Characterization of Aminoglycoside Modifying Enzymes Producing E. coli and Klebsiella pneumoniae Clinical Isolates

Antimicrobial resistance gene profile characterization and dissemination offer useful detail on the possible challenge in treating bacteria. The development of aminoglycoside modifying enzymes (AMEs) is considered as the primary mechanism of resistance to aminoglycosides, in addition to the 16S rRNA methylases. This study aimed at isolation and characterization of aminoglycosides resistant clinical isolates of enterobacteriaceae family from different clinical samples. Over a period of 24 months, thirty samples were collected and 49 clinical isolates of E. coli [n=25], Klebsiella [n=13], Enterobacter species (n=7) and Proteus species (n=4) were isolated from Egyptian clinical laboratories. The identities of the cultures were confirmed following standard microbiological procedures. Resistance of the isolates to aminoglycosides was determined by the disc diffusion method and isolates with highest resistance (n=9) were selected and investigated for 16S rRNA methylase and AMES encoding genes by polymerase chain reaction (PCR) and sequencing. In general, aminoglycoside resistance was found in 95% of the isolates; the isolates displayed the highest rate of resistance to netilmicin (75%) and kanamycin (55%), while resistance to gentamycin (18%) and tobramycin (16%) was low. A total of 9 isolates have the highest aminoglycoside resistant rate, showed the highest appearance for aac(6′)-Ib as well as ant (3")-Ia resistant genes, with aac (3)-II (44%) and ant (4′)-IIb (34%) following closely. The high prevalence of AMEs observed among resistant isolates in this study suggests the urgent need for more efficient treatment designs to mitigate the selection burden as well as improved care of patients who have been infected with these drug-resistant organisms.


iNtRODuCtiON
Gram-negative bacteria (particularly E.coli and Klebsiella) have long been suspected major causes of nosocomial infections, including infections of the urinary tract (UT), respiratory system and bacteremia. 1,2 Resistance of antibiotics, particularly to Gram-negative bacteria, has arisen as worldwide epidemic in the Twenty-First Century. Their resistance to diverse antibiotic classes limits available therapeutic options for their control. In particular, antibiotic-resistant pathogens isolated from hospitals are becoming increasingly common, which is a most health concern around the world. 3 A report published in 2014 revealed that, antimicrobial resistance causes more than 700,000 deaths worldwide per year, with that Fig. expected to increase to 10 million by 2050. 4 There have been a recent development in the management of severe infections by older antibiotics, especially aminoglycosides. 5 This is because the relatively low use of older antibiotics may have helped maintain their effectiveness against certain bacterial isolates that became more resistant to newer antibacterial agents. 6 Aminoglycoside molecules bind irreversibly to the ribosome 30S subunit, leading to complete protein synthesis inhibition and final bacterial death. Moreover, the mRNA translation interference by these antibiotics contributes to misreading of the codons of mRNA. 7 The study of molecular and genetic resistance determinants has a key role in the interpretation, controlling and distribution of resistance pathogens. Antimicrobial resistance mechanisms involving aminoglycosides include the following: addition of aminoglycoside-modifying enzymes leads to the antibiotic being inactive, Changes in ribosomal high affinity sites, the down-regulation of porin genes has decreased antibiotic uptake and efflux pumps. 8,9 Resistance to aminoglycosides may be caused by different mechanisms, particularly enzymatic modification, which is the most significant pathway and is classified into three categories; aminoglycoside nucleotidyl transferase (ant), aminoglycoside acetyltransferase (aac) and aminoglycoside phosphoryl transferase (aph); production of these enzymes is encoded by genes located on bacterial chromosomes or plasmids. 10 Aminoglycoside modifying enzymes catalyze the modification at -OH or -NH 2 groups of the 2-deoxystreptamine nucleus or the sugar moieties and can be acetyltransferases (AACs), nucleotidyl-tranferases (ANTs), or phosphotransferases (APHs). 11 Overall, aac (3)-Ia, aac (6')-II, aac (6')-Ib and aac (3)-II are among the common aminoglycoside in-activating enzymes in several Gram-negative clinical isolates, such as Klebsiella pneumoniae, whereas, aph (3'), ant(3)I or ant(4)Пb are less common. 12,13 Generation of 16S rRNA methyltransferase (16S RMTase) is another pathway of aminoglycoside resistance, which methylates the drug's binding site, making bacteria more resistant to clinically significant members of this group of antibiotics like gentamicin, tobramycin and amikacin. 14 The 16S rRNA methylases (16S-RMTase) have been shown to be responsible for high-level resistance against a range of aminoglycosides in Gram-negative bacilli. 16S rRNA methylases have emerged as a novel resistance mechanism to aminoglycosides. 15 Eight 16S-RMTase genes have been discovered in numerous enterobacteriaceae species and identified as npmA, rmtF, rmtE, rmtD, rmtC, rmtB, rmtA and armA. Particularly, rmtB and armA genes have been found to be the most predominant and extensively widespread throughout Asia. 16 However, in Egypt, little research has been reported on the existence of AMEs and the genes that encode enzymes for aminoglycoside resistance. This study is therefore intended to determine levels of and genes encoding aminoglycoside resistance among E. coli and K. pneumoniae isolates from patients with various infectious diseases.

