Molecular Characterizations of Aminoglycoside Modifying Enzymes of Proteus mirabilis Isolated From Patients in Al-Qadisiyah Governorate

 

Najlaa Abdallah D. AL-Oqaili¹, Majed Kadim AL-Shebli², Azhar N. H. Almousawi³

¹College of Biotechnology, University of Al-Qadisiyah, Iraq

²College of Education, University of Al-Qadisiyah, Iraq

³College of Pharmacy,University of Al-Qadisiyah, Iraq

 

ABSTRACT:

The aim of this study was evaluate the presence of modified enzymes for aminoglycosides antibiotics in Proteus mirabilis. Total of  64 clinical isolates were collected from various sources included urine, middle ear, wounds, burns, high cervical and endometrium cervical, which was taken from in patients and outpatients in Maternal, and General Teaching Hospital in AL- Diwaniya city during the period from 1/3/2015 till 30/9/2015. Select the minimum inhibitory concentration of a number of antibiotics for aminoglycosides for resistant bacterial isolates which are anti-Gentamycin, Tobramycin, Kanamycin, Streptomycin, Netlimicin the isolates showed their ability to grow in high concentrations ranging from (32-1024)µg/ml, also resisted anti-aminoglycosides and with different rates of  Gentamycin, Amikacin ,Tobramycin , Kanamycin, Streptomycin, and Netlimicin, it was in varying proportions (54.68, 31.25, 76.56, 81.25, 85.93, 81.25) %, respectively .

 

 

 

INTRODUCTION:

Proteus spp. are widely distributed in nature. They can be found in polluted water, soil and manure, where they play an important role in decomposing organic matter of animal origin [1-3]. Proteus is a genus of Gram-negative bacteria; many of which cause infections in humans [4], and about 90% of these infections are caused by P.mirabilis [5] and is mostly found in people with compromised immune system (6). It is an opportunistic pathogen that is able to cause severe invasive diseases [7] in critically ill and immunocompromised patients [8]. P. mirabilis strains can acquire vario­us genes for antibiotic resistance via mobile genetic elements: plasmids, integrons, gene cassettes, and transposons [9].

 

 

The most common mechanism of aminoglycoside resistance is enzymatic modification of antibiotic molecule [10]. After modification, the aminoglycoside isnot able to bind to the aminoacyl site of 16S rRNA within the bacterial 30S ribosomal subunits and inhibit protein synthesis [11, 12]. Aminoglycoside - mo­difying enzymes (AME) have been assigned to three classes: aminoglycoside acetyltransferases (AAC), aminoglycoside phosphotransferases (APH), and aminoglycoside nucleotidyltransferases (ANT) [13]. A more specific classification includes the division into subclasses according to the region specificities for aminoglycoside modifications and spectrum of aminoglycoside resistance [14]. To the subclasses with clinical significance among Enterobacteriaceae rods belong following groups: AAC(3)-I, AAC(6’)-I, ANT(4’)-II, ANT(2”)-I, and APH(3”)-I.The subclass AAC(3)-I contributes to the resistance to gentamicin, sisomicin, and astromicin, while AAC(6’)-I shows activity against amikacin and gentamicin. Enzymes from ANT(4’)-II subclass confer resistance to amika­cin, isepamicin, and tobramycin, and AME belonging to ANT(2”)-I mediate resistance to dibekacin, gen­tamicin, kanamycin, sisomicin, and tobramycin. The subclass of APH(3”)-I enzymes shows activity against streptomycin [10]. The other mechanisms, which also contributes in resistance to aminoglycosides, can co­exist in one bacterial cell along with AME [15]. These mechanisms include methylation of 16S rRNA [16], reduced outer membrane permeability [17], efflux pumps and mutations of the ribosomal proteins or 16S rRNA [16].

 

MATERIALS AND METHODS:

Specimens’ collection:

Different clinical samples such as urine, wound swab, burn swab, high cervical, endometrium cervical and ear swab, which was taken from in patients and outpatients in Maternal, and General Teaching Hospital in AL- Diwaniya city during the period from 1/3/2015 till 30/9/2015. The collection process has been conducted according to [18].

 

Identification of Bacterial Isolates:

The isolates were identified according to [19] by using traditional microscopic examination (Gram’s stain), colony morphological features on MacConkey agar and

blood agar, and standard biochemical tests.

 

DNA extraction:

The total genomic DNA of the P.mirabilis was isolated using the DNA extraction and purification kit (Geneaid, USA) according to the manufacturer instructions. DNA preparations were then analyzed by electrophoresis in 1.5% agarose gel.

