Incidence, Antimicrobial Susceptibility, and Toxin Genes Possession Screening of Staphylococcus aureus in Retail Chicken Livers and Gizzards

Few recent outbreaks in Europe and the US involving Campylobacter and Salmonella were linked to the consumption of chicken livers. Studies investigating Staphylococcus aureus in chicken livers and gizzards are very limited. The objectives of this study were to determine the prevalence, antimicrobial resistance, and virulence of S. aureus and MRSA (Methicillin-Resistant Staphylococcus aureus) in retail chicken livers and gizzards in Tulsa, Oklahoma. In this study, 156 chicken livers and 39 chicken gizzards samples of two brands were collected. While one of the brands showed very low prevalence of 1% (1/100) for S. aureus in chicken livers and gizzards, the second brand showed prevalence of 37% (31/95). No MRSA was detected since none harbored the mecA or mecC gene. Eighty seven S. aureus isolates from livers and 28 from gizzards were screened for antimicrobial resistance to 16 antimicrobials and the possession of 18 toxin genes. Resistance to most of the antimicrobials screened including cefoxitin and oxacillin was higher in the chicken gizzards isolates. While the prevalence of enterotoxin genes seg and sei was higher in the gizzards isolates, the prevalence of hemolysin genes hla, hlb, and hld was higher in the livers ones. The lucocidin genes lukE-lukD was equally prevalent in chicken livers and gizzards isolates. Using spa typing, a subset of the recovered isolates showed that they are not known to be livestock associated and, hence, may be of a human origin. In conclusion, this study stresses the importance of thorough cooking of chicken livers and gizzards since it might contain multidrug resistant enterotoxigenic S. aureus. To our knowledge this is the first study to specifically investigate the prevalence of S. aureus in chicken livers and gizzards in the US.

producing strains of S. aureus specially that most of these enterotoxins are heat stable. The objectives of this study were to determine the prevalence of S. aureus and MRSA in retail chicken livers and gizzards collected in Tulsa, Oklahoma and to characterize the recovered isolates for their antimicrobial susceptibility and possession of toxin genes. To our knowledge this study is the first to specifically investigate the prevalence of S. aureus in chicken livers and gizzards in the US.

Isolation of Staphylococcus aureus from Retail Chicken Livers and Gizzards
Chicken livers and gizzards samples were collected from several different grocery stores in the Tulsa, Oklahoma area for a period of six months starting January of 2010. A total of 195 chilled retail chicken liver and chicken gizzard samples were used in this study (156 chicken livers and 39 chicken Gizzards) ( Table 1). Meat samples were purchased from nine grocery stores that belong to six different franchises chains at variable locations in the city. The chicken livers and gizzards belonged to two major brands, which are designated brand A and brand B (Table 1). Samples were selected to be as variable as possible with different expiration and production dates. Chicken livers and gizzards samples were added to 10 mL of buffered peptone water (BPW) (BPW; EMD, Gibbstown, NJ, USA) in sterile plastic bags (VWR Scientific, Radnor, PA, USA) and massaged by hand for approximately 5 min. Ten milliliters was then transferred from the bag and added to 10 mL of enrichment broth of 2× Trypticase Soy Broth with 10% sodium chloride and 1% sodium pyruvate, then incubated at 37 °C for 24 h. A loopful was then streaked to Baird Parker (BP) selective media plates and incubated at 37 °C for 48 h [33]. Four suspected S. aureus colonies (those that have black colonies surrounded by 2 to 5 mm clear zones) were selected and streaked to Trypticase Soy Agar (TSA) plates and subcultured for confirmation on MSA (Mannitol Salt Agar) plates. Pure prospective S. aureus cultures were kept at −80 °C until PCR confirmation.

DNA Extraction
DNA was extracted from the prospective S. aureus strains using the single cell lysing buffer (SCLB) method [38]. One day old colonies were picked and suspended in 40 μL of single cell lysing buffer (SCLB) solution (1.0 mL of TE buffer (10 mM Tris-HCL and 1 mM EDTA) and 10 μl of 5 mg/mL proteinase K) in a 0.2 mL microtube. In a thermocycler, bacterial cells were lysed by initial incubation at 80 °C for 10 min, followed by 55 °C for 10 min, and then 95 °C for 10 min [38]. DNA extracted by the above mentioned method was stored at −20 °C until used as a DNA template for PCR.

