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الرئيسية / Labs and Analysis for Water Quality / Rapid detection of methicillin-resistant staphylococcus aureus gene (MRSA) among non staphylococci strains isolated from different water resources by multiplex PCR

Rapid detection of methicillin-resistant staphylococcus aureus gene (MRSA) among non staphylococci strains isolated from different water resources by multiplex PCR

AbdelRehim Khalid A. A.1*, Hassanein A. M.1, Sabry Younis1a, Y2 Abd El- Azeiz Heikal A.1,3

1Botany Department, Faculty of Science, Sohag University, Sohag-82524, Egypt

2microbilogojy Department, Faculty of Agriculture, Sohag University, Sohag-82524, Egypt

3Regional Laboratory for water research, Central Health Laboratories, Sohag-82524, Egypt

* Corresponding author: AbdelRehim Khalid A. A, Botany Department, Faculty of Science Faculty of science, Sohag University, Sohag-82524, Egypt, +20934601159. bhkhalid1969@yahoo.de

Abstract

One hundred and fourteen water samples were collected from River Nile, treated tap water and groundwater (36, 36 and 42), respectively. Thirty tow strains were isolated and identified as Shigella flexneri, Shigella sonnie, Salmonella serovar Newport, Pseudomonas aeruginosa and E. coli strains according to standard methods. Rapid and sensitive method for detection of methicillin-resistant Staphylococcus aureus (MRSA) gene was developed among non staphylococci strains. . PCR identified 32 methicillin-resistant non staphylococci negative samples. All strains were subjected to different antibiotics and susceptibility was recorded.

Keywords: water, Bactria, MRSA-PCR, antibiotics,

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important hospital associated (nosocomial) bacterial pathogens worldwide. In most routine microbiological settings, the detection of methicillin resistance among staphylococcus isolates is based on phenotypic assays. Genetic confirmation of positive findings based on the detection of mecA gene (Murakami et al., 1991). Meticillin resistance in staphylococcus aureus is primarily mediated by the low-affinity penicillin –binding protein 2a or 2′ (PBP2a or PBP2′) encoded by the chromosomal structural mecA which have reduced the affinity for beta-lactam antibiotics  (Hartman and Tomaz, 1984; Matshashi et al., 1986). However, mecA is also widely distributed among coagulase-negative Staphylococci (CNS), and is associated with methicillin resistance of CNS (Ubukata et al., 1990).

Thus PCR assay targeting mecA alone can’t be discriminated between MRSA and methicillin-resistant CNS. The FemB gene locus is distant from the mecA gene on the chromosome, and it is involved in cell wall pentaglycin side chain and interpeptide bridge formation FemB is highly conserved in S. aureus but it is not found in CNS and non-Staphylococci (Berger-Bachi et al., 1992; Henze et al., 1993 and Kobayashi et al., 1994). Therefore both mecA and FemB are employed for the rapid detection of MRSA by PCR (Kobayashi et al., 1994; Joans et al., 1999; Towner et al., 1998 and Perez-Roth et al., 2001).  This study described the performance of this technique in a different water samples which contain pathogenic microorganisms such as E. coli, Shigella spp, Salmonella spp and Pseudomonas aeruginosa. Moreover the test was investigated by determine the positive readings due to coamplification of mecA from the previous species. In this study the detection of mecA gene (characteristically encoding for methicillin resistance in staphylococci) in a single rapid test, was investigated. All strains were subjected to different groups of antibiotics and resistance was recorded as percentage (%). The aims of the study were the isolation of pathogenic strains from water samples, identification of pathogenic strains, effect of antibiotics on these strains and detection MRSA gene among the isolated strains by using PCR technique.

MATERIALS AND METHODS

Thirty two strains were isolated from different water resources and identified according to Bergey’s Manual (1989). All isolates of Salmonella, Shigella, pseudomonas and E. coli were tested for susceptibility by the Kirby-Bauer’s disk diffusion method (1966) against the  ten following antibiotics: E-Moxiclav (30 µg), Rifamycin (30 mcg), Cefalexin (30 mcg), Erythromycin (15 mcg), Ceftazidim (30 mcg), Norfluxacin (10 µg), Ciprofluxacin (5 µg), Cefepim (30 mcg),    Ertapenem( 10 µg) and Cefoxitin (30 mcg). Identified Staphylococcus aureus starian was selected as standard. Genomic DNA isolation method was modified from Experimental Techniques in Bacterial Genetics (Jones and Bartlet, 1990)

DNA samples were diluted to concentration of 20 ng/µl prior to amplification.  Five µl aliquot of bacterial genomic DNA was combined with 12.5 µl of master mix (500 mM KCl, 100 mM tris- HCl [pH at 25 0C], 15 Mm MgCl2, 1% Triton x-100), 1 µl of a deoxynucleoside triphosphate (dNTP) mixture (concentration of dNTP, 5 mM), and 2 µl each of two 15-base oligonuclutied primers (primer G1 and L1 [concentration, 50 ng/µl]) 3.5 µl deionized water; the final volume was 25 µl.

