Molecular detection of β-lactamases and aminoglycoside resistance genes among Escherichia coli isolates recovered from medicinal plant

Cross contamination from composting farm manure and surface run-off waters or the application of untreated sewage, or manure can be sources of pathogenic microorganisms that contaminate fruits and vegetables in the field. Therefore, the current study was aimed to detect the incidence of Escherichia coli isolates from herbs samples and the molecular detection of β-Lactamases and aminoglycoside resistance genes among E. coli isolates recovered from medicinal plant. 58 strains of E. coli (28.43%) were isolated and serotyping of the recovered E.coli isolates revealed 18 strains O125 (31.03%), 12 strains were (20.69%) O86, 10 strains were (17.24%) O112, 8 strains were (13.79%) O127, 6 strains were (10.34%) O128 and 4 strains were (6.90%) O44. The E. coli isolates were tested by PCR for detection of aacC2 gene; all isolates showed amplification of 896 bp fragments specific for aacC2 gene. Multiplex PCR showed amplification of 516 bp fragments specific for blaTEM gene in all isolates (100%). Moreover, 38 of the E. coli isolate (65.52%) had a band compatible with blaSHV gene (392 bp fragments) while, the presence of blaOXA_1 gene was not detected in all E. coli isolates.


INTRODUCTION
Bacterial food-borne pathogens are the most important food safety issue worldwide.During the past decade, outbreaks of human illness associated with the consumption of fresh fruits and vegetables have increased in the United States (Beuchat, 1996;Brackett, 1999;Blank et al., 2008;CDC, 2008;Berger et al., 2009).The risk of human illness associated with raw produce can be better predicted by monitoring microbial contamination at points of potential contamination in the field during harvesting, during processing and distribution, or in retail markets (Beuchat and Ryu, 1997;Brackett et al., 1993).One major factor that can influence the microbial quality of produce is the type of irrigation used and the water source (Beuchat, 1996;Woodford et al., 2006).For example, cross-contamination from composting farm manure, flooding, and surface run-off *Corresponding author.E-mail: imoussa1@ksu.edu.sa,moussaihab@gmail.com.Tel: 00966560749553.Fax: 00966-14678456.
waters can be sources of pathogenic microorganisms that contaminate fruits and vegetables in the field (Beuchat and Ryu, 1997;Brackett, 1999, Bowen et al., 2006).The presence of enteric human pathogens in the soil may also be largely due to the application of faeces, untreated sewage, or manure either by chance or design.As a result, contamination of fresh produce by fecal coliforms is well documented and recognized as a potential public health problem (Backer et al., 2000;Campbell et al., 2001;Erickson et al., 2007).Several recent outbreaks of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp.have been associated with fresh produce (Backer et al., 2000;Brackett, 2001;Campbell et al., 2001;Itoh et al., 1998).The rapid and accurate identification of bacterial pathogens from food samples is important, both for food quality assurance and to trace outbreaks of bacterial pathogens within the food supply.Growing concerns regarding the safety of fresh produce warrant a greater emphasis on the development of more rapid, specific, and highly sensitive detection methods.Advances in biotechnology have permitted more reliable microbial identification and surveillance (Feng, 1997).Antimicrobial therapy is one of the measures taken to control the diseases caused by E. coli (Blanco et al.,1997), as a result, multi-resistant E. coli isolates may emerge and become a worldwide public health problem, including direct impact on food safety.Antimicrobial resistance determinants are carried mostly by mobile genetic elements such as plasmids, transposons, and integrons (Rowe-Magnus and Maael 2002).
Therefore, fresh or minimally processed vegetables which are often eaten raw or minimally processed, can compromise consumer health safety.The most common bacterial enteropathogens associated with fruits and vegetables are Salmonella spp.(Thunberg et al., 2002) and E. coli O157:H7 (Rangel et al., 2005;Friesema et al., 2008;Pakalniskiene et al., 2009).They are considered among the organisms that are most likely to cause an outbreak and therefore need to be studied for produce safety (Buck et al., 2003).Salmonella is responsible for a localized, self-limiting bacterial infection of the intestinal epithelium, known as ''non-typhoid salmonellosis'' or ''gastroenteritis'' and a systemic infection known as ''typhoid'' or ''enteric fever'' with severe consequences.The incidence of typhoid salmonellosis is stable, with very few cases in developed countries, but cases of nontyphoid salmonellosis are increasing worldwide (Garcı´a del Portillo, 2000).E. coli O157:H7 is the most toxigenic serotype of the genus and is an important cause of diarrhoea, hemorrhagic colitis and haemolytic-uremic syndrome worldwide, associated with high mortality rates of between 10 and 40% (Palermo et al., 2009).Therefore, the main objective of this study was the molecular detection of the resistance caused by βlactamases (blaTEM, blaSHV and blaOXA_1 genes) and the resistance caused by aminoglycoside resistance genes responsible for resistance to gentamicin (aacC2 gene) in resistant E. coli isolates recovered from medicinal plants.

