Abstract: A polymerase chain reaction was standardized for the identification of Aeromonas sp. using primers against 16S rRNA gene and aerolysin gene. The primers used were found to be highly specific for Aeromonas sp. and did not give positive result with other bacteria including other Gram positive and Gram-negative bacteria. The minimum detection level of PCR was found to be 102 and 104 cells mL-1 in case of 16S rRNA and aerolysin gene targeted assay, respectively. Suitability of the enrichment broth (Alkaline peptone water-cephalothin, APW-C) when tested to detect Aeromonas from the spiked samples gave good results on direct usage of the broth for template preparation without any subsequent treatment. The kinetics of the spiking study indicated that a minimum of 24 h enrichment was required for the detection of Aeromonas by cultural and PCR method. Among two PCR assays detection limits achieved by PCR targeting16S rRNA gene were better than aerolysin gene PCR assay. The results were comparable to cultural method. A total of 100 samples comprising of 50 each of chicken and fish samples were screened by cultural and PCR methods for the presence of Aeromonas. Two chicken samples and three fish samples turned out to be positive by both cultural method and PCR targeting 16S rRNA. From this study it was concluded that PCR assay targeting 16S rRNA gene can be used for the rapid detection of Aeromonas from chicken and fish samples after one step enrichment in APW-C.
INTRODUCTION
The genus Aeromonas is widely distributed in aquatic environment and increasingly reported as a primary pathogen of human and lower vertebrates. Aeromonas has been reported as an etiological agent in a variety of human infections including gastroenteritis and extra intestinal infections (Janda and Abbott, 1998). In India, Aeromonads are common contaminants in a wide spectrum of foods namely fishes and other seafoods, raw and cooked meat, poultry, vegetables, milk and milk products (Agarwal et al., 2000; Bachhil et al., 2002; Khurana and Kumar, 1997). These foods play an important role in the dissemination of the pathogenic Aeromonas to humans. The traditional classical microbial procedures for the detection of Aeromonas sp. are laborious and time consuming or do not allow quantitative assessment of there organisms (ICMSF, 1996). In this context the detection of the pathogen requires rapid and specific methods, which assist in the control of pathogenic organisms from various food sources to human population, which are mostly regarded as the main transport vector to human population. Although PCR is very effective with pure culture, its application to food samples is limited by the complex composition of food matrices that can inhibit the assay. In addition, PCR cannot differentiate the DNA from live cells or dead cells. To overcome these problems, enrichment of sample and a sample preparation step prior to the PCR analysis is necessary.
Keeping in view the above points this study was envisaged to develop a rapid, sensitive and specific PCR based assay to detect Aeromonas from chicken and fish samples and also to assess the comparative efficacy of PCR assay vis-a-vis conventional isolation and identification method for detection of Aeromonas from artificially and naturally contaminated chicken and fish samples.
Materials and Methods
Bacterial Strains
The bacterial strains used in this study are listed in Table
1.
Oligonucleotide Primers
The primers for conserved regions 16S rRNA gene (Graf, 1999) and aerolysin
gene (Santos et al.,1999) of Aeromonas used in this study were
got synthesized from Bangalore Genei, Bangalore. The details of the primers
used are as follows:
| Primer set No. | Product size | ||
| 16 S rRNA (Graf,1999) | |||
| 1. | Forward primer Reverse primer |
5’-TCATGGCTCAGATTGAACGCT-3’ 5’-CGGGGCTTTCACATCTAACTTATC-3’ |
599 bp |
| Aerolysin gene (Santos et al., 1999) | |||
| 2. | Forward primer Reverse primer |
5’-GCAGAACCCATCTATCCAG-3’Reverse 5’-TTTCTCCGGTAACAGGATTG-3’ |
252 bp |
Standardization of Polymerase Chain Reaction
Template DNA Preparation
Three different methods of cell lysis and release of DNA as described below
were compared for their use in PCR assay.
Sodium Dodecyl Sulphate (SDS) Treatment
About 1 mL of the overnight incubated culture of Aeromonas was centrifuged
and the pellet was suspended in about 20 μL of 0.05% SDS and heated in
a boiling water bath for 5 min and snap chilled. From this about 5 μL was
used as template in PCR.
Sonication
About 1 mL of the overnight incubated culture of Aeromonas was centrifuged
at 5000 g for 20 min and the pellet was resuspended in distilled water, subjected
to sonication (MSE Sanyo Sonicator) 10 μ amplitude, 60 sec/cycle, 5 cycles
and was used in PCR.
Boiling and Chilling
In this method about 0.5-1.0 mL of the overnight-incubated culture of Aeromonas
was subjected to vigorous heating in a boiling water bath for 10 min and
then snap chilled. From this about 5 μL was used as template in PCR.
PCR Protocol
The PCR was set up in 50 μL reaction volume. The reaction mixture was
optimized as follows 5.0 μL of 10x Taq DNA polymerase buffer, 1.5 mM MgCl2
(3 μL), 0.22 mM of dNTP mix (6 μL), 10 pM of forward and reverse
primer (2 μL each), 1U of Taq DNA polymerase, 5 μL of cell lysate.