PCR Identification
A multiplex PCR reaction was used to identify the isolated suspected S. aureus by using specific primers for S. aureus and MRSA to amplify a 108 bp and a 312 bp fragments respectively (Table 4). Twenty microliters PCR reactions, which included 10 µL of Qiagen Master Mix (Qiagen Inc., Valencia, CA, USA), 4 µL of sterile water (Qiagen), 1 µL of each forward and reverse primer (IDT, Coralville, IA, USA), and 2 µL of DNA template, were performed. The PCR protocol was as follows: initial denaturing at 95 °C for 5 min (followed by 35 cycles of denaturing at 95 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min) and ending with extension at 72 °C for 10 min. PCR amplicons were subjected to agarose gel electrophoresis and DNA bands were visualized and recorded using a gel documentation system. Isolates showing resistance to cefoxitin and/oxacillin were subjected to PCR confirmation using a second set of MRSA primers that amplify a 533 bp mecA fragment and two other variant MRSA mecA primer sets (also known as mecC) that amplify 356 bp and 1800 bp fragments to confirm the MRSA phenotype (Table 1).

Antimicrobial Susceptibility Testing
A total of 115 S. aureus recovered isolates (87 chicken liver isolates and 28 chicken gizzard isolates) were subjected to antimicrobial resistance profiling against sixteen different antimicrobials that belong to ten different antibiotic classes ( Table 2). Isolates were grown on Mueller-Hinton (MH) agar (Difco) and incubated for 48 h at 37 °C . 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 the appropriate antimicrobial at different concentrations (Table 2) including the breakpoint established for each antimicrobial according to the Clinical and Laboratory Standards Institute (CLSI) when available [39]. Plates were then incubated at 37 °C for 48 h and results were read for growth or no growth and denoted as resistant or susceptible, respectively according to the breakpoints for each of the tested antimicrobials ( Table 2).

Detection of Toxin Genes
A total of 115 Staphylococcus aureus isolates (87 chicken liver isolates and 28 chicken gizzard isolates) were screened for eighteen different toxin genes that belong to six different toxin gene groups (Table 3). Multiplex PCR was used to detect 18 different toxin genes of S. aureus isolates that include enterotoxins, toxic shock syndrome toxin 1, exfoliative toxins, leucocidins, Panton-Valentine leucocidin (PVL), and hemolysins (Table 3). Three multiplex reactions (A, B, and C), each of which included six toxin genes, were performed ( Table 3). The multiplex PCR targeting the toxin genes were performed in a 20 µL reaction solution that contained 10 µL of Green Master Mix (Promega), 2µL of sterile water, 2 µL of the DNA template and 0.5 µL of each of the toxin gene primers. The PCR protocol included an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation (94 °C for 1 min), annealing (57 °C for 1 min), and extension (72 °C for 1 min), ending with an extension at 72 °C for 7 min. PCR amplicons were subjected to agarose gel electrophoresis and DNA bands were visualized and recorded using a gel documentation system. The expected amplicon band sizes of S. aureus toxin genes are shown in Table 3. Several representative amplicons of each positive toxin were sequenced in house using the same amplifying primers to confirm PCR accuracy. Table 3. Multiplex PCR primers, reaction sets, references, and toxin groups for the screened toxin genes.

Molecular Typing Using spa Genotyping
A subset of the recovered Staphylococcus aureus isolates were subjected to molecular typing using spa typing. Molecular typing using spa was done according to published primers and protocols [43] and spa types were assigned using the BioNumerics Software (Applied Math, Austin, TX, USA) through the Ridom Spa Server.

Prevalence of Staphylococcus aureus and MRSA in Chicken Livers and Gizzards
A total of 195 chilled retail chicken liver and chicken gizzard samples were purchased from several Tulsa area grocery stores starting January 2010 for a period of 6 months. The number of chicken liver samples was 156 and the number of chicken gizzard samples was 39 ( Table 1). The chicken livers and gizzards collected in this study belonged to two major brands, which were designated brand A and brand B ( Table 4). As shown in Table 4, the overall prevalence of S. aureus in chicken livers and gizzards including the two brands together was 36/195 (18.5%). While 27/156 (17.3%) of chicken livers were contaminated with S. aureus, 9/39 (23.1%) of chicken gizzards were positive for the bacterium. The prevalence of S. aureus in brand A chicken livers was 26/71 (36.6%) while it was 9/24 (37.5%) in chicken gizzards of the same brand (Table 1). Only one out of 85 chicken liver samples (1.2%) of brand B was positive for S. aureus and none of the chicken gizzards of this brand was positive for S. aureus. No isolates of chicken livers and gizzards were positive for MRSA since none of them carried mecA or mecC genes.
Even though the overall prevalence of S. aureus in chicken livers and gizzards was 36/195 (18.5%) in our study, the 36.6% and the 37.5% prevalence in brand A chicken livers and gizzards respectively is alarming (Table 4). While there were no available studies in the literature that specifically determined the prevalence of S. aureus in chicken livers and gizzards, a study in Turkey reported contamination in 9/30 (30%) of chicken giblets as a part of a larger study on chicken meat [10]. A second study in Japan reported a higher prevalence of S. aureus in chicken livers (63.8%) and chicken gizzards (58.1%) after enrichment while it was 47.9% and 22.6% respectively before enrichment [9]. The higher prevalence in the Japanese study might be due to differences between the US and the Japanese retail poultry markets. It can also be due to the methods used for identification since the Japanese study used only biochemical tests for identification of the S. aureus strains while molecular identification was used in our study. S. aureus was isolated from 56% of ground turkey collected from Maryland, USA [32] and was found in 25% of retail chicken in Detroit, Michigan where 3.9% were MRSA [31]. In another study in Iowa, 17.8% of retail chicken was contaminated with S. aureus [30], while in North Dakota a higher prevalence of S. aureus (67.6%) in retail chicken was reported [26].
The big difference between the overall prevalence of S. aureus in chicken livers and gizzards in brand A in our study (36.8%) and only 1% in brand B (Table 4) might be due to the difference in food safety and microbiology quality control handling protocols at the two production companies. While not conclusive, this data might suggest that chicken livers and gizzards contamination with S. aureus most probably occurs during handling at the slaughter house or at the retail packaging step.

