1. Background
Listeria monocytogenes, the most important species of Listeria, is commonly present in food, water, feed, soil, and sewage. This food-borne pathogen causes acute complications such as septicemia, meningitis, and encephalitis in humans, especially infants, pregnant women, elderly people, and immune-compromised individuals (1-4). Listeria can grow at low temperatures, with an optimum pH requirement of 5.4 - 9.6 (5, 6).
The antimicrobial resistance of pathogenic microorganisms is a worldwide public health concern because of increasing global trade and travel (7). However, L. monocytogenes is naturally susceptible to various antibiotics targeting Gram-positive bacteria (8), and it has been indicated that clinical strains of L. monocytogenes are sensitive to a wide range of antibiotics. Typically, invasive infections have been treated with a combination of ampicillin or amoxicillin and gentamicin (9). Considering the increasing number of new antibiotic resistant strains of L. monocytogenes reported worldwide, it seems that this bacterium likely obtains various antibiotic resistance genes by horizontal gene transfer, some of which may come from the commensal microorganisms found in food (10).
According to the high mortality rate of L. monocytogenes (about 30%), the presence of this bacterium in food is considered to be a major health problem (11). Moreover, a wide range of food types have been implicated in its transmission, including meat, dairy, fish, and vegetable products. The occurrence of Listeria spp. (including L. monocytogenes) in meat and raw meat products has been investigated in several countries (12-15). To the best of our knowledge, few studies regarding the prevalence and antimicrobial susceptibility of Listeria in foodstuffs in Iran have been documented (3-5, 16). Therefore, the present study was conducted to determine the occurrence and antimicrobial resistance of Listeria spp. isolated from Iranian beef burgers distributed in Ahvaz city.
2. Objectives
The present study was conducted to evaluate the occurrence and antibiotic resistance of Listeria spp. in Iranian beef burgers distributed in Ahvaz city.
3. Materials and Methods
3.1. Sample Collection
In this cross-sectional survey, during a five-month period (February - June of 2014), a total of 150 frozen beef burger samples, made by 10 Iranian production companies located in different provinces, were purchased from local markets in Ahvaz. The burger boxes were checked to be completely frozen and within the shelf-life periods. The samples were put in a cold box and transferred to the laboratory. After defrosting, they were microbiologically analyzed on the same day.
3.2. Microbial Culture
In the first step, the samples were analyzed for the detection of Listeria spp. using enrichment, selective, and isolation protocols, as recommended by the US food and drug administration (17). For each individual sample, 25 g was aseptically removed, blended in 225 mL of Listeria enrichment broth (Merck, Germany), and incubated at 35°C for 48 hours. The enrichment cultures were streaked onto Oxford agar (Merck, Germany) plates, incubated at 35°C for 48 hours, and examined for Listeria (black colonies with black halos). All of the suspected Listeria colonies were subjected to standard biochemical tests, including Gram staining, catalase, and motility testing at 25°C. For further confirmation, other biochemical reactions showing acid production from maltose, mannitol, rhamnose, and xylose, as well as β-hemolytic activity on 5% sheep’s blood agar (Merck, Germany) and MRVP testing were performed.
3.3. Polymerase Chain Reaction Procedures
In the next step, for the identification and confirmation of L. monocytogenes, the suspected colonies were tested via PCR assay. The template DNA was obtained by boiling from a pure culture of the suspected isolate, which was grown in Tripticase Soy Broth (TSB; Merck, Germany) at 30°C overnight. Briefly, the overnight culture was centrifuged, the pellet was re-suspended in 1 mL of deionized water (dH2O), and the sample was boiled at 100°C for 10 minutes. After heating, the obtained suspension was centrifuged at 14,000 rpm for 10 minutes, and then the supernatant was used as the PCR template. In addition, the DNA of standard L. monocytogenes (ATCC 7644) and distilled water were used as positive and negative controls, respectively. Each PCR tube contained 50 μL of the reaction mixture, consisting of the PCR buffer (10 X, 5 µL), MgCl2 (50 mM, 1.5 µL), Taq DNA polymerase (5 U/µL, 0.5 μL), dNTPs Mix (10 mM, 1 µL), primers (100 pmol/µL, 1 µL each), ddH2O (35 μL), and 5 μL (100 ng) of the template DNA. The primers used (Table 1) were from the P60-protein-coding gene (iap-P60), according to Kim et al. (18).
