Molecular Characterization of Resistance Genes in MDR-ESKAPE Pathogens

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Importance of ESKAPE in clinics
The ESKAPE pathogens able to get away from antibiotics biocidal action and generally show new example in pathogenesis, transmission trace and resistance pattern.The ESKAPE group contains Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp 1 Recently, understanding of virulence, resistance, transmission and pathogenicity of these microbes cause to innovative strategies for the progress of new antimicrobial options.Directing attention towards ESKAPE will help to concentrate antimicrobial resistance (AMR) challenge and allow efficient critical assessment of new antimicrobial drugs.Unfortunately, increasing resistance to treatment in ESKAPE has been recognized in recent years 2 Enterococcus faecium (E) One of the parts of normal intestinal flora of most humans is Enterococcus genus 3,4 The two most significant species, Enterococcus faecalis and Enterococcus faecium, cause various human infections such as septicemia, bacteremia, urinary tract infections (UTI), endocarditis, neonatal sepsis meningitis, and wound infections 5,6 Some resistant species e.g.High-level aminoglycoside-resistant (HLAR) enterococci and vancomycin-resistant enterococci (VRE) have been emerged and cause great difficulties in treatment [7][8][9] There are nine vancomycin resistance genes contain van A, B, C, D, E, G, L, M, and N. The most predominant types in worldwide are vanA and vanB [10][11][12] .Gene vanA, relates to a high degree resistance to teicoplanin and vancomycin, is mostly conferred to vancomycin resistant Enterococcus faecium 13 .Gene vanB relates to a vancomycin high level resistant but susceptibility to other glycopeptides such as teicoplanin whereas previous antibiotics able to induce the vanB resistance type 14 .

Staphylococcus aureus (S)
A common agent of the skin microbiota and generally is isolated from moist anatomical areas is S. aureus.Nearly 60% of the human population harbor S. aureus irregularly as intermittent carriers; whereas around 20% of individuals almost always carry a single S. aureus strain as persistent carriers.Even, S. aureus is a common wound pathogen, cause both acute and chronic infections by biofilm formation.
After the first clinical reports of methicillin resistance S.aureus (MRSA) in 1960s, via mecA expression which encodes a low affinity penicillinbinding protein (PBP2a), MRSA has expended resistance against b-lactam antibiotics including all blactams such as penicillins, cephalosporins, and carbapenems.First-line drug of choice for infections due to MRSA is mostly glycopeptide antibiotics such as vancomycin or teicoplanin .Unfortunately, being intensive selective pressure has caused to emerge vancomycin-intermediate S. aureus (VISA) ,and vancomycin-resistant S. aureus (VRSA) 2

Klebsiella pneumonia (K)
Being common isolated strain in health care setting, emergence of MDR K. pneumoniae and spread easily makes this microorganism as a main nosocomial pathogen to cause infections such as bacteremia, septicemia and urinary tract infections (UTIs) in children .Typically, MDR K. pneumoniae have been resistant to several different classes of antibiotics such as aminoglycosides, quinolones , b-lactams, and b-lactamase inhibitors.With the passage of time by means of the generation of their new mutant strains, resistant to antimicrobials drugs in MDR K. pneumoniae will become more and more 15 As a results, carbapenem-resistant Enterobacteriaceae especially carbapenemresistant K. pneumoniae (CRKP),are increasingly implicated in sporadic worldwide outbreaks due to multiple combinations of extended-spectrum b-lactamases (ESBLs) and carbapenemases by means of the dissemination of mobile genetic platforms related to encoding every class of b-lactamase.(2)