Samples
During the summer of 2010, a total of 204 herbs samples; 34 basil, 40 dry mints, 28 marjoram, 72 chamomile, 22 fennel and 8 calendula flower samples were collected and analyzed for detection and isolation of E. coli.Ten negative control field samples were collected from farms continuously monitored for E. coli by standard microbiological techniques.
All samples were transported to the laboratory under refrigerated conditions where they were processed and bacteriologically examined immediately.

Isolation and identification of E. coli
Surface technique was used according to modified NMKL, 125-1996 and ISO/FDIS 7218-2007 and membrane filtration method ISO 9308-1, 2000).All samples were primarily cultured on MacConkey agar medium and incubated aerobically at 37°C.After overnight incubation, a part of single typical well isolated lactose fermenting colonies was tested for sorbitol fermentation by culturing on sorbitol MacConkey agar and sorbitol phenol red agar media and incubated at 37°C overnight.Morphological, cultural and biochemical examination were carried out according to methods described by Quinn et al. (2002).

Resistance gene detection by PCR technique
PCR was conducted with primers described for the detection of BlaTEM, BlaSHV, BlaOXA_1 and aacC2 genes.The primers sequences and the amplified fragments for the resistance genes are illustrated in Table 1.

Extraction of DNA of E. coli samples by QIAamp DNA Mini Kit (Promega-catalogue number 51304)
The QIAamp DNA Mini Kit provides silica-membrane-based nucleic acid purification from tissues, swabs, CSF, blood, body fluids, or washed cells from urine.The spin-column procedure does not require mechanical homogenization, so total hands-on preparation time was only 20 min.Purification of DNA using the QIAamp DNA Mini Kit can be automated on the QIA cube.

DNA amplification
Temperature and time conditions of primers during PCR were carried out according to the method of Palermo et al. (2009) and Friesema et al. (2008).PCR amplifications were performed in a final volume of 50 l in micro-amplification tubes (PCR tubes).The reaction mixtures consisted of 5 l of the DNA template, 5 l 10x PCR buffer (75 mM Tris-HCl, pH 9.0, 2 mM MgCl 2 , 50 mM KCl, 20 mM (NH 4 ) 2 SO 4 ), 1 l dNTPs (40 M), 1 l 1U Ampli Taq DNA polymerase, 1 l (25 pmol) from the forward and reverse primers of both primer pairs and the volume of the reaction mixture was completed to 50 l using double distilled water (DDW).The thermal cycler was adjusted as follows: Initial denaturation at 94°C for 5 min, followed by 35 cycles of (denaturation at 94°C for 30 s, annealing at 56°C for s, and extension at 72°C for 1 min).Final extension was carried out at 72°C for 10 min and the PCR products were stored in the thermal cycler at 4°C until they were collected.

Agarose gel electrophoresis
The PCR products were tested for positive amplification by agarose gel electrophoresis as previously reported by Sambrook et al. (1989) using suitable molecular weight markers.

Antimicrobial susceptibility testing
The 58 herbs strains has resistance pattern against erythromycin, penicillin, amoxicilline clauvenic and vancomycine with a percentage of 100% and gentamicin, ampicillin, chloramphenicol, oxytetracycline, norfloxacin, trimethoprim and nalidixic acid with percentages of 75.9 65.5, 51.7, 48.3, 17.2, 6.9 and 6.9% respectively and non of the herbs strain had resistance against ciprofloxacin and ceftazidime.

