ERIC-PCR
Several studies have used ERIC-PCR and other PCR-based typing methods to differentiate between clonally related and unrelated bacterial strains to investigate epidemic clones circulated in the population and responsible for outbreaks (Struelens 1998). However, the main objective of this study was to investigate the genetic relationship between E. coli strains isolated from imported frozen beef and shrimp and humans suffering from gastroenteritis in the Eastern Province of Saudi Arabia using ERIC1R, ERIC2, and a combination of ERIC1R+ERIC2 to generate genetic fingerprints from extracted genomic DNA. The study was designed to assess the discriminatory power of ERIC primers and to find out the genetic linkage between E. coli strains isolated from humans and strains isolated from frozen beef imported from India and frozen shrimp imported from China and Vietnam, respectively. Several studies have shown bacterial pathogens from these sources have been one of the major public health and societal concerns due to changes in meat animal and fisheries production and the overuse, misuse, and sub-therapeutic application of antimicrobials in humans and animals (Elbashir et al., 2018). Therefore, recent outbreaks of human gastroenteritis have been linked to consuming contaminated seafood and meat products. Globally, foodborne diseases are recognized as responsible causes of morbidity and mortality (Kuchenmüller et al. 2009; Stein et al. 2007). These diseases are transmitted to humans by contaminated foods and result in outbreaks and sporadic cases. In recent decades, pathogens of foodborne diseases are rapidly transported across international borders due to food trades and supply (Kirk et al. 2015). Therefore, several national governments and international organizations established food safety control systems to eradicate the burden of foodborne diseases. Moreover, in 2007, the World Health Organization (WHO) established out a Foodborne Disease Burden Epidemiology Reference Group (FERG) to assess and evaluate the global burden of foodborne diseases (Kirk et al. 2015). FERG highlights the huge foodborne infection estimates and the significance of food handling safety all throughout the food chain, especially in Africa, Southeast Asia, and other greatly affected regions.
ERIC-PCR genotyping
Three ERIC primers were evaluated to investigate the genetic relationship among 38 strains of E. coli isolated from different sources and geographic origins, as shown in Table 1. The dendrogram of the obtained ERIC-PCR fingerprint pattern results was constructed using GelJ software version 2.0 to determine the strain’s genetic relatedness. Among the 38 strains of E. coli analyzed by ERIC1R primer were generated fingerprint bands ranging in size from 250 to 1400 bp, and each profile consisted of three to ten bands (Fig.1-A). The DNA band patterns produced by ERIC1R primer were analyzed using the Dice coefficient. Numerical analysis of ERIC profiles revealed eight identical clusters (A-1 to A-8), and 26 genotypes (A1-26A) were obtained, as presented in Table 1 and Fig. 2. Of the 38 strains, 20 (52.6%) strains were grouped within these eight clusters, and the highest number of strains were seen in clusters A-5, A-6, and A-7. Other clusters from A-1 to A-4 and A-8 were composed of two strains each (Fig. 2). Among the eight clusters, cluster A-2 was composed of two identical strains (ECSC30 and ECSS31) isolated from frozen shrimp imported from China and a human strain isolated from humans suffering from gastroenteritis in the Eastern Province of Saudi Arabia.
On the other hand, 14 (36.8%) strains that showed a genetic similarity value greater than the 90% cut-off were considered genetically related (Fig. 2). Among these strains, the ECSC21 isolated from frozen shrimp imported from China and ECSS33 isolated from the human origin in Saudi Arabia shared 97% genetic similarity. Moreover, the ECSS32 strain of human origin shared 95% genetic similarity with two strains in cluster A-4 isolated from frozen beef imported from India (Fig. 2). Additionally, the ECSS34 strain of human origin showed 92% genetic similarity to two strains in cluster A-2. It is noteworthy that the ECSS36 of human origin shared 93% genetic similarity with the ECSI37 strain isolated from frozen beef imported from India (Fig. 2). Among the ERIC type patterns generated by ERIC1R primer, four strains (ESC14, ECSS35, ECSV3, and ECSV29) were found to be diverse, with single lineages and similarities below 90% and these strains were considered genetically unrelated as indicated by the dice coefficient (Fig. 2). Szczuka and Kaznowski (2004) indicated that strains with a similarity rate below 90% were considered genetically unrelated.
