A Higher Prevalence Rate of Campylobacter in Retail Beef Livers Compared to Other Beef and Pork Meat Cuts

The objectives of this study were to determine the prevalence of Campylobacter jejuni and Campylobacter coli in retail beef, beef livers, and pork meats purchased from the Tulsa (OK, USA) area and to further characterize the isolates obtained through antimicrobial susceptibility testing. A total of 97 chilled retail beef (50 beef livers and 47 other cuts), and 100 pork samples were collected. The prevalence of Campylobacter in beef livers was 39/50 (78%), while no Campylobacter was isolated from the other beef cuts. The prevalence in pork samples was 2/100 (2%). A total of 108 Campylobacter isolates (102 beef livers isolates and six pork isolates) were subjected to antimicrobial resistance profiling against sixteen different antimicrobials that belong to eight different antibiotic classes. Of the six pork Campylobacter coli isolates, four showed resistance to all antimicrobials tested. Among the beef liver isolates, the highest antibiotic resistances were to tetracyclines and β-lactams, while the lowest resistances were to macrolides, aminoglycosides, lincosamides, and phenicols. Resistances to the fluoroquinolone, macrolide, aminoglycoside, tetracycline, β-lactam, lincosamide, and phenicol antibiotic classes were significantly higher in Campylobacter coli than Campylobacter jejuni isolates. Multidrug Resistance (MDR) among the 102 Campylobacter (33 Campylobacter jejuni and 69 Campylobacter coli) beef liver isolates was significantly higher in Campylobacter coli (62%) than Campylobacter jejuni (39%). The high prevalence of Campylobacter in retail beef livers and their antimicrobial resistance raise concern about the safety of these retail products.

than livers were from different cuts such as, steak, stew, shoulder, and bone in. Samples were selected to be variable as possible with different expiration dates and production codes. Meat samples were purchased from nine grocery stores that belong to six different chains at variable locations in the city. The collected beef samples belonged to nine different brands while the pork ones were from seven brands. The retail meat samples were transported to the laboratory in ice boxes and processed immediately upon arrival. Samples were rinsed with Buffered Peptone Water (BPW; EMD, Gibbstown, NJ, USA) in sterile plastic bags (VWR Scientific, Radnor, PA, USA) and massaged briefly by hand for five minutes. Next, 10 mL of the rinsate was added to 10 mL of 2 Bolton broth supplemented with blood and the appropriate antibiotic supplementation. The enrichment was incubated at 42 °C for 48 h and then plated onto Campylobacter Charcoal Desoxycholate Agar (CCDA; Remel, Lenexa, KS, USA) plates with the appropriate antibiotic supplementation. The plates were incubated at 42 °C for 48 h in gas jars containing microaerophilic gas-generating kits (Mitsubishi Gas Chemical, New York, NY, USA). Four to six suspect Campylobacter colonies of each sample from the CCDA plates were then transferred onto Mueller-Hinton (MH; Difco, Sparks, MD, USA) agar plates supplemented with blood and incubated at 42 °C for 48 h under microaerophilic conditions, then purified by sub culturing and kept at −80 °C freezer for preservation until subjected to molecular identification.

DNA Extraction
Bacterial DNA extracts used in polymerase chain reaction (PCR) were prepared from Campylobacter cultures using the single-cell lysing buffer (SCLB) method [19]. Isolates were removed from −80 °C storage, struck to Mueller-Hinton agar (MH; Difco), and incubated at 42 °C for 48 h under microaerophilic conditions. One colony was picked from the plate and suspended in 40 µL of SCLB solution in a 0.2 mL microtube. The SCLB solution consisted of 10 µL of 5 mg/mL proteinase K (Amresco, Solon, OH, USA) and 1.0 mL of TE buffer (10 mM Tris-HCl (J.T. Baker, Phillipsburg, NJ, USA) and 1 mM EDTA (Fisher, Fair Lawn, NJ, USA)). The cells were lysed by heating at 80 °C for 10 min, followed by cooling to 55 °C for 10 min, using a Mastercycler Gradient thermocycler (Eppendorf, Eppendorf, Germany). The suspension was diluted 1:2 in double distilled water and centrifuged in a Microfuge (Clover Laboratories, Waterville, OH, USA) at 4,500 ×g for 30 s to remove cellular debris. The supernatant was used as DNA template for PCR. All DNA extract samples were stored at −20 °C .

