Applied Biosystems, Carlsbad, CA, USA) as follows: 96 o C for 5 min; 35 cycles of 1 min at 96 o C, 1 min at a specific temperature for each primer, and 1 min at 72 o C; and a final extension step of 10 min at 72 o C. Amplification mixture was prepared in a total volume of 25μl including 12.5μl Taq DNA polymerase 2× Master Mix with 1.5mM MgCl 2 (Ampliqon, Odense, Denmark), 0.5μM of both forward and reverse primers, 9μl nuclease-free water, and 2.5μl DNA template (50pg concentration). PCR products were electrophoresed on a 1% agarose gel at 100V, stained with ethidium bromide solution, and finally visualized with a gel documentation system (UviTec, Cambridge, UK).

DNA Sequencing
PCR products were sent to National Instrumentation Center for Environmental Management for Sequencing (Seoul, Korea). bla VEB sequences were compared with those available in the GenBank database (National Center for Biotechnology Information, Bethesda, MD,  respiratory tract (22%) and urine (18.51%) (24). This slight difference can be attributed to geographical factors and also the duration of the experiment. In our findings were found 29 (72.5%) as MBLs producer of P. aeruginosa as in Table 2 higher than a study (23.62%) done by Kamble, 2015 (25), and higher than a study done in India by Agrawal et al (26) which showed only 8.05%. A study conducted in India, Brazil and Iran revealed 32.9%, 30%, 48.3% MBL production respectively (27,28) reported that the percentage of metallo-β lactamase producers of P. aeruginosa was 68%. It is a nosocomial pathogen of particular clinical concern not only because of its extraordinary resistance mechanisms but also for its formidable ability to adapt very well to the hospital environment. In our finding, 80% ESBL production by P. aeruginosa was observed and corroborated with a study in Egypt where 95% of P. aeruginosa isolates were beta-lactamase producers, ESBL producing P. aeruginosa isolates were mostly detected in clinical isolates in a hospital set up according to the university hospital microbiology report. ESBL (29). We found coexisting ESBL + MBL enzymes in 21(52.5%) of the isolates, which was higher than that reported by (30) in which 14.36% of the isolates co-produced ESBL and MBL enzymes.
Biofilm production is a major property of P. aeruginosa enabling the bacterium to cause chronic infections. The remarkable trait of a mature biofilm is the formation of an extracellular matrix and increased resistance to antibiotics (31). In our study, 75% of the isolates were able to form biofilms under in vitro conditions, which is lower than 95% reported by (32). (33) showed the high potential of biofilm formation by clinical isolates of P. aeruginosa regardless of the sample source. In this research, 43.5% of P. aeruginosa burn isolates formed biofilms. Production of ESBL, MBL by non-biofilm and biofilm producers is presented in Table 2. Despite the maximum ESBL producers being biofilm positive, no significant association (P > 0.05) was found between isolates, which is similar to the findings reported by (34). Furthermore, there was no significant association between MBL production and biofilm formation. Among the biofilm-producing isolates, 10 (25%) were coproduction with ESBL and MBL producers, Production of MBL and ESBL in biofilm-isolates mostly occurred in burn samples 10 (25%) for each. As observed, coexisting between MBL and ESBL and biofilm-positive strains 10(25%) did significantly not vary higher.
Although most of the ESBL and MBL producers were resistant to antibiotics, as in Table 3, the association was not statistically significant (P > 0.05). Both Imipenem and Meropenem were found to be effective against ESBL producers. In contrast, a statistically significant association (P≤ 0.05) was established between MBL production and antibiotic resistance, and the resistance reached between 72 and 100% to 5 antibiotics; the finding agrees with the study of (35). Besides, various studies have demonstrated MBL producers as pan drug-resistant strains signifying an alarming threat (36). No significant difference was observed between antibiotic susceptibility profiles in biofilm-positive and biofilm-negative in isolates of P. aeruginosa. The association between the potential to form strong biofilms by P. aeruginosa and antibiotic resistance has also been reported elsewhere (37). The correlation between biofilm formation and antibiotic resistance was significant for aminoglycosides, fluoroquinolones, cephalosporins, imipenem, and piperacillin. Higher antibiotic resistance    in biofilm producers compared to non-producers has been reported in many studies (36).
