Prevalence and Loads of Fecal Pollution Indicators and the Antibiotic Resistance Phenotypes of Escherichia coli in Raw Minced Beef in Lebanon

Meat is an important source of high biological value proteins as well as many vitamins and minerals. In Lebanon, beef meats, including raw minced beef, are among the most consumed of the meat products. However, minced beef meat can also be an important source of foodborne illnesses. This is of a major concern, because food safety in Lebanon suffers from well-documented challenges. Consequently, the prevalence and loads of fecal coliforms and Escherichia coli were quantified to assess the microbiological acceptability of minced beef meat in Lebanon. Additionally, antibiotic resistance phenotypes of the E. coli were determined in response to concerns about the emergence of resistance in food matrices in Lebanon. A total of 50 meat samples and 120 E. coli isolates were analyzed. Results showed that 98% and 76% of meat samples harbored fecal coliforms and E. coli above the microbial acceptance level, respectively. All E. coli were resistant to at least one antibiotic, while 35% of the isolates were multidrug-resistant (MDR). The results suggest that Lebanon needs to (1) update food safety systems to track and reduce the levels of potential contamination in important foods and (2) implement programs to control the proliferation of antimicrobial resistance in food systems.


Introduction
Meat is an integral component of a balanced diet, providing proteins, essential amino acids and various micronutrients such as vitamin B12, niacin, vitamin B6, iron, zinc and phosphorous [1]. Consequently, meat production has increased rapidly over the past 50 years worldwide [2]. Cattle meat production has more than doubled since the 1960s, increasing from 28 million tons in 1961 to 68 million tons in 2014 [2]. In Lebanon, beef meat is among the most consumed meats [3]. The average consumption of beef meat was 39.63 kg/capita in Lebanon [2] and the mean intake was 47.6 g/day in surveyed consumers in Beirut, the capital of Lebanon [3]. Lebanon produced 47,484 tons of beef meat in 2014 [2]; however, beef meat in the Lebanese market is mainly imported from Brazil and India [4]. Beef meat constitutes a popular and economically important food in Lebanon. Notably, beef meat can also be consumed raw as part of famous Lebanese dishes. However, Lebanon has been witnessing challenges in food safety that have received media attention and raised national public health concerns. This was not surprising, because food safety can severely impact the health of a population [5], especially in developing countries with weakened infrastructure and limited resources [6]. Foodborne illnesses have been estimated to affect almost 1 in 10 people (~600 million individuals), while 420,000 die every year [5]. Notably, children less than 5 years old constitute a third of the deaths associated

Sampling of Minced Beef
During the spring and summer of 2018, a total of 50 samples of raw minced beef were collected from 50 different butcheries and grocery stores in Beirut, Lebanon. The samples were immediately placed on ice, transported to the laboratory, and processed within 2-3 h of collection.

Enumeration of Fecal Coliforms and E. coli
For each sample, minced beef meat was weighed, and 25 g were aseptically placed in a sterile stomacher bag (Fisher Scientific, New Hampshire, USA). The sample was then diluted 1:10 with 225 mL of sterile buffered peptone water (BPW) (Oxoid, Hampshire, UK) and homogenized for 1 min using a stomacher (Thomas Scientific, New Jersey, USA). The suspension was then serially diluted (10-fold) in BPW and 3 dilutions (10 −1 , 10 −2 , 10 −4 ) were plated on RAPID'E. coli 2 agar plates (BioRad, Hercules, California, USA) in duplicates. The plates were incubated at 44 • C for 18-24 h under aerobic conditions. Colony forming units (CFU) that matched the diagnostic phenotypes (violet to pink E. coli colonies and blue to green colonies for other fecal coliforms) were counted and bacteria densities were determined by averaging the counts from the duplicates. Data were reported as averages of CFU per gram of raw minced beef. BPW without meat was used as control throughout the experiment. E. coli DH5α was used to test the quality of the RAPID'E. coli 2 agar plates. Average CFU counts of E. coli and fecal coliforms were compared using the Student's t-test. A p < 0.05 was used to identify statistically significant differences.