Acinetobacter baumannii (A)
A great challenge for physicians and clinical microbiologists is management of MDR Acinetobacter spp.infections.Capability to survive in a health care setting makes it a common agent for healthcare-associated infections and lead to multiple outbreaks.(16-18) spectrums of infections due to MDR Acinetobacter spp.contain bacteremia, meningitis, pneumonia, UTI, and wound infection [19][20][21][22][23][24] A. baumannii is intrinsically resistant to antibiotics constitutively due to express active efflux pump systems, the low-quantity expression of small-aperture outer membrane porins; possess a resistance island, which includes a cluster of genes encoding antibiotic and heavy metal resistance which impart resistance to ammonium-based disinfectants.The broad acquisition of ESBLs in some isolates confers with resistance to all known antimicrobials, containing imipenem and colistin.This combination of intrinsic virulence and multiple resistance factors, makes A. baumannii as symbol the superbug .So current clinical demand for novel antimicrobials is necessary 25,26

Pseudomonas aeruginosa (P)
There are some differences in the medical society to the definition of MDR, so the real incidence of MDR P. aeruginosa is not well proved 27 Most of the time, MDR was described as resistance to at least three antimicrobial drugs from a different classes of antibiotics, mostly penicillins, cephalosporins, aminoglycosides, carbapenems, antipseudomonal, and fluoroquinolones.Annually, different geographic places and centers limit the ability to determine the right percentage of MDR P. aeruginosa spread 28,29 Current articles have emphasized that above mentioned agents may or may not be as impressive as first-line drugs, but may as well as be pertaining to more considerable adverse effects (i.e.ototoxicity ,nephrotoxicity ,and neurotoxicity) [30][31][32][33][34][35][36][37][38] Antibiotic therapy may induce expression or select for stably depressed mutants, resulting in resistance to ticarcillin, piperacillin and thirdgeneration cephalosporins.Most common resistant mechanisms are usually due to the metallo-blactamases or MBLs (such as IMP, VIM, SPM and GIM), combination of low outer membrane permeability and multidrug efflux systems, overexpression of nodulation-cell division (RND) family of transporters (e.g., MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM), detriment of the outer membrane protein (porin) OprD, and other mechanisms such as enzyme production and target mutations.Expression of acetyltransferases, nucleotidyl transferases and phosphotransferases (enzymes related to aminoglycoside-modifying and aminoglycoside resistance), are common too  .

Enterobacter Species (E)
Enterobacter spp., most commonly cause the urinary and respiratory tracts, bloodstream and serious nosocomial infections, displaying broad MDR via plasmid-encoded ESBLs and carbapenemases, such as KPC, verona integronencoded metallo-b-lactamase, OXA and even metallo-²-lactamase- 66,67 .In addition to colistin and tigecycline, few antimicrobials drugs are effective against these resistant organisms .There are little or no drugs in the 'pipeline' that are known to be capable of effectively addressing this mounting health crisis 2,68 In veterinary medicine, fluoroquinolone resistance is become to increase.This resistance is occurred by both chromosomal and plasmidmediated fluoroquinolone resistance (PMQR) mechanisms which accompany with other antimicrobial resistance genes containing b-lactamases.The genes' relationship with PMQR can cause resistance to fluoroquinolone when joined with topoisomerase mutations and efflux pumps 69,70 So surveillance studies about ESKAPE can help governments to provide a public health plan to decrease use of improper antibiotics in infections caused by ESKAPE.In the last decade, along with the problem associated with nosocomial infections, MDR bacteria in community and hospitals have exceed.The objective of this study is to molecular approaches to resistance genes in ESKAPE pathogens.

Sample collection
In this descriptive study, 384 bacteria were isolated from clinical samples (such as trachea, urine, wound, discharges, ascites fluid, pleural fluid, blood, synovial fluid, and catheter) in Loghman-Hakim Hospital, Tehran, Iran.