| Target Gene | Primer Sequence | Product Size, bp | Reference |
|---|---|---|---|
| iap | 454 | (18) | |
| Forward | 5'-CTGGCACAAAATTACTTACAACGA-3 ́ | ||
| Reverse | 5'-AACTACTGGAGCTGCTTGTTTTTC-3 ́ |
The Target Gene, Sequence of Primers, and Product Size (bp) Used for the Detection of L. monocytogenes
The cycling conditions used in the thermal cycler (Bioer, China) were as follows: first, denaturation at 94°C (3 minutes, 1 cycle), denaturation at 94°C for 45 seconds, annealing at 60°C for 45 seconds, and extension at 72°C for 1 minutes. After 35 cycles, a final cycle comprised of a 5 minutes extension step at 72°C was conducted. The amplified PCR products were detected by agarose gel electrophoresis (Paya Pajoohesh Pars, Iran), stained, and visualized under UV light illumination (Kiagen, Iran).
3.4. Antimicrobial Susceptibility Testing
According to the methods of the Clinical and Laboratory Standards Institute (CLSI) (19), antimicrobial susceptibility tests were carried out using the Kirby-Bauer disk diffusion method. The tested antimicrobial agents were ampicillin, amikacin, erythromycin, streptomycin, penicillin, tetracycline, gentamicin, chloramphenicol, co-trimoxazole, and vancomycin. A swab was taken from each bacterial suspension (1 × 107 CFU/mL) and stroked on Mueller-Hinton agar (Merck, Germany), and then antibiotic discs (Padtan Teb, Iran) were placed on the agar. After incubation at 35°C for 24 hours, the diameter of the inhibition zone was measured for each antibiotic based on the appropriate schedule. Then, the isolates were classified as resistant, intermediate (reduced susceptibility), or sensitive.
3.5. Statistical Analysis
The results were analyzed via the chi-square test and Fisher’s exact test using SPSS 16.0 software. The mean values were considered to be statistically different at 95% confidence levels.
4. Results
Out of 150 examined beef burger samples, on Oxford agar, black colonies were observed on 23 culture plates, with similarity to Listeria spp. colonies. Using biochemical tests, two samples (1.33%) were positive for L. innocua, and the rest were negative. Moreover, none of the suspected colonies were L. monocytogenes positive via the PCR assay (Table 2).
| Company | Sample Number | Number of Suspected Listeria | Listeria spp. Positive | Confirmed Species |
|---|---|---|---|---|
| 1 | 17 | 4 | 0 | - |
| 2 | 17 | 0 | 0 | - |
| 3 | 16 | 4 | 1 | L. innocua |
| 4 | 12 | 2 | 0 | - |
| 5 | 18 | 3 | 0 | - |
| 6 | 16 | 4 | 0 | - |
| 7 | 10 | 2 | 0 | - |
| 8 | 14 | 0 | 0 | - |
| 9 | 17 | 1 | 1 | L. innocua |
| 10 | 13 | 3 | 0 | - |
| Total | 150 | 23 | 2 | 2 |
Number of Samples; Suspected and Positive Listeria spp
The statistical analysis showed no significant difference in the prevalence of Listeria spp. between the different companies (P > 0.05).
The data revealed that one of the isolates was resistant to tetracycline, and the other was resistant to co-trimoxazole. Both of the isolates showed intermediate susceptibility to chloramphenicol; however, they were sensitive to the other tested antibiotics.
5. Discussion
Many incidences of listeriosis have resulted from the consumption of contaminated foodstuffs, such as dairy, meat, vegetables, and seafood (3, 4). The presence of L. monocytogenes in raw meat increases the risk of listeriosis in people who consume undercooked meat or meat by-products, such as burgers. Therefore, to ensure food safety, this pathogen should be absent in 25 g of foodstuff (20). Overall, 1.33% (2 of 150) of all of the beef burger samples in the present study were contaminated with Listeria spp., while L. monocytogenes was not detected. It is worth noting that L. innocua is not a pathogen, but since both species share ecological niches, the presence of the bacterium could be an indicator of probable contamination with L. monocytogenes (21). Thus, when looking for sources of L. monocytogenes, the presence of other species, especially L. innocua, could be managed as equally significant.