Prevalence and Loads of Fecal Coliforms and E. coli
We assessed the acceptability of the minced beef samples in Lebanon by quantifying the prevalence and loads of fecal indicators, namely fecal coliforms and E. coli. Fecal coliforms were present in 49 of 50 (98%) raw minced beef samples, while E. coli was detected in 38 samples (76%). Fecal coliform CFU counts in positive samples ranged between 6.3 × 10 4 CFU/g and 1.62 × 10 7 CFU/g, while E. coli CFU counts ranged between 4.5 × 10 3 CFU/g and 3.48 × 10 6 CFU/g ( Figure 1a). The visual distribution of the CFU counts of fecal coliforms in comparison to those of E. coli is presented in Figure 1b, which also shows the median, quartiles (25% and 75%), minimum and maximum values, and outliers.
According to LIBNOR standards, the acceptable limit of fecal coliforms in minced beef is 100 CFU/g [21], which is similar to other countries such as New Zealand that has a limit ranging from 100 to 1000 CFU/g [32], while the USA uses 1000 CFU/g of total coliforms as a critical limit for ground beef [32]. Therefore, using the LIBNOR standards and those from other countries, 98% of the tested minced beef samples in Lebanon exceeded the microbiological criterion based on fecal coliform counts ( Figure 1a). The LIBNOR standard [21] for minced raw meat does not include limits on E. coli counts. However, there are many countries and researchers who established an E. coli limit to determine the acceptability of meat. For example, the USA uses 500 E. coli CFU/g as a critical limit for boneless beef [33]. Other studies showed that some states have more strict limits. For example, Oregon State (USA) has set a maximum of 50 CFU/g of E. coli [34], while New York State (USA) adopted 10 CFU/g of E. coli for assessing the acceptability of minced meat products [34]. Furthermore, the European Union (EU) Commission Regulation (EC) No. 2073/2005 set the limit of E. coli at 50 CFU/g [35]. When considering the aforementioned limits, it can be concluded that 76% of the tested minced beef samples in Lebanon exceeded the microbiological criterion based on E. coli counts.
Foods 2020, 9, x FOR PEER REVIEW 4 of 14 linkage method [31]. A graphical presentation of the matrix (heat map) was produced, where the upper limit (1) was colored green, while midpoint (0) and lowest limit (−1) were colored black and red, respectively [31].