Primer

MecA1            (5′ –GTA GAA ATG ACT GAA CGT CCG ATA A)

MecA2              (5′- CCA ATT CCA CAT TGT TTC GGT CTA A)

The PCR cycling conditions were as follows: initial denaturation at 940C for 4 minutes. Followed by 30 cycles of 45 s at 940C, 45 s at 500C, and 60 s at 720C. With the final extension step at 720C for 2 minutes. Ten microleter were loaded onto agarose gel electrophoresis.

Results

The different isolates were identified at the genus level by means of standard procedures described in Bergey’s manual of systematic microbiology and by standard laboratory procedures (E. coli, Shigella flexneri , shigella sonnie, Salmonella serovar Newport and Pseudomonas aeruginosa).

Table (1): Antibiotic Susceptibility.

Lane 1: MRSA- positive control with the 310 bp mecA amplicons. Lane 2: DNA Marker. Lanes (3-4): Shigella flexneri strains isolated from Groundwater at Sohag. Lanes (5-6): Shigella flexneri strains isolated from Nile water at Sohag. Lanes 7: Shigella flexneri strains isolated from Nile water at Akmim. Lanes 8: Shigella flexneri isolated from Nile water at Tahta. Lanes 9: Shigella flexneri isolated from Nile water at Sakolta. Lanes (10-12): Shigella flexneri isolated from Nile water at Girga.

  

Lane 1: MRSA- positive control with the 310 bp mecA amplicons. Lane 2: DNA Marker. Lanes (3-4): Shigella flexneri strains isolated from Groundwater at Sohag. Lanes (5-6): Shigella flexneri strains isolated from Nile water at Sohag. Lanes 7: Shigella flexneri strains isolated from Nile water at Akmim. Lanes 8: Shigella flexneri isolated from Nile water at Tahta. Lanes 9: Shigella flexneri isolated from Nile water at Sakolta. Lanes (10-12): Shigella flexneri isolated from Nile water at Girga.

Lane 1: DNA Marker. Lanes (2-3): E. coli strains isolated from Nile water at Sohag station. Lane 4: E. coli strains isolated from Nile water at Akmim station. Lanes 5: E. coli strain isolated from Nile water at Girga station. Lane 6: E. coli strains isolated from Nile water at Dar-Elsalam station. Lanes (7-8): Pseudomonas aeruginosa strains isolated from Nile water at Sohag station. Lanes 9: Pseudomonas aeruginosa strains isolated from Nile water at Akmim station. Lanes 10: Pseudomonas aeruginosa strains isolated from Nile water at Girga station. Lanes 11: Pseudomonas aeruginosa strains isolated from Nile water at Dar-Elsalam station. Lanes 12: Pseudomonas aeruginosa isolated from Nile water at Tahta station. Lane 13: MRSA- positive control with the 310 bp mecA amplicons.

 Lane 1: MRSA- positive control with the 310 bp mecA amplicons. Lane 2: DNA Marker. Lanes 3: Salmonella serovar Newport isolated from Nile water at Sohag. Lanes 4: Salmonella serovar Newport isolated from Nile water at Akmim.

Lane 1: MRSA- positive control with the 310 bp mecA. Amplicons.  Lane 2: DNA Marker. Lane 3: E. coli strains isolated from Nile water at Tahta. Lane 4: E. coli strains isolated from Nile water at Sohag. Lane 5: E. coli strains isolated from Groundwater at Tahta. . Lanes (6-7): E. coli isolated from Groundwater at Tema.  Lanes (8-9):Shigella sonnie strains isolated from Nile water at Tahta. Lane 10:  Shigella sonnie strains isolated from Nile water at Sohag. Lane 11: psedumonas aerugimosa strains isolated from Nile water at Tahta.

Antibiotic susceptibility test:-

A total of 32 bacterial isolates (23 were isolated during the hot season and 9 isolates during the cold season). Results obtained using 10 antibiotics are represented in table 32. The percentage of resistant strains to antibiotics were recorded in ( Table 1and 2)

Salmonella serovar Newport:

Two isolates were isolated at hot season and identified as Salmonella serovar Newport. As shown in (Table 1& 2), these isolates were resistant to the tested antibiotics, since 33.33% of them were sensitive and 20 % have shown medium sensitivity to the tested antibiotics. The tested strains isolated from Nile water at Sohag site were resistant to Cefalexin, Cefepim, Rifamycin, Ertapenem, and Cefoxitin. It was sensitive to Ciprofluxacin, Norfloxacin, Ceftazidim. Also, it shows low sensitivity to Erythromycin and E-Moxiclav.