The present study showed 20(50%) frequency of MDR P. aeruginosa as presented in Table 4. In comparison (38) reported a 22.7% incidence in Islamabad; while 29% prevalence of MDR P. aeruginosa was found by the study conducted in Peshawar (39) reported. The overall prevalence of antibiotic-resistant P. aeruginosa increases, with up to 10% of global isolates found to be multidrug-resistant (40).
P. aeruginosa (PDR) strains are the most challenging organisms for clinicians to be overcome. According to the results obtained in the present study, 2 (5%) of the total isolates were PDR, which is lower than that reported in Nepal (41). In a study carried out in a tertiary care hospital in Nepal, PDR was also isolated from the clinical samples. This indicates the alarming situation of MDR pseudomonal infections in ICUs of various parts of Nepal (42). Out of 66 isolates, 6.1% were PDR isolates; this sort of complete (or almost complete) lack of treatment options is an increasingly common and desperate occurrence in tertiary care medical centers. The resistance pattern is increasing in such a way that the evolution of PDR P. aeruginosa occurred (43). XDR percentage in the present research (45%) was much more than reported by (44) who reported the value of 26% in samples from Latin America between 2004 and 2015. Countries with the highest MDR were Guatemala, Venezuela, Honduras and Brazil (43.8, 32.6, 31.9 and 31.5%), respectively. So, rational use of antibiotics should be practiced, the prevalence of MDR/XDR P. aeruginosa is certainly on the rise worldwide. Although important geographical differences exist (45), the prevalence of MDR P. aeruginosa has increased in the past decade and currently ranges from 15% to 30% in many areas (46).
On the other hand, in our investigation indicates that burn samples were exhibited the majority of the three types of resistance 6 (42.8%) MDR and 6 (42.8%) XDR, and 2 (14.2%) PDR. It has been reported that patients suffering from background infections such as blood stream infection (septicemia), bronchial infection or diabetic foot ulcers are more susceptible to MDR/ XDR P. aeruginosa strain; whereas patients with other types of infection (urine, sputum, ear swabs, eye, and vagina) were 5 times less likely to be infected by an MDR/ XDR strain. In this sense, patients treated at the Intensive Care Unit were 3.4 times more likely to become infected by an MDR/XDR P. aeruginosa strain (47).
In addition, interesting results were obtained in the present study regarding coproduction of MDR, XDR and PDR with MBL&ESBL& Biofilm 14 (35%), 5 (12.5%), 2 (5%) respectively in clinical isolates. (10%) of MBL producers in our study were MDR and resistant to most of the antibiotics used as in Table 5. A similar result was obtained by (48). This may be attributed to the fact that MBL genes are often carried along with other resistance genes resulting in multi-drug resistance limiting treatment options (Table 5,6) (49).
The rising antibiotic resistance against commonly used drugs is of great concern. PCR assay was performed to reveal the prevalence of ESBLs genes among 40 isolates of P. aeruginosa (bla VEB-1 and bla GES-5-) genes as in Table 6 with amplified size bla GES-5 643pb, bla-VEB-1 642pb as depicted in Figure 2.