Prevalence and Loads of Fecal Coliforms and E. coli
We assessed the acceptability of the minced beef samples in Lebanon by quantifying the prevalence and loads of fecal indicators, namely fecal coliforms and E. coli. Fecal coliforms were present in 49 of 50 (98%) raw minced beef samples, while E. coli was detected in 38 samples (76%). Fecal coliform CFU counts in positive samples ranged between 6.3 × 10 4 CFU/g and 1.62 × 10 7 CFU/g, while E. coli CFU counts ranged between 4.5 × 10 3 CFU/g and 3.48 × 10 6 CFU/g (Figure 1a). The visual distribution of the CFU counts of fecal coliforms in comparison to those of E. coli is presented in Figure 1b, which also shows the median, quartiles (25% and 75%), minimum and maximum values, and outliers.
According to LIBNOR standards, the acceptable limit of fecal coliforms in minced beef is 100 CFU/g [21], which is similar to other countries such as New Zealand that has a limit ranging from 100 to 1000 CFU/g [32], while the USA uses 1000 CFU/g of total coliforms as a critical limit for ground beef [32]. Therefore, using the LIBNOR standards and those from other countries, 98% of the tested minced beef samples in Lebanon exceeded the microbiological criterion based on fecal coliform counts ( Figure 1a). The LIBNOR standard [21] for minced raw meat does not include limits on E. coli counts. However, there are many countries and researchers who established an E. coli limit to determine the acceptability of meat. For example, the USA uses 500 E. coli CFU/g as a critical limit for boneless beef [33]. Other studies showed that some states have more strict limits. For example, Oregon State (USA) has set a maximum of 50 CFU/g of E. coli [34], while New York State (USA) adopted 10 CFU/g of E. coli for assessing the acceptability of minced meat products [34]. Furthermore, the European Union (EU) Commission Regulation (EC) No. 2073/2005 set the limit of E. coli at 50 CFU/g [35]. When considering the aforementioned limits, it can be concluded that 76% of the tested minced beef samples in Lebanon exceeded the microbiological criterion based on E. coli counts.
(a) Taken together, the wide prevalence and high loads of fecal indicator bacteria on minced beef samples in Lebanon are concerning, perhaps suggesting the need for better hygienic practices in the minced beef production chain. Admittedly, assessing the hygienic practices and possible sources of contamination of raw minced beef in a sustainable manner can be challenging. Specifically, monitoring must encompass (1) different food iterations that may contain raw beef in Lebanon (a notable example is minced beef with mixed herbs, Middle Eastern spices and onions that are consumed raw or cooked), (2) different types of indicator organisms and pathogens that might be present in each product, (3) differences in the handling and/ or processing procedures in foodservice establishments, butcher shops, and meat processing plants, and (4) a variety of sampling and testing procedures that require specialized laboratories, equipment and trained personnel [36,37]; all of which are difficult to secure in a country that is experiencing severe political and economic crises. However, despite the challenges, the safety of meat as well as other essential foods is critical to maintain public health and should be prioritized by the Lebanese government.
Both fecal coliforms and E. coli are effective indicators of fecal pollution. The latter suggests the presence of a variety of pathogens that can negatively impact human health [23,38]. In this study, 98% and 76% of the meat samples were rejected based on counts of fecal coliforms and E. coli, respectively, showing a difference in the rate of rejection based on the tested indicator. Subsequently, Lebanon would benefit from studies that screen for the best indicators of meat acceptability. This will reduce monitoring costs and will perhaps better reflect the acceptability of the meat. LIBNOR, along with other specialized stakeholders, should work on modifying and updating the current safety guidelines to enable proper assessment of the microbial quality of raw meat products in Lebanon.  Table 1). All isolates were sensitive to AMC, CFM, IPM, and MEM. Furthermore, 35% of the E. coli were classified as multi-drug Taken together, the wide prevalence and high loads of fecal indicator bacteria on minced beef samples in Lebanon are concerning, perhaps suggesting the need for better hygienic practices in the minced beef production chain. Admittedly, assessing the hygienic practices and possible sources of contamination of raw minced beef in a sustainable manner can be challenging. Specifically, monitoring must encompass (1) different food iterations that may contain raw beef in Lebanon (a notable example is minced beef with mixed herbs, Middle Eastern spices and onions that are consumed raw or cooked), (2) different types of indicator organisms and pathogens that might be present in each product, (3) differences in the handling and/ or processing procedures in foodservice establishments, butcher shops, and meat processing plants, and (4) a variety of sampling and testing procedures that require specialized laboratories, equipment and trained personnel [36,37]; all of which are difficult to secure in a country that is experiencing severe political and economic crises. However, despite the challenges, the safety of meat as well as other essential foods is critical to maintain public health and should be prioritized by the Lebanese government.

AMR Profiles of E. coli Isolated from Minced Beef Meat
Both fecal coliforms and E. coli are effective indicators of fecal pollution. The latter suggests the presence of a variety of pathogens that can negatively impact human health [23,38]. In this study, 98% and 76% of the meat samples were rejected based on counts of fecal coliforms and E. coli, respectively, showing a difference in the rate of rejection based on the tested indicator. Subsequently, Lebanon would benefit from studies that screen for the best indicators of meat acceptability. This will reduce monitoring costs and will perhaps better reflect the acceptability of the meat. LIBNOR, along with other specialized stakeholders, should work on modifying and updating the current safety guidelines to enable proper assessment of the microbial quality of raw meat products in Lebanon.  Table 1). All isolates were sensitive Foods 2020, 9,1543 6 of 13 to AMC, CFM, IPM, and MEM. Furthermore, 35% of the E. coli were classified as multi-drug resistant (resistance to ≥three classes of antibiotics) [39] (Table 1). Notably, three and 12 isolates exhibited resistance to six and five classes of antibiotics, respectively (Table 1). Hierarchal clustering organized the AMR profiles into four distinct clusters (Figure 2). Resistance to CIP and NOR was notable in the AMR profiles of isolates in Cluster 1 (C1). Similarly, resistance to STR-TET and resistance to LEX were notable in C2 and C4, respectively (Figure 2). The majority of multi-drug resistant (MDR) E. coli were grouped in C1 and C2, while C3 and C4 were mainly comprised of E. coli that were resistant to one or two antibiotics (Figure 2).

