Emergence of Multidrug-resistant Carbapenemases and MCR-1 Producing Pseudomonas aeruginosa in Egypt

Pseudomonas aeruginosa is an expedient Gram-negative bacterium, which is characterized by its ability to acquire antimicrobial resistance. In this study, 56 unrepeatable carbapenem-resistant P. aeruginosa isolates were gathered from various clinical sources from hospitals in Cairo and Mansoura universities. The isolates exhibited diminished susceptibility towards carbapenems, quinolones, aminoglycosides and chloramphenicol by using disc diffusion method. Carbapenemase production was confirmed among the isolates, where all the 56 P. aeruginosa isolates harboured carbapenemase genes including bla VIM (43 isolates), bla KPC (38 isolates), bla NDM-1 (17 isolates), bla IMP (16 isolates) and bla OXA-48 (15 isolates). Among the isolates, 13 carried only one carbapenemase gene, while 43 isolates carried multiple carbapenemase genes. MCR-1 production was confirmed in 10 of the tested isolates by detecting the mcr-1 gene encoding for the colistin resistance. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) evaluation showed that the tested isolates were unrelated to each other. Therefore, this study rises the danger of emergence of MDR P. aeruginosa resistant to carbapenems coupled with other antimicrobials including colistin, which is regarded as the last reservoir for the management of infections caused by MDR Gram-negative pathogens. Early inspection of resistance patterns in MDR organisms is an important tool to control and prevent infections via limiting the spread of these pathogens.


INTRODUCTION
P s e u d o m o n a s a e r u g i n o s a i s a threatening human pathogen that is usually linked to nosocomial infections. 1,2Commonly, this pathogen is multidrug-resistant (MDR) due to its extraordinary capacity to acquire resistance to a wide range of antimicrobials. 3Usually, carbapenems are considered the first treatment choice against infections brought about by P. aeruginosa.Because of the widespread use of carbapenems, there has been a global increase in reports regarding P. aeruginosa carbapenem resistant isolates. 4It is also notable that isolates of P. aeruginosa have started to depict resistance to the antibiotics that are used as a last choice for therapy including colistin. 5ndeed, the primary mechanism for pathogens to resist carbapenems is the production of carbapenemases, which are mainly plasmid mediated and considered as a type of β-lactamases. 6Carbapenemases have a wide range of hydrolytic capabilities against antimicrobials including carbapenems, cephalosporins and penicillins. 7,8]12 The most significant developing mechanisms of β-lactam resistance in P. aeruginosa are those arbitrated by extended-spectrum β-lactamases (ESBLs) and MBLs.β-lactamases inhibitors do not inhibit MBLs, which have a significant capacity to hydrolyze carbapenems efficiently and other β-lactams. 10,11,13,14The KPC type is primarily present on pKpQIL plasmid, which occurs in Klebsiella pneumonia. 15The OXA-48 type is frequently found on carbonen 62-kb IncL/M conjugative plasmids of Acinetobacter baumannii. 12otably, polymyxins are typically considered the final treatment line against MDR Gram-negative bacteria.The polycationic peptide called colistin (also named to as polymyxin E) binds to anionic lipopolysaccharide molecules in the outer membrane of Gram-negative cell walls and disrupts them by competing with Ca2 + and Mg2 + cations for these molecules. 16,17Unfortunately, colistin resistance has recently developed because of the drug misuse. 16The horizontal transfer of phosphoethanolamine transferase enzymeencoding gene mcr-1 was discovered in 2015 and it has a key role in the colistin resistance. 18his enzyme has the ability to modify the lipopolysaccharides of the outer membrane lipid A of Gram-negative pathogens. 17,19he horizontal transfer of plasmidmediated resistance has two risks.First, the plasmids can confer the resistance to a variety of drugs.5][6] The rise of carbapenemresistant pathogens all over the world with identical mobile genetic elements suggests that genes for carbapenemases have been spread horizontally. 6The MBL-producing genes bla VIM , bla NDM-1 and bla IMP as well as bla KPC (class A) and bla OXA-48 (class D) genes can transfer horizontally via plasmids and can spread quickly to other bacteria. 4,5,7,8,10,11,20The same case is for mcr-1, which is plasmid mediated and has the ability to spread quickly over the globe. 17,19ased on this context, the objective of the present investigation was to estimate the dominance of MBLs (VIM, NDM-1 and IMP) as well as class serine enzymes (the KPC type and OXA-48 type), which are responsible for carbapenemases activity in the isolates of P. aeruginosa.Moreover, to investigate the prevalence of MCR-1 among the isolates.After all, the clonal clustering of the tested isolates was established utilizing (ERIC)-PCR.