PCR Identification
All Campylobacter suspect isolates were tested for the identification of Campylobacter genes by multiplex PCR reaction using primers specific for Campylobacter jejuni and Campylobacter coli [20] ( Table 1). The PCR was carried out in 25 µL reactions. Each 25 µL reaction contained 12.5 µL Taq Polymerase Master Mix (Qiagen Inc., Valencia, CA, USA), 7.5 µL sterile water (Qiagen), 1 µL (25 pmol) each primer (IDT, Coralville, IA, USA), and 3 µL of template DNA. The cycling conditions were set as follows on the Mastercycler Gradient thermocycler, initial denaturing at 95 °C for 5 min followed by 30 cycles of denaturing at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 2 min, followed by final extension at 72 °C for 10 min (modified from Konkel, et al. [21]).
At the completion of the Polymerase Chain Reaction, reactions were held at 4 °C until gel electrophoresis. The expected amplicon sizes were 160 bp for C-1 gene, 400 bp for the cadF gene, and 894 bp for ceuE gene (Table 1). Campylobacter jejuni ATCC #33560, and Campylobacter coli strain #96121033 (Oklahoma Animal Disease Diagnostic Laboratory, OSU, Stillwater, OK, USA) were used as positive controls, and sterile water was used as a negative control.

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was performed using the agar dilution plate technique for a total of 108 Campylobacter isolates against sixteen antimicrobials belonging to eight different antibiotic classes (Table 2). Isolates were grown on Mueller-Hinton (MH) agar (Difco) supplemented with 5% laked horse blood (Hemostat Laboratoties, Dixon, CA, USA) and incubated for 48 h at 42 °C at microaerophilic conditions. Cultures were then added to Mueller-Hinton broth (Difco), adjusted to turbidity equal to a 0.5 McFarland standard, and inoculated onto 6-inch MH agar plates supplemented with 5% blood and antimicrobials at different concentrations (Table 2) including the breakpoint established for each antimicrobial according to the Clinical and Laboratory Standards Institute (CLSI) (ciprofloxacin, erythromycin, doxycycline, and tetracycline), National Antimicrobial Resistance Monitoring System (NARMS) (gentamicin, clindamycin, azithromycin, and nalidixic acid), and other published articles (ampicillin, streptomycin, and chloramphenicol [24], amoxicillin [25], cephalothin [26], kanamycin [27], tilmicosin [28], and oxytetracycline [29]) . Campylobacter jejuni ATCC #33560 was used as a quality control strain. The plates were incubated at 42 °C for 48 h under microaerophilic conditions. The plates were read for growth or no growth and denoted as resistant or susceptible, respectively according to the breakpoints for each of the sixteen tested antimicrobials ( Table 2). Multidrug Resistance (MDR) was defined as resistance to three or more antibiotic classes [30]. Kruskal-Wallis test was used to compare resistance of Campylobacter jejuni and Campylobacter coli to the 16 tested antimicrobials. Fisher's exact test was used to compare MDR between the two species.