The cycling conditions included an initial denaturation at 94°C for 5 min
followed by 30 cycles each of 1 min denaturation at 94°C, 1 min annealing
at 55°C and 1 min elongation at 72°C. It was followed by a final extension
for 5 min at 72°C. The samples were analysed in 1.5% agarose gel electrophoresis
with ethidium bromide.
| Table 1: | List of bacterial strains |
Specificity of PCR Assay
The specificity of primer set No. 1 (16S rRNA) and set No. 2 (aerolysin
gene) were tested with different Gram positive and Gram-negative bacteria as
listed in Table 1
Sensitivity of the PCR
Trials were conducted to evaluate the lowest quantum of bacterial cells
that would produce a visible signal by the PCR assay. The standard strain of
Aeromonas (MTCC 646) was inoculated in BHI broth and grown overnight
at 37°C. The cells were pelleted by centrifugation at 6000 rpm for 10 min
and the cell concentration was adjusted to an approximate concentration 108
cells mL-1 based on spectrophotometer absorbance reading of 0.6 at
540 nm and then ten fold serial dilutions were made and subjected to PCR assay
according to standardized protocol. The total viable count (log cfu mL-1)
of the bacterial suspension was confirmed by spread plate method.
Evaluation of Enrichment Broth
Suitability of the enrichment broth (APW-C) was assessed to test whether
any ingredient of medium will hinder the PCR. The organism was grown overnight
in the broth and1 ml aliquots was taken in triplicate from the broth and were
subjected to three different treatments as described in Table
2 before it was used in PCR.
Experimental Inoculation/spiking Studies
The experimental inoculation studies were carried out to assess the efficacy
of the standardized PCR method for the detection of Aeromonas from the
chicken meat samples after one-step enrichment of samples. The meat samples
(chicken) were collected randomly from different parts of the carcass from the
local markets (Retail outlets) in and around Barielly. Adjustment of concentration
to 1x108cells mL-1 and further serial ten fold dilutions
of standard Aeromonas culture was done as described above. The spiking
of meat samples was carried out as shown in flow diagram (Fig.
1). The experimental inoculation of chicken meat and subsequent identification
of Aeromonas was repeated five times to ascertain the repeatability of
the cultural and PCR results Table 3.
Detection of Aeromonas from Natural Samples
A total of 100 samples comprising of 50 meat (chicken) and 50 fish samples
(gills) were purchased from the local market (Retail outlets) of Barielly. The
samples were subjected for isolation/ detection of Aeromonas by cultural
method as well as PCR method as described above, after one-step enrichment.
| Table 2: | Suitability of enrichment broth for detection of Aeromonas
from foods |
| Fig. 1: | Flow diagram for experimental inoculation studies |
Results and Discussion
Comparison of different protocols for DNA extraction revealed that sonication and heat lysis method gave a clear-cut amplicon of 599 and 252 bp length product on PCR assay targeted for 16S rRNA and aerolysin genes, respectively. Template prepared by lysis buffer also gave positive amplicon but it was lesser in intensity than other methods. Among snap chilling after boiling and sonication method, the former method was used in all subsequent experiments of study as it was found to be very simple, rapid and gave good results. Some previous workers have also successfully used this method for the release of DNA (Gonazalez-Rodriguez et al., 2002; Surendran et al., 2003). The PCR protocol was standardized by optimizing annealing temperature, primer, MgCl2 concentrations, template volume and cycling conditions. Electrophoretic analysis of the PCR product revealed the specific amplification of 599 and 252 bp fragments without any spurious product for both the primers targeted against 16S rRNA and aerolysin genes (Fig. 2). The specificity of the standardized PCR assay was tested by subjecting to different Gram positive and Gram-negative organisms (Table 1) to PCR assay. Only Aeromonas cultures yielded a specific PCR product of desired length i.e., 599 and 252 bp. These findings are in confirmation with the results obtained by Gonazalez-Rodriguez et al. (2002) who have reported no cross-reactions with other bacteria using the primer against aerolysin gene. These findings are in confirmation with the results obtained by Gonazalez-Rodriguez et al. (2002). The sensitivity of the PCR assay was evaluated by subjecting serial ten fold dilutions of pure culture of Aeromonas ranging from 109 to 10 cells mL-1. The minimum detection level was found to be 102 cells mL-1 for 16S rRNA gene and 104 cells mL-1 for aerolysin gene (Fig. 3 and 4). Similar sensitivity of PCR has been reported in literature (Khan and Cerniglia, 1997; Gonazalez-Rodriguez et al., 2002).
| Fig. 2: | Standardization of PCR for detection of Aeromonas.