Identification ESKAPE pathogens
Laboratory identification of Enterococci faecium was done by Gram staining, growth on blood agar, hydrolysis of esculin with blackening of bile esculin agar, negative catalase production, and growth on 6.5% sodium chloride [71][72][73] The isolates were identified as Staphylococcus aureus based on morphologic and biochemical tests such as: Gram stain, catalase, coagulase, hot-cold ²-hemolysin on blood agar, DNAase and mannitol salt agar fermentation 74 All the strains were screened for methicillin resistance by means of oxacillin (1 µg) and cefoxitin (30 µg) examination by disk diffusion test method, based on the standard guidelines 75 For identification of Klebsiella pneumonia and Enterobacter Spp., the samples were cultured on nutrient agar, MacConkey agar, blood agar and eosin methylene blue (EMB) agar (All of media were purchased from Hi Media Company, India).After incubation of plates at 35°C for 24 h , the pure isolates identified based on Gram stain and biochemical tests such as; catalase, oxidase, indole production, citrate utilization, sugar metabolism reaction on triple sugar iron agar, urea test, orthonitrophenyl-²-galactoside (ONPG) test, and methyl red Voges-Proskauer(MRVP), as described in standard bacteriological methods.All of the above chemicals and media were purchased from Sigma-Aldrich, Germany 76,77 For identification of Acinetobacter baumannii, we used standard bacteriologic and biochemical methods, which contained Gram staining, catalase tests, oxidase tests, motility, oxidation/fermentation (O/F) tests, citrate utilization tests, and capability to grow at 37 and 44 o C 78 Pseudomonas aeruginosa is a nonfermenting Gram negative rod which often related to human infection.It is a strict aerobe with a growth temperature range of 5-42°C and colonies with characteristic grape-like smell of aminoacetophenone.The blue-green appearance of pus or of an organism culture is pertaining to the combination of pyocyanin (blue pigment) and pyoverdin (fluorescein, yellow pigment).Other pigments such as pyorubin (red) or pyomelanin (brown) was produced by several strains.Almost all strains are motile by means of a single polar flagellum.At least six colonial types were produced by P. aeruginosa on nutrient agar after 24hr aerobic incubation at 37°C.Colonies isolated on selective or blood agar identified by a positive oxidase reaction, Gelatinase positive reaction and characteristic pigment production as 'P.aeruginosa' 79

Resistant Gene detection in ESKAPE pathogens
By AccuPrep Genomic DNA extraction kit (cat.no.k-3032 lot no.1008J, BIONEER) DNA was extracted from all GBS isolates.PCR amplification profile comprised a 300 nM concentration of each oligonucleotide primer (Eurofins MWG Operon); 200 mM (each) deoxynucleoside triphosphates dCTP, dGTP, dATP, and dUTP; 0.125 U of Taq DNA polymerase; and 5.5 mM MgCl2 (from GENET BIO, prime Taq TM DNA polymerase,URL:www.genetbio.com) The PCR products were analyzed by gel electrophoresis on 1.5% BIONEER agarose gels in 1X TBE buffer (890 mM of boric acid, 890 mMTris, 40ml of 0.5 M EDTA, pH 8.0) at 100 V for 60 min.Green loading buffer with DNA stain(Jena Bioscience ,Lot:111.034)was used during loading the samples and ladder.The sizes of the PCR products were determined by comparison with the molecular size standard (50bp-1Kb linear scale; low range DNA ladder or 100bp-3Kb linear scale and mid-range DNA ladder, Jena Bioscience.The rPSL gene was used for each reaction as housekeeping control gene primer sequences used in this study were presented in table 1.