AMR Profiles of E. coli Isolated from Minced Beef Meat
In comparison, 55% of E. coli isolated from raw beef preparations in Northwest Spain were resistant to TET [40]. Additionally, 6.67% of the E. coli isolated from beef muscles in Ghana were resistant to AMC, CHL, and GEN, respectively [41]. In the same study, 40% and 0% of the isolates were resistant to TET and CIP, respectively [41]. In Egypt, 25% of the E. coli isolated from raw samples were resistant to TET, STR, and AMP, respectively [42]. This suggested that the resistance in Lebanon was comparable to those in other countries. However, it should be noted that beef growing operations are limited in Lebanon in comparison to the aforementioned countries. Furthermore, the size of Lebanon and its human population are much smaller in comparison to these countries. Taken together, this suggested that (1) there is an overreliance on the use of antibiotics in Lebanese farming [17,43,44] and/or (2) the resistance percentages are masked by the beef imported from other countries. Regardless, given that E. coli is an indicator for monitoring antibiotic resistance in foods, the AMR detected in Lebanese raw minced beef samples is a cause of concern. Therefore, the prevalence of multi-drug resistant E. coli isolates in the raw beef meat samples in Lebanon should be monitored periodically to assess the dissemination of these bacteria and the potential proliferation of transmissible genetic determinants of resistance [43][44][45]. Given the potential negative impacts on consumers, environment, and farmed animals, decreasing the incidences of multi-drug resistance in cattle and particularly in beef is a necessity.
Many studies have reported a high number of MDR pathogens associated with foods, food-animals and food-environment, including E. coli, Campylobacter spp., and Salmonella spp. [42][43][44][45][46][47]. The emergence and proliferation of resistance have been linked to the improper use of antibiotics in medical and agricultural practices [16,17,48,49]. In the latter, AMR bacteria and genes encoding resistance can be transmitted to humans via (1) the food chain, (2) direct farm exposure, and/or (3) environmental contamination with farm waste and products [48][49][50]. As a result, recalcitrant AMR infections associated with food and/or agriculture have become a global risk [11,[16][17][18][19]. This is predicted to be more severe in developing countries that have challenges in infrastructure and antimicrobial stewardship [17][18][19]. Lebanon is one of these countries that have also been affected by wide-spread pollution. Therefore, the emergence and dissemination of resistance in Lebanon should receive national and global attention, because AMR bacteria and genes can spread locally and beyond national borders, affecting other countries [43,51].

Conclusions
This study revealed a high occurrence of fecal coliforms and E. coli in raw minced beef in the Lebanese market. The high loads of these indicator bacteria are worrying, especially because beef meat is a major food item in the Lebanese cuisine and is consumed raw or minimally cooked in many instances. The occurrence of multi-drug resistant E. coli in the meat highlights the importance of adopting and implementing policies for reducing the use of antibiotics in food animal production in Lebanon and countries of importation. Furthermore, it might be beneficial to investigate and monitor the genetic bases of AMR in E. coli isolated from minced beef in order to assess the possibility of the persistence and dissemination of resistance to other vital matrices in Lebanon. Here, we call for updating national food safety systems in Lebanon and for investing in infrastructure and expertise that allow sustainable monitoring of food safety and the spread of AMR. This will greatly benefit public health and the economy in Lebanon.