Shigella flexneri:

Ten isolates were obtained at hot season and identified as Shigella flexneri. As shown in (Table 1& 2). 62% from the Shigella flexneri isolates were resistant to the tested antibiotics, 34% of them were sensitive and 4 % have shown medium sensitivity to the tested antibiotics.  The isolates from Nile water in Tahta were resistant to Cefalexin (30 mcg), Erythromycin, Cefepim, Rifamycin, Ceftazidim, but were sensitive to E-Moxiclav, Ciprofluxacin, Norfluxacin, Ertapenem, Cefoxitin. Also the tested strains isolated from groundwater at Sohag city were 100% resistant  to Erythromycin, Rifamycin, Cefalexin, Ceftazidim,  Cefoxitin,  resistant with 50% to Cefepim, Norfluxacin  and Ertapenem. Also, it were sensitive (100%) to Ciprofluxacin and were 50% sensitive to E-Moxiclav, Norfluxacin, Ertapenem.These strains showed low sensitivity to E-Moxiclav  and Cefepim. The strains isolated from Nile water at Sohag site were resistant with 100% to Erythromycin, Cefalexin, Ceftazidim, Rifamycin, Cefepim, resistant with 66.67% to Cefoxitin, E-Moxiclav  and resistant to 30 % to Ertapenem. Also, it can be significant from the table (29) that these strains were sensitive with 100% to Ciprofluxacin, Norfluxacin, sensitive (66.67%) to Ertapenem, sensitive (33.33%) to Cefoxitin and E-Moxiclav. The strains isolated from Nile water at Girga site were resistant with 100% to Erythromycin, Cefalexin, Rifamycin, resistant (66.67%) to Cefepim, Ertapenem. Ceftazidim, resistant (33.33%) to Ciprofluxacin, Norfluxacin), E-Moxiclav and Cefoxitin. Also, these strains were sensitive (66.67%) to Norfluxacin, E-Moxiclav, sensitive (33.33%) to Cefepim, Ertapenem, Ceftazidim, Cefoxitin and Ciprofluxacin. These strains showed low sensitivity (33.33%) to Cefoxitin and Ciprofluxacin .

Shigella sonnei:

Three isolates were isolated at the cold season and identified at genus level as  Shigella sonnei. As shown (Table 1& 2) that 60% from the Shigella sonnei isolates were resistant to the  tested antibiotics, 33.33% of them were sensitive and 6.7 % showed low sensitivity to the tested antibiotics. Also,   as seen  table in table (27) the strains were isolated from Nile water at Tahta site were resistant with 100% to antibiotics Erythromycin, Cefalexin, Ceftazidim, Rifamycin, Cefepim, Cefoxitin, Norfluxacin, resistant with 50% to  E-Moxiclav, Ciprofluxacin  and Ertapenem. These strains were sensitive with 50% to E-Moxiclav, Ciprofluxacin and Ertapenem. The strain K13 which was isolated from Nile water at Sohag site showed the highest resistance to Rifamycin, sensitive to Cefalexin, Ceftazidim, Cefepim, Norfluxacin, E-Moxiclav, Ciprofluxacin, Ertapenem, low sensitive to Erythromycin and Cefepim .

  1. coli:

Five isolates were isolated at the hot season, five other were isolated at the cold season and identified at genus level as E. coli. As shown in (Table 1& 2) that 26% from the E. coli isolates were resistant to the tested antibiotics, 66% of them were sensitive and 8% have shown medium sensitivity to the tested antibiotics. In details it can be significant that the isolates DK1, DK2 and DK3 were isolated from Nile water at Sohag site during the hot season were resistant with100% to Cefepim, Ceftazidim, resistant (33.33%) to E-Moxiclav, Cefoxitin, sensitive with 100% to Cefalexin, Rifamycin, Ciprofluxacin,  Norfluxacin, Ertapenem, sensitive (66.67%) to Erythromycin, E-Moxiclav, Cefoxitin  and moderate sensitive (33.33%)to Erythromycin . The isolate DK4 and DK5 were isolated from Nile water at Girga site dring the hot season were resistant with 100% to Rifamycin, Ertapenem , resistant with 50% to Erythromycin, E-Moxiclav, Cefepim, Ceftazidim, sensitive with 100%to Ciprofluxacin,  Norfluxacin, Cefoxitin, sensitive with 50% to Cefepim, Ceftazidim. These isolates were moderate sensitive with 100% to Cefalexin, low sensitive to Erythromycin and E-Moxiclav. The isolate DK6 was isolated from groundwater at Tema site during the cold season was resistant to Cefepim, Ceftazidim, sensitive with to Cefalexin, Rifamycin, Ciprofluxacin,  Norfluxacin, Ertapenem, Cefoxitin, Erythromycin, Ceftazidim. The isolate DK7 and DK9 were isolated from groundwater at Tahta site during the cold season were resistant with 100% to  Cefepim, Ceftazidim, resistant with 50% to Cefoxitin, sensitive with 100% to  Cefalexin, Rifamycin, Ciprofluxacin, Norfluxacin, Ertapenem, Erythromycin, Erythromycin, E-Moxiclav and sensitive with 50% to Cefoxitin. The isolate DK8 was isolated from Nile water at Tahta site was resistant to Cefepim, sensitive to Cefalexin, Rifamycin, Ciprofluxacin, Norfluxacin, Ertapenem, Erythromycin, E-Moxiclav and moderate sensitive to Cefoxitin, Ceftazidim. The isolate DK10 was isolated from Nile water at Sohag site during the cold season was resistant to Cefepim, Ceftazidim, sensitive to sensitive to Cefalexin, Cefoxitin, Ciprofluxacin, Norfluxacin, Ertapenem, Erythromycin, E-Moxiclav and moderate sensitive to Rifamycin.

Pseudomona aeruginosa:

six isolates were isolated  at the hot season and cold season and identified at genus level as  Pseudomona aeruginosa. As shown in (Table 1& 2). that 62.85% from the Pseudomona aeruginosa isolates were resistant to the tested antibiotics, 34.28% of them were sensitive and 2.85% have shown medium sensitivity to the tested antibiotics. At more accurately analysis it can be obvious that the isolate PSI1 was isolated from Nile water at Tahta site during the hot season was resistant to Cefepim, to Cefalexin , Rifamycin, Ciprofluxacin, Norfluxacin, Ertapenem, Erythromycin, E-Moxiclav, Cefoxitin, Ceftazidim and sensitive to Ciprofluxacin,  Norfluxacin. Also, the isolates PSI2, PSI3 and PSI4 were isolated from Nile water at Sohag site were resistant(100%) to Erythromycin, Cefalexin ,resistant (66.67%) to Ceftazidim, Cefepim, Cefoxitin, resistant (33.33%) to E-Moxiclav, Ertapenem. These isolates were sensitive (100%) to Ciprofluxacin,  Norfluxacin, sensitive (66.67%) to Ertapenem, sensitive (33.33%) to Ceftazidim, Cefepim, E-Moxiclav, Cefoxitin and moderate sensitive (33.33%) to E-Moxiclav. The isolate PSI5 and PSI6 were isolated from Nile water at Girga site during the hot season were resistant (100%) Erythromycin, Cefalexin, Ceftazidim, Cefepim, Cefoxitin, Rifamycin, sensitive (100%) to E-Moxiclav, Ciprofluxacin, Norfluxacin and Ertapenem. The isolate PSI7 was isolated from Nile water at Tahta site during the cold season was resistant (100%) to Erythromycin, Cefalexin, Ceftazidim, Cefepim, Cefoxitin, Rifamycin, Ertapenem, sensitive (100%) Ciprofluxacin , Norfluxacin and moderate sensitive to E-Moxiclav.

Detection of MRSA gen by PCR amplification

Methicillin-resistant gene was undetected (negative) in E. coli, Salmonella spp, shigella spp. and pseudomonas sp as shown in fig.87- 90.

Discussion

Staphylococcus aureus is a remarkable pathogenic organism that has acquired resistance to all classes of antimicrobials and thus is a continuing threat in both the hospital and community health care units (Biedenbach, et al., 2004). The acquisition of the mecA gene, which confers resistance to methicillin, spawning so-called methicillin-resistant S. aureus (MRSA), has resulted in a highly resilient pathogen that has reached epidemic levels in many parts of the world (Rennie, et al., 2004). All isolated strains had broad spectrum resistance for all tested antibiotics especially quinolones (Ciprofluxacin) and B-Lactams (E-moxclav) as shown in (Table 1& 2). Also,  as significant in figures (2-5) all strains were negative for MRSA-PCR this is due to all strains belonging to Enterobactericea unless P. aeruginosa and all of them did not have mecA gene.  Our results were in agreement with (Sekigushi et al., 2004 and kawai et al., 2009).

 

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