Bacterial isolates and media
This study was carried out after the approval of the research ethics committee of Faculty of Pharmacy, Tanta University, Egypt (code: TP / RE /12-21-M-002).The current study included 56 unrepeatable carbapenem-resistant P. aeruginosa isolates.These isolates were gathered from several clinical sources from hospitals in Cairo and Mansoura universities.The examined isolates were collected, identified and stored using the standard microbiological procedures. 21All isolates were cultured at 37°C in Luria Bertani medium (LB broth; tryptone 1% w/v, yeast extract 0.5% w/v and NaCl 1.0% w/v), otherwise specified, and stored in 50% glycerol/LB broth at -80°C.During the culture and sensitivity testing, the isolates were cultured on modified Muller-Hinton agar (MHA) supplemented with all antimicrobials, except for colistin as described before. 22For colistin, to improve its diffusion, the modified MHA containing 30% agar was used.

Carbapenemase phenotypic examination
Modified Hodge test (MHT) was carried out to test carbapenemase production as described before. 25A commercial disc containing 10 μg of imipenem was placed in the middle of a MHA plate that had been inoculated with E. coli ATCC 25922.The tested P. aeruginosa isolates were considered β-lactamase positive if they enabled E. coli ATCC 25922 strain to resist the imipenem giving a cloverleaf-like indentation.During these experiments, K. pneumoniae ATCC BAA-1705 and ATCC BAA-1706 strains were used as positive and negative controls, respectively.
EDTA synergistic test was used to evaluate the MBLs activity as indicated before. 26his test involves challenging the tested isolate with one disc containing anhydrous EDTA (292 μg) (Sigma Chemicals in St. Louis, MO) and two imipenem discs (10 μg each).The discs are spaced 25 mm apart in the MHA plate.Positive MBL production was demonstrated by an increase in the inhibition zone width of more than 4 mm around the imipenem-EDTA disc in comparison to the imipenem disc alone.As a control, E. coli ATCC 25922 standard strain was used during these experiments.
Furthermore, KPC enzyme synthesis evaluation was established by the boronic acid test as specified before. 27Antimicrobial discs (cefepime, meropenem or imipenem) and a KPC

Polymerase chain reaction (PCR)
The QIA amp® DNA miniprep kit (Qiagen, Germany) was utilized to extract plasmid DNA from P. aeruginosa isolates.9][30][31][32] The PCR reactions were established in the volume of 25 μL.Each PCR reaction consisted of 3 μL of template DNA, 1 μL forward primer (10 μM), 1 μL of reverse primer (10 μM), 12.5 μL Dream Taq PCR master mix 2x (Fermentas, USA) and 7.5 μL nuclease free water.Tubes with no template DNA were used as a negative control.The PCR cycling was established as follows; primary denaturation for 5 min at 95°C, followed by 35 cycles of (denaturation for 30 s at 95°C, annealing for 30 s at 52°C for bla VIM , bla IMP , bla NDM-1 and mcr-1, while at 48°C and 58°C for bla KPC and bla OXA-48 , respectively and extension for 1 min at 72°C) and last extension for 5 min at 72°C.
Results of PCR were inspected using gel electrophoresis, where the products were run on 1.2% agarose gel and visualized utilizing ethidium bromide (MP biomedicals, France).The size of the obtained amplicons was matched to DNA ladder (Thermo Scientific, UK).The tested gene was considered positive if a single sharp band of its expected size appeared beside the matched band of the DNA ladder reflecting its size.

Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR)
The clonal relatedness of the P. aeruginosa isolates was done using ERIC-PCR.The banding profile was generated using the oligonucleotide primer ERIC-2 (Table 1) 33 according to the method previously described. 34The resultant patterns attained from ERIC-PCR were construed using the software Past® (version 4.01). 35The similarities among the fingerprints were calculated based on Pearson correlation (optimization, 1%; position tolerance, 1%).By using the UPGMA algorithm, the fingerprints were sorted based on their similarity to generate their corresponding dendrograms. 36solates with a resemblance of more than 85% were deemed clonal.

Clinical features and antimicrobial sensitivity patterns of the tested isolates
A l l t e s t e d P. a e r u g i n o s a w e r e carbapenemase-producing isolates and harboured carbapenemase genes.According to the clinical source as shown in Table 2, 22 isolates (39.28%) were gathered from urine, 13 isolates (23.21%) from wounds, 9 isolates (16.1%) from burns, 6 isolates (10.71%) from sputum, 4 isolates (7.14%) from ear swabs and 2 isolates (3.57%) from eye infections.In a 1:1 ratio, samples were drawn from both males and females.Regarding the antimicrobial susceptibility patterns of the tested isolates as shown in Table 3, the highest resistance percentage was observed with sulbactam/ amoxicillin, cefoperazone, cefotaxime, ampicillin and nalidixic acid, where 100% of the isolates were resistant.Moreover, the resistance percentage to ceftazidime and ceftriaxone was 94.6%, followed by carbenicillin (91.1%) and both tobramycin and gentamicin (80.35%).The resistance percentages to meropenem and imipenem were 96.4% and 75%, respectively.On the other hand, colistin was shown to have the lowest resistance rate (17.8%), while amikacin and norfloxacin resistance was (44.6%).In addition, the resistance percentages to levofloxacin, ciprofloxacin and chloramphenicol were 55.3%, 46.4% and 62.5%, respectively.The examined isolates U25 and B56 showed complete resistance to all antimicrobials used in this study.

Phenotypic and genotypic detection of carbapenemases and MCR-1 among the tested isolates
According to Table 2, 31 isolates (55.35%) were MHT positive.Furthermore, the EDTA synergistic test was positive in 50 isolates (89.23%).In addition, the boronic acid test gave positive findings with 38 isolates (67.85%).