The ERIC2 primer produced fingerprint bands ranging in size from 150 to 1200 bp, and each fingerprint profile consisted of five to ten bands (Fig. 1-B). Numerical analysis of ERIC profiles revealed that seven distinct clusters (B-1 to B-7) and 24 genotypes (B1 to B24) were resolved, as shown in Table 1 and Fig. 3. Among the 38 strains analyzed using ERIC2 primer, 21 (55.3%) grouped within these seven clusters were identical and shared a 100% genetic similarity (Fig. 3). Moreover, 11 (28.9%) out of 38 strains showed a genetic similarity greater than the 90% cut-off value, and six strains were revealed as single lineages below 90% genetic similarity and considered genetically unrelated (Fig. 3). These strains (ECSC14, ECSV4, ECBI37, ECSS36, ECSS34, and ECSC18) exhibited genetic similarity below 90% and were considered genetically unrelated (Fig. 3). The highest number of strains were grouped in clusters B-3, B-4, and B-5, while other clusters were composed of between two to three strains in each cluster (Fig. 3). Interestingly, cluster B-3 is composed of four identical strains and two of these strains (ECSV22 and ECSC30) were isolated from frozen shrimp imported from Vietnam and China, and other strains ECBI42 and ECSS35 were isolated from beef imported from India and humans suffering from gastroenteritis in Eastern Province of Saudi Arabia (Fig. 3).
Concerning the combination of ERIC1R+ERIC2 primer, two to five bands were obtained for each fingerprint and sizes ranging between 200 to 1200 bp (Fig. 1-C). Among the 38 strains, six strains were not typeable by ERIC1R+ERIC2 primer (Table 1). The ERIC1R+ERIC2 primer revealed 16 genotypes (C1 to C16) and six clusters (C-1 to C-6) among the 32 genotyped strains, as shown in Figure 4. Of the 32 strains, 22 (57.9%) were grouped within these six clusters, and the highest number of strains were composed in cluster C-3 (Fig. 4). Eighteen strains of E. coli isolated from frozen shrimp imported from China and Vietnam were grouped in identical clusters (C-3, C-4, C-5, and C-6) and showed 100% genetic similarities. Moreover, all strains isolated from frozen beef imported from India were grouped in separated clusters (C-1 and C-2), and except for strain, ECSI38 exhibited a single lineage cluster below 90% genetic similarity (Fig. 4). Among the five strains of human origin genotyped by ERIC1R+ERIC2 primer, only one strain, ECSS31, shows 93% genetic similarity to strain ECSC30 isolated from frozen shrimp imported from China. The other four strains of human origin exhibited single lineages and revealed genetic similarity below 90% and were considered genetically unrelated (Fig. 4).
Evaluation of ERIC-PCR as a typing tool
The calculated discrimination indexes obtained by ERIC-1R, ERIC-2, and the ERIC-1R+ERIC-2 combination were 0.976, 0.965, and 0.903, respectively (Table 2). The Discrimination Index obtained by all evaluated ERIC primers was considered acceptable and above the confidence value (Szczuka and Kaznowski 2004). Among the three evaluated ERIC primers, the best discriminatory ability for epidemiological concordance was obtained by ERIC-1R (DI = 0.976) and ERIC-2 (DI = 0.965), and both primers revealed a 100% typeability value (Table 2). Among the three investigated ERIC primers, the least typeability of 84% was obtained by the ERIC1R+ERIC2 primer pair (Table 2). Our results suggest that the ERIC1R and ERIC2 primer are effective and suitable in differentiating genetic relatedness of E. coli isolated from different sources, and ERIC-PCR should be adopted as a molecular typing technique for tracking monitoring transmission during pathogenic E. coli outbreaks. In this study, the ERIC1R and ERIC2 primers had the best discriminatory ability and typeability value and proved to be suitable for epidemiological investigation and genetic analysis among the population of E. coli strains. A similar study conducted elsewhere evaluated ERIC-PCR primers as PCR-based tools to discriminate against different bacterial strains and concluded that the ERIC2 primer set had shown a more reliable ability to discriminate against the analyzed strains (Zara et al. 2006). In this study, the ERIC1R+ERIC2 primer combination had a discriminatory power value of D = 0.903 for differentiating E. coli strains (Table 2). However, several published articles used a combination of ERIC1R+ERIC2 primer as recommended in previously published studies elsewhere (Versalovic et al. 1991; Woods et al. 1993). Therefore, our obtained results in this study suggesting the use of ERIC1R and ERIC2 primer without combination will generate a more discriminative value index than the combination of the two primers. Furthermore, ERIC primers should be optimized to identify which primer will give an excellent typeability and discriminatory power, as were claimed in some published articles using the ERIC primer without specifying the exact primer used in ERIC-PCR protocol.