Antimicrobial Susceptibility of the Recovered Isolates
A total of 115 Staphylococcus aureus isolates (87 chicken liver isolates and 28 chicken gizzard isolates) were subjected to antimicrobial resistance profiling against sixteen different antimicrobials that belong to ten different antibiotic classes (Tables 2 and 5). As shown in Table 5, the percentage of resistance of the 115 S. aureus isolates from chicken livers and gizzards to the sixteen tested antimicrobials were as follow: ampicillin (88.9%), tetracycline (71.3%), doxycycline (63.5%), penicillin (60.9%), erythromycin (45.2%), azithromycin (40,9%), vancomycin (39.1%), oxacillin with 2% NaCl (32.2%), ciprofloxacin (29.6%), trimethoprim/sulfamethazole (24.3%), rifampin (23.5%), cefoxitin with 2% NaCl (19.1%), clindamycin (12.2%), kanamycin (12.2%), chloramphenicol (10.4%), and gentamicin (10.4%). As shown in Table 5, the percentage of resistance to the sixteen tested antimicrobials varied between chicken livers and chicken gizzards isolates. The percentage of resistance found in the chicken gizzards was higher than chicken livers isolates for the following 10 antimicrobials: azithromycin, ciprofloxacin, oxacillin, cefoxitin, tetracycline, vancomycin, doxycycline, penicillin, kanamycin, and erythromycin (Table 5). On the other hand, for gentamycin, ampicillin, trimethoprim/sulfamrthoxazole, clindamycin, rifampin, chloramphenicol the chicken livers isolates showed a higher resistance (Table 5). This variability in antimicrobial resistance between isolates from chicken livers and gizzards might be attributed to the concentration of different antimicrobials in the liver and/or the fact that chicken gizzards often have more fats that would make some highly lipid soluble antimicrobials like azithromycin get to higher concentrations in the gizzards. Overall 35/115 (30%) of the screened isolates from chicken livers and gizzards were multidrug resistant to more than seven antimicrobials (data not shown) which is worrisome.
Resistance to vancomycin was relatively high in isolates from chicken livers and gizzards in our study (Table 5).Vancomycin Resistant Enterococcus faecium (VRE) was previously reported in swine in Michigan, USA and was thought to be widespread despite the historical absence of the use of agricultural glycopeptides like avoparcin. Screening our phenotypically vancomycin strains for the presence of the vanA gene is currently underway as a part of a larger study focusing on vancomycin resistant S. aureus strains isolated from various US retail meats. Chicken livers and gizzards isolates in our study were highly resistant to ampicillin, tetracycline, doxycycline, penicillin, and erythromycin ( Table 5). The literature is lacking data about antimicrobial resistance of S. aureus strains isolated from chicken livers and gizzards. One study in Turkey [10] reported that S. aureus isolates from chicken giblets were resistant to penicillin G (22.2%) and erythromycin (33.3%). S. aureus recovered strains in our study that showed resistance to cefoxitin and/or oxacillin (highly prevalent in chicken gizzards as shown in Table 5) were subjected to additional PCR protocols to check for the presence of a mecA homologue ( Table 1). None of these isolates showed the presence of mecA gene or it homologues (mecC). Phenotypic MRSA isolates that do not harbor the mecA gene were reported [32,44]. This might be due to over production of Beta-lactamase enzymes or the presence of a variant mecA gene that does not amplify with the known PCR primers. The recent advancement in whole genome sequencing through next generation sequencing can help identifying such homologues.
The high number of multidrug resistant S. aureus detected in our study is alarming. It raises concerns about inappropriate practices including the use of antimicrobials as growth promotors in food animal production and the frequent use of antimicrobials in poultry husbandry. Genes coding for antimicrobial resistance can move through horizontal gene transfer to clinical pathogenic strains and contribute to the creation of superbugs. Death in hospitals are often attributed to sepsis resulting from infections caused by multidrug resistant pathogens like MRSA, Pseudomonas aeruginosa or Candida albicans rather than the original cause of the hospitalization.