Lane M: 100 bp ladder, Lane 1: Product obtained with primer for 16S rRNA
gene, Lane 2: Product obtained with primer for Aerolysin gene |
| Fig. 3: | Minimum detection levels of Aeromonas cells by using
primer for 16S rRNA gene. Lane M: 100 bp DNA ladder plus, Lane 1: 10 cells
mL-1, Lane 2: 102 cells mL-1, Lane 3: 103
cells mL-1, Lane 4: 104 cells mL-1, Lane
5: 105 cells mL-1, Lane 6: 106 cells mL-1,
Lane 7: 107 cells mL-1, Lane 8: 108 cells
mL-1 |
Better detection limits with PCR for 16S rRNA gene than aerolysin gene PCR may be attributed to the multiple copies of ribosomal RNA that are present in a single bacteria (Rahman, 2002, Gonzalez-villasenor and Manak, 1998). It has been previously reported that certain media components inhibit PCR reaction (Rossen et al., 1992). Hence suitability of enrichment medium viz., APW-C and three different treatments for extraction and concentration of target organisms in small volume were tested. The results revealed that all the three treatments yielded PCR products of equal intensity indicating no PCR inhibitory substance was present in the medium. Our results are in agreement with previous findings of (Sachan and Agarwal, 2000) for the isolation of this organism.
Spiking studies in meat (chicken) were done to determine the ideal enrichment protocol, which could detect the least concentration of inoculum at the earliest by PCR assay and also compare the results with that of cultural method. As reported by several workers (Wang et al., 1997; Boer and Beumer, 1999; Olsen, 2000), we opted for an enrichment step prior to PCR to avoid the possibility of false negative result. In order to further increase the validity and reproducibility of the experimental inoculation studies, a total of five trials were conducted as shown in Table 3.
| Fig. 4: | Minimum detection levels of Aeromonas cells by using
primer fsor. Aerolysin gene, Lane M: 100 bp DNA ladder plus, Lane 1: 10
cells mL-1, Lane 2: 102 cells mL-1, Lane
3: 103 cells mL-1, Lane 4: 104 cells mL-1,
Lane 5: 105 cells mL-1, Lane 6: 106 cells
mL-1, Lane 7: 107 cells mL-1, Lane 8: 108
cells mL-1 |
| Fig. 5: | PCR result for screening of natural samples against 16S rRNA
gene. Lane M: 100 bp DNA ladder plus, Lane 1: Meat isolate, Lane 2: Meat
isolate, Lane 3: Fish isolate, Lane 4: Fish isolate |
| Table 3: | Results of detection of Aeromonas from spiked meat
samples |
| Numerator-No. of positive. Denominator-No. of trials | |
The results of detection of Aeromonas from spiked samples by cultural method revealed that 103 cells g-1 (4 out of 5) were positive from the post enrichment period of 12 and 18 h, whereas only some samples showed positive results from the samples spiked with the lower concentration (<102cells g-1) at both 12 and 18h enrichment period. On further incubation at 24 h, the detection limit of the assay was increased and it was able to detect samples (3 out of 5) with initial concentration of upto 10 cell g-1 of meat, whereas only 2 out of 5 samples spiked with 1 cell g-1 of Aeromonas showed positive results on cultural examination. The results of detection of Aeromonas from the spiked samples by PCR indicated the superiority of PCR assay using primer for 16S rRNA over PCR assay targeting aerolysin gene. A good correlation was observed between results, PCR assay with 16S rRNA and cultural method.
From the kinetics study in milk samples it was observed that at 12 and 18 h enrichment the detection limits were 103 and 104 cells mL-1 for 16S rRNA and aerolysin gene, respectively. Enrichment for up to 24 h increased the detection limits of the 16S rRNA PCR assay, where it was able to detect 3 samples out of 5 which were spiked with as less as 10 cells mL-1, whereas the detection limits remained same incase of aerolysin gene targeted PCR. Inconsistent results from the samples spiked with lower concentrations of Aeromonas sp. by both PCR and cultural method, may be attributed to the over growth of other competing micro flora (Lee et al., 1990). Similar detection limits from spiked food samples had been previously reported (Khan and Cerniglia, 1997; Gonzalez-Rodriguez et al., 2002).
Based on the results obtained from experimental inoculation/spiked studies, natural samples were screened for the Aeromonas sp. 50 chicken and 50 fish samples were screened for the presence of Aeromonas. Two samples from 50 meat and three samples from 50 fish samples turned out to be positive by cultural and PCR method targeting 16S rRNA (Fig. 5). This study of detection of Aeromonas from natural samples by cultural and PCR method has substantiated the results obtained from the spiking studies. A good correlation was observed between the cultural method and 16S rRNA targeted PCR assay. Thus from the study it is concluded that polymerase chain reaction targeting 16S rRNA can be used for rapid detection of Aeromonas from meat (chicken) and fish samples after single step enrichment in APW-C for 24 h.
Acknowledgements
We express our gratitude to the Director, Indian Veterinary Research Institute for providing the necessary facilities. This research as carried out under the project Quality assurance and monitoring of foods from livestock and poultry supported by National Agricultural Technology Project, Indian Council of Agricultural Research and New Delhi, India.