Results of Multiplex PCR for detection of βlactamases (blaTEM, blaSHV and blaOXA_1 genes) and single PCR detection of aminoglycoside resistance genes responsible for resistance to gentamicin (aacC2 gene) in resistant E. coli isolates
Multiplex PCR was carried out to detect the resistance genes against ampicillin, amoxicillin clavulanic acid and gentamicine as shown in Table 3 and Figure 1.
A total of 58 E. coli isolates recovered by bacteriological examination were tested by PCR for detection of aacC2 gene responsible for the gentamicinresistant.All the isolates showed amplification of 896 bp fragments specific for aacC2 gene as shown in Table 3 and Figure 2. A multiplex PCR was designed to detect BlaTEM, BlaSHV and BlaOXA_1 genes in amoxicillinclavulanic acid and ampicillin resistant E. coli isolates.Among the 58 amoxicillin-clavulanic acid and ampicilline resistant isolates, multiplex PCR showed amplification of 516 bp fragments specific for BlaTEM gene in all isolates (100%).Moreover, 38 of the E. coli isolate (65.52%) had a band compatible with BlaSHV gene (392 bp fragments) while, the presence of BlaOXA_1 gene was not detected in all E. coli isolates as shown in Table 3 and Figure 1.

DISCUSSION
In recent years, the growing consumption of fresh, minimally processed vegetables has lead to an increase in the number of outbreaks of food-borne disease linked to fresh produce (Anon, 2002;Blanck et al., 2008;Berger et al., 2009;Pakalniskiene et al., 2009;Palermo et al., 2009).Agricultural irrigation with wastewater that can be raw, treated and/or partially diluted, is a common practice worldwide and constitutes the main source of pathogen contamination (CDC, 2006;CDC, 2008;Friesema et al., 2008).In the present investigation, 204 herbs samples: 34 basil, 40 drymint, 28 marjoram, 72 chamomile, 22 fennel and 8 calendula flower samples were investigated bacteriologically to detect and isolate E. coli.
The in vitro sensitivity test of 58 E. coli strains isolated from herbs in this study against 13 antibiotics agents revealed that all of the tested strains were highly resistant  to penicillin, vancomycine, amoxicillin clauvinic acid and erythromycin with percentage 100% and this also had been reported by Nazir et al. (2005) who found that all the E. coli isolates were found resistant to penicillin G (94.45%).
These results agrees with those of Wani et al. ( 2004) who found that the in vitro sensitivity profiles revealed that all isolates were resistant to penicillin and erythromycin which indicated the need for judicious use of antibiotics.
Due to the emergence of large numbers of different blaSHV and blaTEM, b-lactamases isoelectrofocusing appears not to be the method of choice for establishing an ESBL phenotype (Hernandez et al., 2005).The solution to this problem has been to characterize ESBLencoding genes using molecular biology techniques with specific primers for blaSHV, blaTEM, and blaCTX-M genes, followed by DNA sequence analysis of the PCR amplicons (Boyd et al., 2004).Recently, multiplex PCR assays for detection of bla-TEM, blaSHV and blaOXA-1 gene have been described (Woodford et al., 2006).
All the 58 E. coli isolates recovered by bacteriological examination were tested by PCR for detection of aacC2 gene responsible for the gentamicin-resistant.All the isolates showed amplification of 896 bp fragments specific for aacC2 gene as shown in Table 3 and Figure  2. The obtained results confirm the conclusion of Ho et al. (2010) who stated that the gentamicin-resistant isolates was PCR positive for the aaC2 gene.Among the 58 amoxicillin-clavulanic acid and ampicillin resistant isolates, multiplex PCR showed amplification of 516 bp fragments specific for BlaTEM gene in all isolates (100%).Moreover, 38 of the E. coli isolate (65.52%) had a band compatible with BlaSHV gene (392 bp fragments).The predominant resistance genes were: ampicillin, blaTEM1-like and gentamicin aaC2 genes (Guerra et al., 2003).
Therefore, in conclusion, this study show that multiplex PCR is a suitable tool for rapid screening of plasmid-encoded L-lactamases which are known to confer amoxicillin-clavulanic acid resistance in E. coli.

Figure 1 .
Figure 1.Multiplex PCR showing positive amplification of 516 base fragment specific for BlaTEM gene and 392 base pair fragments specific for BlaTSHV gene.

Figure 2 .
Figure 2. Agarose gel electrophoresis showing amplification of 896 aacC2 gene responsible for the gentamicin resistant.

Table 1 .
The list of primers used in the present study.

Table 2 .
Serotypes of E. coli isolates recovered from the examined herbal samples.

Table 3 .
Results of the resistance gene detection among the E. coli isolates