 

Polymerase chain reaction:

Polymerase chain reaction was used to amplify the entire sequences ofthe genes studied in this research. The specific primers (Bioneer, Korea)used for the amplification of these genes [20] were shown in (table 1). The PCR mixtures contained: Top DNA polymerase 1U, dNTP (dATP, dCTP, dGTP, dTTP) each: 250µM, Tris-HCl (pH 9.0) 10mM, KCl 30mM, MgCl2 1.5mM, Stabilizer and tracking dye .The cycling parameters of amplification were: Initial denaturation 95°C for 5min, Denaturation 95°C for 30 sec., Annealing (52°C) for 30 sec., Extension 72°C for 1min., Final extension 72°C for 5min.

 

Agarose gel electrophoresis:

The products were separated in 1.5% agarose gel in TBE buffer (pH 8), stained with ethidium bromide, and photographed in ultraviolet light [21]. The electrophoresis result noticed by using gel documentation system.

 

 

 

 

Table (1): Sequences of oligonucleotide primers used in PCR assays for amplification of genes encoding AME

 

Antimicrobial susceptibility test:

Susceptibility to antimicrobial agents was determined by using the disk diffusion method [22]. The following antimicrobial agents (obtained from Bioanalyse Turkyi, Oxoid England) were used: gentamycin (GM), amikacin (AK), Tobramycin (TOB), Kanamycin (K), Netilmicin (NET) and Streptomycin (S).

 

Susceptibility to antimicrobial agents was determined by using the minimum inhibitory concentrations (MICs) [23]. The following antimicrobial agents (obtained from Kocaeli Turkyi, Hamburg Germany) were used: gentamycin (GM), Tobramycin (TOB), Kanamycin (K), Netilmicin (NET) and Streptomycin (S).

 

The inoculums were prepared by growing the various Proteus mirabilis on separate agar plates and colonies from the plate were transferred with inoculating loop into 3 ml of normal saline in a test tube. The density of these suspensions was adjusted to 0.5 McFarland standards. The surface of Muller-Hinton agar (Himedia India) plate was evenly inoculated with the organisms using a sterile swab. The swab was dipped into the suspension and pressed against the side of the test tube to remove excess fluid. The wet swab was then used to inoculate the Muller-Hinton agar by evenly streaking across the surface. The antibiotic discs were applied to the surface of the inoculated agar and the plates were incubated overnight at 37ºC. The diameter of zone of growth - inhibition observed was measured and compared to the chart provided by National Committee for Clinical Laboratory Standards (NCCLS).

 

RESULTS AND DISCUSSION:

Isolation and identification:

Sixty four isolates were identified as P.mirabilis consisted of 32 isolates (12.8%) obtained from urine, 17 isolates (9.18%) obtained from middle ear, 3 isolates (7.5%) obtained from wounds, 4 isolates (4.7%) obtained from burns and 8 isolates (8.88%) obtained from high cervical and endometrium cervical (table 2).

 

 

 

Table (2): Distribution of P.mirabilis among various clinical sources.

Type of samples	No. of samples (%)
Urine	32 (12.8)
Middle ear	17 (9.18)
Wounds	3 (7.5)
Burns	4 (4.7)
High cervical and endometrium cervical	8 (8.88)
Total	64 (9.84)

 

We analyzed resistance to 6 antibiotics aminoglycosides all isolates showed micro­biological resistance (table 3), Gentmycin about (54.68%) this result agrees with (24) about (50%) but not agrees with [25] about (33%), also Amikacin (31.25%) this result agrees with [26] about (38.4%) but this current result not agrees with [27,24] about (1.6,5)% , respectively. Tobramycin (76.56%)this result agrees with [28] about (81%) but this current result not agrees with [29] about (33.3%). also antibiotics Kanamycin, Streptomycin and Netlimicin about 81.25, 85.93, 81.25%, respectively.

 

 

 

 

Table (3): Percentage (%) of antimicrobial resistance and sensitivity of P.mirabilis .

 

 

 

We analyzed resistance to five antibiotics for 20 isolates under study were subjected to test the minimum inhibitory concentrations (MICs) in (table 4), value of MIC to Gentamycin were (32-512)µg/ml this result agrees with [30 ] were MIC value (8 ≤ 512-)µg/ml, but this current result not agrees with [31] were MIC value (0.5-256) µg/ml, also Tobramycin were MIC (32-512) µg/ml which is a break point (8 ≤ ) µg/ml, this result agrees with (32) were MIC value for Tobramycin (16-512) µg/ml but this current result not agrees with [31] were MIC value (0.5-256) µg/ml , also study [30] were MIC value for Tobramycin (4-1024) µg/ml, Kanamycin in the current study were MIC vaule (32-1024) µg/ml which is a break point (16 ≤ ) µg/ml , this result approach with [31] were MIC value (2-1024) µg/ml, while Streptomycin and Netilmicin in the current study were MIC value (128-1024)µg/ml which is a break point (32 ≤ , 16 ≤ ) µg/ml, respectively.