DISCUSSION
Health crisis of ESKAPE pathogens seems overwhelming.It is necessary that the last remaining antimicrobial agents be protected against intellectual choice and ameliorated infection control.Selection of suitable guidelines is readily accessible and accurate prescribing protocols have been successfully implemented worldwide 2 Briefly, healthcare-associated, community-acquired, and nosocomial infections should be carefully considered.Knowledge of residential antimicrobial resistance can protect the selection of a convenient empirical therapeutic regimen in which diseases due to ESKAPE pathogens 94 For appropriate therapy, knowing the antibiotic resistance pattern in ESKAPE pathogens is essential, therefore we examined the distribution of antibiotic resistance in mentioned microorganisms.To our knowledge, this is the first assessment of the ESKAPE pathogens in Iran.There were different results about the resistant pattern of antibiotics in ESKAPE pathogens.
The worldwide emergence of MRSA is a remarkable challenge for public health based on centers for disease control (CDC) reports, 1% of all Staphylococcal in fections and 50% of healthcareassociated Staphylococcal infections are caused by MRSA 74 There is different prevalence of MRSA in the world.Twelve percent of MRSA strains were detected in 2015 in PIRC Tehran, Iran 74 , 11.3% in Germany, in 2007, (100) and 17.57% west of Iran, in 2013 (101).Compared to studies in Germany (6.5%),The Nether lands (1.4%), Shiraz, Iran (5.3%), Pediatric Infections Re search Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran (3.2%), Switzerland (3.3%), the USA (3.4%), France (6.6%) and the UK (6.7%), the prevalence of MRSA strains were lower than in our study (102-109).Rezaei et al. considered colonization of MRSA and MSSA in atopic dermatitis patients.They found a higher rate (33%) of MRSA colonization in the nasal cavity.The MRSA was one of the most frequent organisms that were found on their skin 110 .The MRSA isolates showed variable resistance to clindamycin, ceftri axone, cefpodoxime, azithromycine, and erythromycin 111 .Resistance to penicillin and clindamycin 111,112 was similar with the other studies.
By definition, all MRSA isolates can take the mecA gene, which allows resistance to all ²-lactam drugs, containg cephalosporins and carbapenems.In our study and in similar studies, several MRSA are susceptible to a number of beta lactams, such as cephalosporins [113][114][115] .
The ESBL propagation in Enterobacter spp. was approximately twofold as high as in compare with the ESBL outbreak in invasive E. coli and Klebsiella pneumonia isolates from the identical period in the Netherlands (4.7% and 6.9%, respectively).A presumably description for this diversity was the absence of a laboratory protocol for ESBL tracing in Enterobacter spp., eventuating in a lack of infection control measures and then an increased likelihood of nosocomial outbreak.Of the ESBL-producing strains, 40% were MDR, i.e., altogether resistant to ciprofloxacin, cotrimoxazole, and tobramycin or gentamicin, versus 3% in the non-ESBL isolates.With enhancing of MDR strains will increase the utilization of carbapenems; an unfavorable expansion in the face of the universal emergence of carbapenemase-producing Enterobacteriaceae strains 123,124 .
It should be mentioned that the clinical response of a patient after receiving antibiotic does not always correlate with the laboratory reports.Even so, it should be noted that description of pathogens antimicrobial resistance patterns needs a consecutive update 125 CONCLUSION Similar to other developing countries, antimicrobial resistance pattern surveillance has not been awarded sufficient regard in our country.Outcomes of present study demonstrated that the rate of antibiotics resistance is growing in Iranian health care setting.So, Iranian health ministry should provide guideline protocol and appropriate programs for antibiotic therapy in hospitals particularly alongside the other physicians for prescribing suitable antibiotics for antibiotic resistance prevention, better remedy and evaluation of the patients soon.In addition, educational and medical systems in Iran need training some well-educated personnel on the prohibition and management of antibiotic resistance.Knowledge of resistance genes associated with ESKAPE pathogens is necessary to prepare useful data about tracing and treatment of infection related to these microorganisms and may be beneficial to clinicians for selection a convenient empirical therapeutic diet in diseases due to ESKAPE pathogens at the bed head.Surveillances of healthcare setting, community-acquired, and nosocomial infections is suggested annually.We must enhance hospital infection-control procedure for restricting resistance spread.This procedure will ensure a steady stream of new antibacterial drugs to meet the needs of current patients.Further research needed in this regards in Iran.

ACKNOWLEDGMENT
This research has been supported by Research center Laboratory of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran.A special thanks to dr.Mir Davoud Omrani, dr Ali Rahimipour for all their assistance.
Statistics: Data were analyzed using IBM TM SPSS 20 software.

Table 2 .
The prevalence of resistance genes related to multiple drug resistance in ESKAPE pathogens NT: not tested M. The clinical importance of emerging ESKAPE pathogens in nosocomial infections.J. Paramed.Sci, 2016; 7: 45-57.2. Pendletona JN, Gormana SP, Gilmore BF .Clinical relevance of the ESKAPE pathogen.