Among the ESBLs, the frequency of distribution of bla VEB-1 gene was 20(50%) and 16(40%) for bla GES-5 gene, respectively. Maximum bla VEB-1 positive isolates of P. aeruginosa were observed among burn samples, and maximum bla GES-5 positive isolates were detected in burn also. In the study by (50) in Tehran, the frequency of the bla VEB-1 gene was 100% while (51) also in Tehran observed that the frequency of bla VEB was 31.34%. Contrary to this value, bla VEB genes with 13.3% prevalence have    been reported in another study (52). Various studies have reported P. aeruginosa carrying bla GES gene (53). Another research conducted in Saudi Arabia confirmed the presence of genes encoding bla VEB and bla GES among extended-spectrum P. aeruginosa clinical isolates from burn patients'(54) Molecular characterization of bla VEB-1 like genes from several P. aeruginosa isolates showed variability among the bla VEB-1 like encoded proteins, as has been found recently in P. aeruginosa isolates from Kuwait (bla VE-1a and bla VEB-1b ) (13) 15 strains were isolated from burn units environment bla VEB-1 was found in 6 (40%) isolates (4) among those isolates, two isolates (4.6%) produced the ESBL GES-5, possessing the ability to hydrolyze imipenem isolated from blood in Brazil (55). It is believed that the clinical isolates with (bla-VEB-1 and bla  genes are associated with the MDR phenotype. Bacterial isolates simultaneously carry multiple β-lactamases of different classes, such as the ESBL20(50%) and MBL 17(42.5%)with biofilm 17(42.5%) and MDR 20(50%) and PDR 9(2.25%) and XDR 2(5%) with gene coexisting and simultaneously encoding ESBLsVeb-1 gene as in Table 7, coexisting bla GES-5 gene also with different classes, ESBL 16(40%) and MBL 13(32.5%) with biofilm 16(40%) and MDR 16(40%) and XDR 9(2.25%), PDR 2(5%) as seen in Table 8. An experiment in Egypt, (9) reported bla VEB-1 was 10.4% in P. aeruginosa with ESBLs and MBLs in 122 P. aeruginosa isolates; but another study (56) revealed that the frequency of MDR and ESBL-positive of the isolates were 19/116 (16.37%), and 8/116 (6.89%), respectively, and 4 isolates (3.4%), amplified bla VEB-1 .on the other hand, the production of bla GES has been shown to be related to expanded spectrum cephalosporin resistance (57). An analysis performed in a hospital in Riyadh, Saudi Arabia (between January to April 2010) indicated that 25 (16%) isolates of P. aeruginosa were ESBL producers, with 5 (20%) carrying blaGES genes (58). The first report in Iran (1) declared that the frequency of bla VEB , and bla GES among the ESBL isolates were 24%, and 0%, respectively. In another study, in contrast with the present study, the results of ESBLs genes detection showed that 78.6% of ESBLs producer of P. aeruginosa strains carried bla GES-gene while bla VEB genes appeared in 10.7% (59). Also, the prevalence of ESBL was 69.44%, and MBL was 42.85% with bla VEB-1 68% and bla GES-5 20% as ESBLs in P. aeruginosa. in Saudi Arabia (54) which was higher than our results. bla  was described originally in multidrug-resistant P. aeruginosa isolates collected from a South African teaching hospital, were associated with a nosocomial outbreak and displayed expanded hydrolysis to carbapenems, the simultaneous production of ESBLs and MBLs by the same isolate is known to further enhance resistance (13). P. aeruginosa XDR clinical isolate co-expresses an MBL and ESBL gene bla GES-1 in Peru (60), which confirmed our results. The genotype of human bacteria is important in causing a variety of abnormalities and cancers (61)(62)(63)(64)(65). Accordingly, research in this regard is valuable and necessary.
To the best of our knowledge, this is the first report of MDR P. aeruginosa clinical isolate that co-expressed an ESBL (bla VEB-1 . and bla  ) genes with MBL and XDR and PDR in Kurdistan.
In the analysis sequence of bla VEB-1 gene from Iraqi bacterial isolate P. aeruginosa as depicted in Figure 3, the selected isolate is registered in the global gene bank. Locally P.aeruginosa isolates in Erbil city from clinical samples don't match with global isolates and due to ge-    netic variation they may represent new isolates, thus the isolates were selected to register as a new isolate in the global gene bank. It is accepted under accession numbers (LOCUS MN900953) of nucleotides sequenced. The phylogenetic trees bla VEB gene encoding ESBL revealed a genotype closely related to others, registered in GenBank Table 9, from India, Iran, France, Brazil, Singapore, Kenya, USA, and China. Similar to the P. aeruginosa gene, gene sequencing revealed a 99% similarity with other isolates deposited in GenBank. Among the ESBL-producing isolates analyzed in the present study, 3 (8.5%) were carrying the bla VEB gene and 32 (91.4%). The phylogenetic trees of the bla-VEB genes isolates were also recorded by Al-Ouqaili et al., 2020 (66), isolated from Al-Karkh General Hospital (Baghdad, Iraq) representing similarity to those isolated elsewhere (24 countries) including the USA, Poland, United Kingdom, France, Germany, South Korea, India, Kenya, and China., with compatibilities of 99%, and showed high similarity with similar isolates from other countries.
Genetic variation in the sequence of this gene which the isolates distributed in the phylogenetic tree. Following the comparison of the sequence of blaVEB-1 in locally and global P. aeruginosa isolates documented in NCBI, the phylogenetic tree showed similarity between the local and global isolates.