DISCUSSION
P. aeruginosa is one of the most prevalent Gram-negative opportunistic bacteria that can result in nosocomial infections.The scenario becomes worse if the infections are linked to MDR pathogens, which limits the treatment options 37 .Indeed, the resistance of Gramnegative pathogens, including P. aeruginosa, to carbapenems is a global health concern. 10his study found that each isolate of the collected P. aeruginosa was at least resistant to one carbapenem (imipenem or meropenem).Moreover, 50 isolates (89.23%) were MBL producers.Previous reports showed similar results, where a study indicated that 12 P. aeruginosa isolates of 80 (26.25%) were carbapenemase producers. 10According to another study, 14 P. aeruginosa isolates of 114 (12.2%) were carbapenem resistant.Thirteen isolates of these 14 isolates (11.4%) exhibited the MBL phenotype. 11Another two studies established in Brazil and Korea demonstrated that 43.9% and 92.7% of P. aeruginosa isolates were resistant to carbapenem, respectively. 20,38In addition, a study that was carried out in Canada depicted that 228 patients were infected with imipenem resistant P. aeruginosa.This study demonstrated that 98 isolates were MBL producers. 39In addition, a study conducted in Iran indicated that 110 isolates out of 122 (90 %) were imipenem resistant and MBL producers. 40ndeed, MHT is a confirmator y phenotypic test for the inspection of P. aeruginosa carbapenemase synthesis.However, it should be noted that the sensitivity and specificity of MHT for detecting carbapenemase synthesis are debatable.Although all the tested isolates generated carbapenemases, 31 isolates (55.3%) were MHT  Former reports stated that the bla VIM gene is the most common produced gene in carbapenem-resistant P. aeruginosa. 10,11,13,14This is in accordance with this study, where bla VIM was harboured by 46 P. aeruginosa isolates (76.78%) as the most common MBL.In a study conducted in Egypt, bla VIM gene was found in 8 (57%) isolates out of 14 P. aeruginosa imipenem resistant isolates 11 .Moreover, this gene was found in 19 (55.88%) isolates out of 34 carbapenem-resistant P. aeruginosa isolates in another report. 10In two Iranian studies, 40 and 44 the prevalence rates of the bla VIM gene were reported to be 1.6% and 55%, respectively.Another study established in Canada indicated that 90 (39.47%) isolates out of 228 imipenem resistant P. aeruginosa isolates harboured bla VIM gene.Moreover, during the year 2003, a nosocomial outbreak was caused by a cluster of bla VIM producing strains. 39Another study in Poland depicted a higher incidence of P. aeruginosa strains harbouring the bla VIM gene (68%). 45n this study, bla IMP gene was found in 16 P. aeruginosa isolates (28.57%).In Egypt, a study demonstrated that 5 isolates out of 14 imipenem resistant P. aeruginosa isolates (35%) harboured bla IMP gene. 11Other studies found lower incidence rates of bla IMP including 1.75% in Canada, 39 and 3% in Iran. 44Another study in Iran, on the other hand, depicted a greater prevalence of bla IMP (55%) through the tested isolates of P. aeruginosa. 40n Egypt, the first report of P. aeruginosa harbouring bla NDM-1 was published in 2014, 14 where the authors showed that 2 P. aeruginosa isolates out of 33 (6%) were carbapenem-resistant and harboured bla NDM-1 .Moreover, another report showed a greater incidence of P. aeruginosa isolates that are resistant to carbapenem carrying bla NDM-1 . 13According to this study, bla NDM-1 gene was found in 17 P. aeruginosa carbapenems and carrying isolates (30.4%) and this percentage is high in relation to the previous reports.
Regarding bla KPC , in Egypt, a study demonstrated that this gene was found in 1 isolate (2.9%) out of 34 P. aeruginosa carbapenemaseproducing isolates, while bla OXA-48 gene was not found in all 34 carbapenemase-producing isolates. 10In the current study, bla KPC and bla OXA-48 occurred in 38 (67.85%) and 15 (26.78%) isolates, respectively.These incidence percentages of both genes are much higher than the previous reports.Another study conducted in Iran indicated that bla KPC presented in 13% of 108 P. aeruginosa isolates, 41 while in Puerto Rico, 99 (4.1%) out of 2415 P. aeruginosa isolates harboured bla KPC gene