 

 

 

 

Table (4): Minimum inhibitory concentrations (MIC) for ant P.mirabilis isolates.

 

Aminoglycosides (Amgs) are highly potent, broad spectrum antibiotics with many desirable properties for the treatment of human infections. Among resistance mechanisms to Amgs inactivation by aminoglycoside-modifying enzymes (AMEs) is the most important both in terms of level and frequency of resistance conferred to the bacterium [33]. There are three family of AMEs including aminoglycoside acetyltransferases (AACs), phosphotransferases (APHs), and nucleotidyl transferases (ANTs). Each group of enzymes consists of different isozymic forms that differ in substrate regiospecificity for their reactions [33]. Amgs modification has emerged in all clinically relevant bacteria of both Gram-positive (Staphylococcus spp., Enterococcus spp.) and Gram negative, including Proteus spp. strains [33, 34, 25, 35]. These enzymes are often plasmid-encoded but may also be associated with transposable elements facilitating the rapid dissemination of the genes in various bacterial populations [33, 36]. In this study we present occurrence of modified enzymes for aminoglycosides antibiotics in clinical strains of Proteus mirabilis. The genes encoding AME were detected in 20 P.mirabilis isolates which represents five genes (Table 5). The results of the current study showed that the presence of the gene aac(3)-Ia in P.mirabilis about (20%) (Fig.1), while a study showed [37] presence the gene aac(3)-Ia about (1.51%). The presence of aac(6’)-Ib about (40%) (Fig.2) this result agrees with [38] about (36.5%), also agrees with [39] about (37.28%), but the result of the current not agree with (40) about (71.43%). While The presence of ant(4’)-IIa about (15%) (Fig.3) this result approach with [37] the presence of ant (4’)-IIa gene was not detected in any of tested isolates.

 

Also the presence of the gene ant(2”)-Ia about (50%) (Fig.4), while a study showed [37] about (80.30%), but not agree with (41) about (36%). Finally, the study showed that the presence of the gene aph(3”)-Ib about (50%) (Fig.5), this result not agree with [37] about (21.21%).

 

 Table (5): Prevalence of genes encoding AME in p.mirabilis isolates

 

Fig.1. Demonstration of aac(3)-Ia gene resistant for antibiotic aminoglycosides with PCR in agarose gel. M: Marker ladder 100-2000bp, Lanes 1-4 positive numbers for the gene aac(3)-Ia of clinical Proteus mirabilis (80mA and 100V) for one hour.

 

Fig.2. Demonstration of aac(6’)-Ib gene resistant for antibiotic aminoglycosides with PCR in agarose gel. M: Marker ladder 100-2000bp, Lanes 1-8 positive numbers for the gene aac(6’)-Ib of clinical Proteus mirabilis (80mA and 100V) for one hour.

 

Fig.3. Demonstration of ant(4’)-IIa gene resistant for antibiotic aminoglycosides with PCR in agarose gel. M: Marker ladder 100-2000bp, Lanes 1-3 positive numbers for the gene ant(4’)-IIa of clinical Proteus mirabilis (80mA and 100V) for one hour.

 

 

Fig.4. Demonstration of ant(2”)-Ia gene resistant for antibiotic aminoglycosides with PCR in agarose gel. M:Marker ladder 100-2000bp, Lanes 1-10 positive numbers for the gene ant(2”)-Ia of clinical Proteus mirabilis (80mA and 100V) for one hour .

 

 

Fig.5. Demonstration of aph(3”)-Ib gene resistant for antibiotic aminoglycosides with PCR in agarose gel. M:Marker ladder 100-2000bp, Lanes 1-10 positive numbers for the gene aph(3”)-Ib of clinical Proteus mirabilis (80mA and 100V) for one hour

CONCLUSION:

The results show that the prevalence of resistant genes for the anti-aminoglycosides was also studied , the results were also variable for genes aac(3)-Ia ,aac(6')-Ib ,ant(4')-IIa ,ant(2")-Ia and aph(3")-Ib ,in percentages ( 20 , 40 , 15 , 50 , 50 ) % ,respectively.

 

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Accepted on 24.11.2017        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(7): 2809-2813.