Prevalence of Campylobacter in Beef and Pork
The overall prevalence of Campylobacter (Campylobacter jejuni and Campylobacter coli) in beef livers was 39/50 (78%), whereby 13/50 (26%) of the samples was contaminated with Campylobacter jejuni, 24/50 (48%) of the samples was contaminated with Campylobacter coli, and 2/50 (4%) of the samples was contaminated with both Campylobacter jejuni and Campylobacter coli. None of the 47 other beef cuts contained Campylobacter. The prevalence of Campylobacter (Campylobacter jejuni and Campylobacter coli) in pork was 2/100 (2%). The pork samples contained only Campylobacter coli (2/100) and no Campylobacter jejuni was found.
The low prevalence of Campylobacter in retail pork meat (2%) is not surprising and in relative agreement with previous studies. Zhao et al. [6] found that the rate of Campylobacter contamination in pork chops was 0.5%. Hong et al. [16] reported a Campylobacter prevalence of only 1.6% in pork meat where Wong et al. [15] found a relatively higher prevalence of 9.1%. Both of the pork samples contained Campylobacter coli. Most of Campylobacter contaminations of pork have generally been found to be Campylobacter coli [31][32][33].
The overall prevalence of Campylobacter in beef livers in our study was 39/50 (78%) while no Campylobacter was isolated from the other beef cuts. Despite their limited number, previous studies have found beef livers to be contaminated with Campylobacter. Strachan et al. [18] found that 69% (22/32) of their beef liver samples were contaminated with Campylobacter. Kramer et al. [32] found that 54.2% of ox livers they tested were contaminated with the bacteria. On the other hand, Enokimoto et al. [34] found a Campylobacter prevalence of only 5% in the beef livers they tested but their beef liver samples were picked up at a meat slaughter center in Japan right after the cattle slaughtering and was surface sterilized to exclude any bacteria on the surface. The high prevalence of Campylobacter in retail beef livers is alarming and might be due to cross contamination since the livers are recovered from several cows and possibly piled up together. Ghafir et al. [17] suggested that the high level of recovery of Campylobacter from livers is probably due to the fact that the liver surface stays moist, which might protect this foodborne pathogen. Fecal carriage of Campylobacter by the slaughtered cows is a possible source of contaminating beef livers in slaughter houses. Liver location makes them easily prone to bile contamination. The risk of this high prevalence of Campylobacter in beef liver could be magnified by cooking livers lightly to avoid overcooking undesired taste.
Out of the 39 beef liver samples that were positive in our study 13 (33%) were Campylobacter jejuni and 24 (62%) were Campylobacter coli and two samples were contaminated with both species. Kramer et al. [32] found in their study that 49% of their Campylobacter isolates from beef livers were Campylobacter jejuni and 2.1% were Campylobacter coli. Ghafir, et al. [17] found that in their beef samples, all of the isolates were Campylobacter jejuni. In a fecal samples study, Nielsen et al. [31] found 90.9% of the isolates from cattle were Campylobacter jejuni and 6.8% were Campylobacter coli. In contrast to those reports, our study showed higher numbers of Campylobacter coli than Campylobacter jejuni. Some of the differences in Campylobacter prevalence discussed above might be due to seasonal or geographic area variations.