There have been several reports regarding the isolation of Listeria spp. and L. monocytogenes from raw meat and meat products worldwide. For example, in Bulgaria, 786 samples (containing 505 samples of fresh meat and 281 samples of raw-dry and raw-smoked sausage) were analyzed for Listeria spp. From the beef and pork samples, 7.7%, 0.6%, 4.6%, and 0.8% were contaminated with L. monocytogenes, L. ivanovii, L. innocua, and L. welshimeri, respectively (22). In another study in Malaysia, L. monocytogenes was detected in 8.57% of the meat product samples (13). According to Wieczorek et al. (15), it was determined that 44 out of 406 hide samples (10.8%) were contaminated with L. monocytogenes, whereas 10 (2.5%) corresponding bovine carcasses were positive for this pathogen in Poland.
There are limited data regarding the prevalence of Listeria spp. in beef products consumed in Iran. For instance, Jalali and Abedi (5) found that Listeria spp. were present in 6.7% of the meat and meat product samples, 1.3% of the dairy samples, 1.2% of the vegetable samples, and 12% of the ready-to-eat samples in Isfahan, Iran. Moreover, Rahimi et al. (4) reported that out of 1,107 different meat samples collected in Iran, 141 samples (12.7%) were positive for Listeria spp. The most commonly recovered species was L. innocua (75.9%), followed by L. monocytogenes (19.1%). In contrast, a low incidence of Listeria spp. (2.7%) and L. monocytogenes (0.66%) in minced beef in Ahvaz city has been reported (16).
Our findings are not in agreement with the high prevalence of Listeria spp. in meat products reported by previous Iranian researchers. This could be due to the season, geographic conditions, sanitation in the meat production chain supply, or methodological differences. The low number of Listeria isolates in this survey may also be due to the actual low incidence in the products, or the presence of live-injured bacterial cells (LIBC) which cannot grow properly on culture media. This is a big concern in public health with regard to the presence of LIBC, because they may be undetectable via regular culture methods, but are potentially pathogenic under favorable conditions (13).
It is known that meat and meat products may be infected at the slaughterhouse due to cross-contamination, which occurs during evisceration, slicing, mincing, and other processing stages. Meat and meat products are stored under refrigeration or freezing, and the absence of competitive microorganisms, along with the suitable water activity and pH values of the food, allow this psychrotolerant pathogen to grow at high levels (23). In preparing minced meat, the release of blood and meat juices during cutting, deboning, and grinding also favor the growth of Listeria, and may cause an increase in contamination during the processing of raw meat products (4, 23).
The results of the antimicrobial susceptibility tests have indicated intermediate susceptibility of the isolates to chloramphenicol. In addition, one of the isolates was resistant to tetracycline and another to co-trimoxazole, and both strains showed susceptibility to the other tested antibiotics. These results are in agreement with previous works (9, 24), which reported that L. innocua isolates from meat products were resistant to tetracycline and co-trimoxazole. According to previous studies, the L. innocua isolated from meat and foodstuffs, and their antibiotic resistance, are indicated in Table 3.
Antibiotic-resistant L. innocua Isolated From Meat Products in Previous Studies
With the increasing number of antibiotic resistant strains of L. monocytogenes reported worldwide, it seems that antibiotic resistance in this bacterium can be acquired or transferred by genes from plasmids and transposons of commensal microorganisms, such as other Listeria spp. and Gram-positive bacteria which may found in foods (10), or mutational events in chromosomal genes (8, 25, 26). The newly acquired resistance protects the bacteria from being disrupted during antibiotic treatment (11).
As previously mentioned, L. innocua and L. monocytogenes are closely related species, and thus genetically very similar. Although L. innocua is not a pathogenic species, it has been reported that the bacterium could be a transferable reservoir of antibiotic resistance for L. monocytogenes (27). Therefore, it could be suggested that the isolation of resistant L. innocua in foodstuffs could be a potential risk to public health.
Although L. monocytogenes strains with resistance to streptomycin, penicillin, and tetracycline have been isolated from food sources (11, 28, 29), resistance to those antibiotics commonly used to treat listeriosis, such as ampicillin, amoxicillin (with or without gentamicin), and trimethoprim-sulfamethoxazole (co-trimoxazole), has rarely been observed (9).
5.1. Conclusions
Our findings showed the presence of antibiotic resistant Listeria strains in Iranian beef burgers, indicating the possible presence of L. monocytogenes. Thus, it is possible for the pathogenic bacterium to obtain various antibiotic resistance genes by horizontal gene transfer in the meat production supply chain. Additionally, there is a potential risk to public health from the consumption of raw or undercooked burgers, which may increase the possibility of the acquisition of resistance to antibiotics.