Construction of phylogenetic trees based on ERIC-PCR fingerprint patterns generated by ERIC1R, ERIC-2, and ERIC1R+ERIC2 primers revealed a total of eight, seven, and six identical unique clusters with 100% similarity index (Fig. 2, 3, and 4). However, the ECSS31 strain was isolated from humans and was found to be identical to the ECSC30 strain isolated from frozen shrimp imported from China, as shown in Cluster A-2 (Fig. 2). Similarly, a phylogenetic tree based on ERIC-PCR fingerprint patterns generated by ERIC-2 primer revealed three strains (ECSV22, ECSC30, and ECBI42) isolated from frozen shrimp and beef imported from Vietnam, China, and India, respectively, showed identical relatedness to human strain (ECSS35), as shown in Cluster B-3 (Fig. 3). A phylogenetic tree generated by a combination of ERIC1R+ERIC2 primer revealed the ECSS31 strain isolated from humans shared 92% genetic similarity with the ECSC30 strain isolated from frozen shrimp imported from China (Fig. 4). However, identical genetic linkage of these strains isolated from imported frozen beef and shrimp to human strains (ECSS31 and ECSS35) associated with gastroenteritis in the Eastern Province of Saudi Arabia indicates contamination of imported frozen beef and shrimp as a possible vehicle source of infection. Our study also revealed a strong genetic relationship between E. coli strains isolated from frozen shrimp imported from China formed identical group clones (Clusters: C-3, C-5, and C-6) with strains isolated from frozen shrimp imported from Vietnam (Fig. 4). Furthermore, we identified E. coli strain ECBI38 isolated from frozen beef imported from India had identical genetic similarity (Cluster: B-6) with strains isolated from frozen shrimp imported from China and Vietnam (Fig. 3). Therefore, genetic identical similarity patterns of these clones of E. coli strains proved that these strains are circulated and exist in these areas and might be spread through imported frozen beef and shrimp, lacking adequate hygiene during handling, storage, transportation, and commercialization the point of sale. A similar study conducted elsewhere demonstrated that contaminated frozen seafood and shrimp might play an important role in disseminating virulent and multi-drug resistant enterobacteria strains. Also, the study found that E. coli isolates harbored major virulence genes and their contamination levels are beyond the recommended limits (Barbosa et al. 2016; Dib et al. 2018).
Our study also revealed that four out of six E. coli strains recovered from humans suffering from gastroenteritis in the Eastern Province of Saudi Arabia were different clones, and only two strains (ECSS31 and ECSS35) were grouped in identical clone clusters with strains isolated from frozen shrimp imported from China as indicated in cluster: A-2 (Fig. 2). Also, the ECSS35 strain formed an identical clone with E. coli strains isolated from frozen beef and shrimp imported from India, China, and Vietnam, as indicated in cluster: B-3 (Fig. 3). E. coli comprise a diverse group of bacteria with pathogenic variants, pathovars responsible for causing morbidity and mortality. These pathogen have low infectious doses and are transmitted through food and water, causing major public health problems (Croxen et al. 2013). Food or water might be contaminated with the feces of infected humans and animals, and this contamination plays a significant role in the transmission of E. coli. Contamination of meat and seafood products often occurs during the processing and packing (García et al. 2010). In addition, the use of manures from animals as fertilizer can contaminate food and irrigation water (Croxen et al. 2013; García et al. 2010). Another area of interest we found in our study was the determination of genetic similarity among strains isolated from frozen shrimp imported from China and Vietnam; these were grouped in identical clone clusters. However, the present study revealed the coexistence of this circulated identical clone of E. coli in these geographical areas.
Over the last two decades, several authors have used ERIC-PCR as a rapid molecular typing method for determining genetic relationships among several bacterial species isolated from food, environmental, and human samples. In this study, ERIC-PCR demonstrated excellent discrimination among unrelated E. coli strains, and this method is a useful DNA fingerprinting technique for distinguishing E. coli isolated from different sources (Ateba and Mbewe 2014; Dorneles et al. 2014; Fendri et al. 2013; Gautam et al. 2022; Igwaran and Okoh 2020; Pakbin et al. 2021; de Sa Guimaraes et al. 2011; Szczuka and Kaznowski 2004). Our results indicate that imported frozen beef and shrimp products might become an important source of enteric pathogenic E. coli in Saudi Arabia. The use of ERIC-PCR typing provides information on the genetic relatedness of E. coli strains isolated from humans to E. coli strains isolated from imported frozen beef and shrimp in the Eastern Province of Saudi Arabia. However, our findings indicate that the use of ERIC1R, ERIC2, and a combination of ERIC1R and ERIC2 is effective in finding genetic relatedness among strains of E. coli isolated from human and retail imported frozen shrimp and beef sources and facilitating a better understanding of the transmission route and tracking the origin of pathogenic E. coli in Saudi Arabia.
The identical overlap of two strains (ECSS31 and ECSS35) isolated from humans suffering from gastroenteritis and strains of E. coli strains isolated from frozen beef and shrimp imported from India, China, and Vietnam highlights the importance of these sources for human enteric pathogenic E. coli in Saudi Arabia. The ERIC-PCR results demonstrated a clear source of contamination that may occur in the harvest area of shrimp and handling or storage of meat and shrimp. However, there is an urgent need for monitoring programs, and rigorous molecular studies are required to track the exact source of contaminants of enteric pathogenic E. coli and other pathogens in imported frozen beef, shrimps, and other frozen seafood and meat products. Consequently, accurate control measures by food authorities must be implemented to warranty a safe quality of imported frozen seafood and meat products for adequate consumer protection.