Antimicrobial Resistance Profiling
A total of 108 Campylobacter isolates (102 beef liver isolates and six pork isolates) were subjected to antimicrobial resistance profiling against sixteen different antimicrobials that belong to eight different antibiotic classes (Tables 2 and 3). Table 3 shows the percentage of resistance of the 102 Campylobacter isolates (33 Campylobacter jejuni and 69 Campylobacter coli) isolated from beef livers to the sixteen tested antimicrobials that belong to eight antibiotic classes. The percentage of resistance to the sixteen tested antimicrobials varied between Campylobacter jejuni and Campylobacter coli isolates. Among the beef liver isolates, resistance to fluoroquinolones (ciprofloxacin) was significantly higher in Campylobacter coli (62%) than in Campylobacter jejuni (39%) ( Table 3). It is also shown in Table 3 that none of the tested Campylobacter jejuni isolates were resistant to all tested macrolides, while only 29% of Campylobacter coli were resistant. Twenty percent of the tested beef liver Campylobacter coli isolates was resistant to the three tested aminoglycosides while none of the Campylobacter jejuni isolates showed resistance (Table 3). Resistance to all tested tetracyclines was significantly higher in Campylobacter coli (97%) than in Campylobacter jejuni (73%). Resistance to phenicols, lincosamides, and β-lactams was significantly higher in Campylobacter coli, and there was no significant difference for resistance to quinolones between the two species (Table 3). Of the six Campylobacter coli pork isolates, four showed resistance to all antimicrobials tested and the other two were resistant to all of the antimicrobials tested except clindamycin, erythromycin, and gentamicin (data not shown). The distribution of the Multidrug Resistance (MDR) which was defined as resistance to three or more antibiotic classes among the 102 Campylobacter (33 Campylobacter jejuni and 69 Campylobacter coli) beef livers isolates was significantly higher in Campylobacter coli (62%) than Campylobacter jejuni (39%) ( Table 3). Among the beef liver isolates, the highest antibiotic resistances were to tetracyclines and -lactams, while the lowest resistances were to macrolides, aminoglycosides, lincosamides, and phenicols ( Table 3). The highest antibiotic resistances of Campylobacter were to oxytetracycline, amoxicillin, cephalothin, doxycycline, tetracycline and ampicillin, while the lowest resistances were to gentamicin and clindamycin (Table 3). Ishihara et al. [35] found that 34% of their Campylobacter jejuni isolates and all three of their Campylobacter coli isolates were resistant to oxytetracycline. They found no ampicillin or erythromycin resistance among their isolates. Chartre et al. [11] found among their cattle isolates, 2.4% to gentamicin, 9% resistance to ampicillin, 18% to erythromycin, 44% to ciprofloxacin, 44% to nalidixic acid and 88.1% to tetracycline. Taremi et al. [36] found no resistance to erythromycin among their Campylobacter isolates. Ishihara et al. [35] found that 1.5% of their Campylobacter coli isolates were resistant to ampicillin, 32% were resistant to nalidixic acid, 48.5% to erythromycin, and 82% to oxytetracycline. In a study of pig farms, Campylobacter coli isolated from pigs were found to contain 20% resistance to ampicillin, 34% to nalidixic acid, 55% to erythromycin, and 79% to tetracycline [37]. Velazquez et al. [38] found that 99% of their isolates were resistant to cephalothin and 16.7% were resistant to ampicillin. Table 3 one can conclude that most of the isolates with the highest rates of antimicrobial resistance were Campylobacter coli. This has been seen previously, where Campylobacter coli carry on average more antimicrobial resistances than do Campylobacter jejuni [6,11,35,39]. Ge et al. [39] found no chloramphenicol resistance in Campylobacter jejuni and only 3% in Campylobacter coli. Our study also found no resistance in Campylobacter jejuni to chloramphenicol but Campylobacter coli had a 32% resistance rate. Their study also found that Campylobacter jejuni had 42% resistance rate to erythromycin while Campylobacter coli had 61% [39]. Our study showed similar pattern in regards to Campylobacter coli where it showed a 54% resistance to erythromycin but Campylobacter jejuni showed only a 3% resistance. Little et al. [40] showed that their Campylobacter coli isolates were resistant to erythromycin 16.7%). They found that their Campylobacter coli pork isolates had higher resistances to tetracycline, nalidixic acid, ciprofloxacin and erythromycin. Another study showed that the rates of resistance among Campylobacter coli to erythromycin was 25%, tetracycline was 96%, ciprofloxacin was about 97%, doxycycline was 98%, and nalidixic acid was 100% [16]. Among Campylobacter jejuni isolates they found resistances of erythromycin was 6%, tetracycline was 94%, ciprofloxacin and nalidixic acid was about 96%, and doxycycline was 98% [16].

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In our study resistances to fluoroquinolones, macrolides, aminoglycosides, tetracyclines, β-lactams, lincosamides, and phenicols antibiotic classes were significantly higher in Campylobacter coli than Campylobacter jejuni isolates. While the overall prevalence of multidrug resistance in Campylobacter in our study is alarming, the fact that Campylobacter jejuni, the predominant species in human infections showed lower resistance to fluoroquinolones, and higher susceptibility to macrolides, and aminoglycosides is encouraging since human treatments are mostly dependent on these classes.

Conclusions
The prevalence of Campylobacter in retail beef livers is significantly higher than in other beef and pork meat cuts. Multidrug resistance was generally higher in the Campylobacter coli isolates than in the Campylobacter jejuni ones. Beef livers should be cooked thoroughly before consumption and should not be fed raw to household pets. The high prevalence of Campylobacter in retail beef livers and their antimicrobial resistance seen in this study raise concerns about the use of antimicrobials and the safety of these retail products.