MATERIAl ANd METhOdS Collection, isolation and identification of bacteria
Thirty clinical samples were obtained from three laboratories located in the Beni-Swef Governorate, Egypt. The ethics committee at the faculty of medicine, Beni-suef University, NU. Beni-suef, Egypt, approved the study. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. All the samples were collected from October 2016 to November 2018. These clinical samples were first grown aerobically on tryptone soya broth (TSB) then streaked on Mac-Conkey agar (Oxoid, UK) and incubated for 24-hours at 37°C. Basic microbiological techniques, such as Gram staining and colony morphology, and some biochemical tests, were used to confirm the identity of the isolates.

PCR detection and Sequencing of AMEs and 16S-RMTases Encoding Genes
Polymerase chain reaction was employed to detect the genes encoding AMEs [ant(4′)-

aac(6')-II, aac(3)-II, aac(6')-aph(2″), aph(3')-IIIa and aph(3')VI]
and 16S rRNA methylases (rmtB and armA) in aminoglycosides resistant isolates. Primers employed are presented in Table 1. Extraction of DNA was performed in accordance to the instructions of the manufacturer of the extraction kit (Bioneer Company, Korea). PCR cycling was done in VeritiTM thermocycler (Applied Biosystems, USA) with initial denaturation at 96°C for 7 minutes followed by 35 cycles of the following sequence; a denaturation step at 96°C for one minute, annealing at 52°C for one minute, chain elongation at 72°C for one minutes, with final extension at 72°C for 10 minutes. An amplification reaction at a complete volume of 25 μl was formulated using 12.5 μl 2X Master Mix (Thermo Fisher Scientific, USA), 2.5 μl of template DNA (50 pg concentration), 0.5 μM of both primers (reverse and forward) and 9 μl of nuclease-free water. Electrophoresis of PCR products was performed at 100 volts for 40 minutes on a 1.5 % agarose gel buffer, then stained by ethidium bromide dye and viewed using an iBrightTM system of gel documentation (UVtec, UK).
A direct sequencing of both strands of pure PCR products was performed for confirmation of the detected genes by the Macrogen Company (Seoul, South Korea). Sequence analysis and alignment were carried out by the BLAST program online (http://blast.ncbi.nlm.nih.gov/Blast), (NCBI).

Isolation rates of bacteria from the clinical samples
Thirty clinical samples from separate laboratories were used to obtain 49 enterobacterial isolates throughout the study period.  Fig. 2 shows the phylogenetic tree. Table 2 shows that, forty-nine isolates (100%) were resistant to rifampicin, 44 isolates   (Table 4 with Fig. s 1 and 3).

DisCussiON
Despite the growing resistance rates and several side effects of aminoglycosides, they are still effective antibacterial agents, particularly to treat bacterial infections. Aminoglycosides (gentamicin, amikacin, and tobramycin) resistance percentage was 78% for K. pneumoniae and 33% for E. coli among cerebrospinal fluid and blood clinical isolates in Montenegro, according to the CAESAR Annual Report 2018. 26 Phylogenetic tree showed that, isolates of this study were closely related to K. pneumoniae and E. coli. Previous studies have reported antibiotic resistance of E. coli and Klebsiella pneumoniae. [27][28][29] This study was done on Gram-negative isolates (particularly E. coli and Klebsiella spp.), which showed resistance to aminoglycosides as amikacin, gentamicin, netilmicin and tobramycin. In addition, the prevalence of resistance genes was assessed.
When the susceptibility of the tested isolates to several aminoglycosides was tested, the high rates of resistance were found for kanamycin (72%) and tobramycin (42%), followed by gentamycin (38%) and netilmicin (24%). These results were in the same line with Eftekhar et al., who discovered that 78.5% of clinical isolates were resistant to kanamycin. 30 Another study done by Estabraghi et al. found that clinical isolates exhibited an elevated resistance rate to gentamicin (24%) and amikacin (93%). 31 One of the most commonly reported aminoglycoside resistance mechanisms in clinical isolates is aminoglycoside-modifying enzymes (AME). 32 In diverse members of resistant bacteria as Acinetobacter baumannii, Enterococcus species, 33 Pseudomonas aeruginosa, 34 and methicillin-

aph(2″)
E. coli resistant Staphylococcus aureus (MRSA), 33 the occurrence of 16S rRNA methylases and AME is becoming increasingly established. Overall, the high rates of aminoglycoside resistance indicate that overuse of these antibiotics in hospitals has resulted in the emergence and spread of resistant isolates. Additionally, the data of the present study emphasize the need for establishing a local and national antimicrobial resistance surveillance system for monitoring the administration of antimicrobials and emergence of antibiotic resistance within the bacterial isolates present  isolates from their studies, in Western Norway. 12 Collectively, these results suggest that aac(3)-II and aac (6')-Ib, are of great importance among aminoglycoside resistance-inducing genes and are globally significant in aminoglycoside resistant isolates located in various geographic regions.

CONClusiON
The current study showed that Klebsiella and Escherichia coli isolated from clinical samples exhibited a high rate of resistance to aminoglycosides. In this study, it was shown that AMEs as well as 16S rRNA methylase encoding genes were positively expressed in E. coli as well as Klebsiella isolates that may be responsible for the aminoglycosides resistance. Moreover, our findings showed that resistance to aminoglycoside was essentially due to AMEs in clinical isolates; with ant (3")-Ia and aac (6′)-Ib being the most prevalent resistance encoding genes. 16S rRNA methyl transferases do not appear to play any role in aminoglycoside resistance among the studied bacteria. Routine aminoglycoside resistance monitoring and antimicrobial management actions can contribute to the reduction of the distribution of resistant bacteria, and the improvement of treatments.