Toxin Genes Possession Screening of the Recovered Isolates
A total of 115 Staphylococcus aureus isolates (87 chicken liver isolates and 28 chicken gizzard isolates) were screened for eighteen different toxin genes that belong to six different toxin gene groups (Tables 3 and 6). As shown in Table 6, the prevalence of toxin genes in the 115 S. aureus isolates from chicken livers and gizzards to the eighteen tested toxin genes were as follow: hla (94.5%), hld (94.5%), hlb (48.7%), sei (42.6%), lukE-lukD (36.5%), seg (29.6%), seh (4.3%), sed (0.9%), sea (0%), seb-sec (0%), sec (0%), see (0%), sej (0%), tst (0%), eta (0%), etb (0%), lukM (0%), and lukS-lukF (0%). S. aureus hemolysin genes were found at a higher percentage in the chicken livers and gizzards than other groups of toxin genes screened. Also no isolates harbored enterotoxin genes sed, sea, sebsec, sec, see, or sej, the toxic shock syndrome toxin 1 gene tst, the exfoliative toxin genes eta, etb, or the Leucocidin gene lukM ( Table 6). The prevalence of enterotoxin genes seg (71.43%) and sei (92.9%) in chicken gizzards was higher than in chicken livers, where seg was 16.1% and sei was 26.4%. One isolate from chicken livers was positive for the entoretoxin gene sed (1.2%) and 23/87 of chicken livers isolates were positive for the entoretoxin gene sei (26.4%). The prevalence of hemolysin genes hla (97.5%), hld (97.5%) and hlb (64.4%) in chicken livers was higher than in chicken gizzards when it was 85.7%, 85.7% and 0% respectively. The Hemolysin gene hlb was present only in the chicken livers. Both chicken livers and gizzards isolates had similar prevalence for lukE-lukD.   The literature is lacking data related to the prevalence of toxin genes in S. aureus isolated from chicken livers and gizzards. Even recent studies discussing S. aureus in US retail poultry in general lacks such toxin genes prevalence data. A study in Japan reported that 25.3% of their chicken liver isolates were enterotoxigenic while 36.4% of their chicken gizzards produced enterotoxins [9]. Even though they have not used PCR to detect enterotoxin genes in the Japanese study, their chicken gizzards strains showed a higher prevalence of enterotoxins that their chicken livers ones which is in agreement with our findings. The higher prevalence of hemolysin genes in chicken livers isolates than in the chicken gizzards ones might be due to the availability of blood in the liver which might select for S. aureus strains with blood lysing abilities. Chicken livers and gizzards should be cooked thoroughly since enterotoxins of S. aureus are known for their heat tolerance. So even if the cooking temperature was high enough to kill the pathogen, enterotoxins produced on the chicken livers or gizzards could tolerate such temperature increasing the risk of food poisoning.

Genotyping Using spa Typing
A subset of Staphylococcus aureus recovered strains (6 from chicken livers, and 5 from chicken gizzards) were subjected to molecular typing by spa typing (Figure 1). As it is shown in Figure 1, spa types were grouped into two major clusters with the majority of isolates in each cluster belonging to one source. As it is also shown in figure 1, the tested isolates showed higher diversity in regards to their spa types since 7 different spa types were detected among a subset of 11 isolates. The detected spa types (t1081, t064, t002, and t091) were not known to be livestock associated and hence, maybe of a human origin [45]. This is in agreement with what we discussed earlier in the introduction section about that the origin of S. aureus strains detected in US retail meats is mostly of a human origin rather than livestock associated as it is the case in Europe.

Conclusions
The prevalence of S. aureus in retail chicken livers and gizzards tested in this study varied between the two brands tested. While one of the brands showed very low prevalence of S. aureus, the second Chicken Liver brand showed prevalence close to 37%. The percentage of resistance to most of the antimicrobials screened was generally higher in isolates recovered from chicken gizzards. While no isolate harbored the mecA or mecC gene, a higher percentage of the chicken gizzards isolates were resistant to cefoxitin and/or oxacillin making them phenotypically similar to MRSA. A high percentage of S. aureus recovered strains particularly from chicken gizzards harbored enterotoxins seg and sei. The lucocidin genes lukE-lukD was equally prevalent in chicken livers and gizzards isolates. The hemolysin hlb gene was only prevalent in the chicken livers strains while hla and hld were prevalent in chicken livers and gizzards strains. Using spa typing, a subset of the recovered isolates showed that they are not known to be livestock associated and hence, maybe of a human origin. Data obtained from this study stress the importance of thorough cooking of chicken livers and gizzards since it might contain multidrug resistant enterotoxigenic S. aureus.