46
. Moreover, a study in Sudan reported that 60% of P. aeruginosa isolates carried bla OXA-48 gene. 47ccording to this study, 43 (76.78%)P. aeruginosa isolates carried more than one carbapenem resistance gene.Compared to another report in Egypt, this percentage is very high.Previous report showed that 1 isolate out of 34 P. aeruginosa carbapenemase-producing isolates harboured both bla VIM and bla KPC . 10Another study depicted that only 4 (28.5%)isolates out of 14 P. aeruginosa carbapenemase-producing isolates carried both bla VIM and bla IMP . 11This gives an indication for the prevalence of carbapenem resistance genes that increased and became a serious problem in Egypt.
According to the data of this study, the lowest percentage of resistance among the tested P. aeruginosa isolates was recorded for colistin (10%).Given the fact that colistin has the potential to be nephrotoxic, it should only be used as a last choice for serious infections that cannot be cured with antimicrobial combinations. 48In this study, all P. aeruginosa colistin resistant isolates harboured mcr-1 gene.These data revealed high colistin resistance percentage compared to previous studies that showed complete sensitivity to colistin 10 and another study that demonstrated that only 1 isolate from 66 P. aeruginosa isolates was resistant to colistin. 28n the present study, the homology analysis (using ERIC-PCR) revealed that plasmidmediated carbapenemases and colistin resistance genes could be easily transmitted across the P. aeruginosa isolates.Most of the tested isolates were unrelated to each other; however, they all included one or more carbapenemase gene.According to these findings, it can be concluded that P. aeruginosa isolates can easily share plasmids that encode antimicrobial resistance markers.These findings support earlier studies that described the horizontal spread of plasmids encoding carbapenemases as well as MCR-1 among Gram-negative bacteria. 4,10,11,13,14,20,38,41,47,49verall, one of the biggest threats to the healthcare systems globally is the appearance of MDR pathogens.This study demonstrated the presence of MDR P. aeruginosa isolates that are resistant to both carbapenems and colistin as well as other antimicrobials.Early inspection of MDR P. aeruginosa isolates with any diminished susceptibility to the carbapenems as well as colistin is essential for the choice of the most proper antimicrobial treatment and the application of effective infection management protocols.Increasing the health awareness and the appropriate use of antimicrobials, particularly carbapenems and cephalosporins, as well as the minimalistic use of colistin may help to prevent the emergence of such resistance patterns.

Figure .
Figure.Dendrogram of ERIC-PCR exhibiting the clonal relatedness of P. aeruginosa isolates.The resulting patterns obtained from ERIC-PCR were interpreted using the software Past® (version 4.01).The similarities between the fingerprints were calculated based on Pearson correlation (optimization, 1%; position tolerance, 1%), and the fingerprints were grouped according to their similarities by using UPGMA algorithm to generate their corresponding dendrograms.Isolates with > 85% similarity were considered clonal.ERIC-PCR, enterobacterial repetitive intergenic consensus polymerase chain reaction

Table 1 .
Oligonucleotide primers used in this study

Table 2 .
Distribution of carbapenem and colistin resistance genes in P. aeruginosa tested isolates and their phenotypic detection Isolate Source Sex Ward Infection bla VIM bla KPC bla NDM-1 bla IMP bla OXA-48 mcr-1 MHT EDTA Boronic

Table 2 .
Cont... Isolate Source Sex Ward Infection bla VIM bla KPC bla NDM-1 bla IMP bla OXA-48 mcr-1 MHT EDTA Boronic B: Burns, BCC: Burns and cosmetics center, BI: Burn infection, CH: Chest hospital, CUH: Cairo university hospital, E: Ear swabs, EI: Eye infection, EY: Eye, F: Female, ICU: Infection control unit, M: Male, MHT: Modified Hodge test, MIH: Mansoura international hospital, MUH: Mansoura university hospital, OC: Ocular center, OM: Otitis media, RTI: Respiratory tract infection, S: Sputum, U: Urine, UNC: Urology and nephrology center, UTI: Urinary tract infection, W: Wound, WI: Wound infection inhibitor (400 μg benzene boronic acid; Sigma-Aldrich, Steinheim, Germany) were employed in this assay.The tests were carried out by inoculating a MHA plate with the tested isolate in the presence of antimicrobial discs with or without boronic acid.Antimicrobial-boronic acid disk is compared to the antibiotic disc alone, and KPC enzyme production is considered positive if the diameter of the inhibitory zone around the former increases by 5 mm or more.The standard strain E. coli ATCC 25922 was used in this test to establish quality control.

Table 4 .
P. aeruginosa isolates that harbour more than one carbapenem-resistant gene accompanied with the colistin resistant gene, mcr-1

Table 5 .
P. aeruginosa isolates that harbour only one carbapenem-resistant gene accompanied with the colistin resistant gene, mcr-1