Charlene R. Jackson

Use of a Genus- and Species-Specific Multiplex PCR for Identification of Enterococci

ABSTRACT The 16S rRNA gene has previously been used to develop genus-specific PCR primers for identification of enterococci. In addition, the superoxide dismutase gene (sodA) has been identified as a potential target for species differentiation of enterococci. In this study, Enterococcus genus-specific primers developed by Deasy et al. (E1/E2) were incorporated with species-specific primers based upon the superoxide dismutase (sodA) gene for development of a multiplex PCR. This assay provides simultaneous genus and species identification of 23 species of enterococci using seven different reaction mixtures. Accuracy of identification of the multiplex PCR was determined by comparisons to standard biochemical testing, the BBL Crystal kit, VITEK, and API Rapid ID 32 Strep. Isolates from swine feces, poultry carcasses, environmental sources, and retail food were evaluated and, overall, results for 90% of the isolates tested by PCR agreed with results obtained using standard biochemical testing and VITEK. Eighty-five percent and 82% of PCR results agreed with results from the API Rapid ID 32 Strep and BBL Crystal tests, respectively. With the exception of concurrence between identification using standard biochemical testing and VITEK (85%) and between BBL Crystal and VITEK (83%), the percent agreement for PCR was higher than or equal to any other pairwise comparison. Multiplex PCR for genus and species determination of enterococci provides an improved, rapid method for identification of this group of bacteria.

Effects of Tylosin Use on Erythromycin Resistance in Enterococci Isolated from Swine

ABSTRACT The effect of tylosin on erythromycin-resistant enterococci was examined on three farms; farm A used tylosin for growth promotion, farm B used tylosin for treatment of disease, and farm C did not use tylosin for either growth promotion or disease treatment. A total of 1,187 enterococci were isolated from gestation, farrowing, suckling, nursery, and finishing swine from the farms. From a subset of those isolates (n = 662), 59% (124 out of 208), 28% (80 out of 281), and 2% (4 out of 170) were resistant to erythromycin (MIC ≥ 8 μg/ml) from farms A, B, and C, respectively. PCR analysis and Southern blotting revealed that 95% (65 out of 68) of isolates chosen from all three farms for further study were positive for ermB, but all were negative for ermA and ermC. By using Southern blotting, ermB was localized to the chromosome in 56 of the isolates while 9 isolates from farms A and B contained ermB on two similar-sized plasmid bands (12 to 16 kb). Pulsed-field gel electrophoresis revealed that the isolates were genetically diverse and represented a heterogeneous population of enterococci. This study suggests that although there was resistance to a greater number of enterococcal isolates on a farm where tylosin was used as a growth promotant, resistant enterococci also existed on a farm where no antimicrobial agents were used.

Genetic mechanisms of antimicrobial resistance identified in Salmonella enterica, Escherichia coli, and Enteroccocus spp. isolated from U.S. food animals

The prevalence of antimicrobial resistance (AR) in bacteria isolated from U.S. food animals has increased over the last several decades as have concerns of AR foodborne zoonotic human infections. Resistance mechanisms identified in U.S. animal isolates of Salmonella enterica included resistance to aminoglycosides (e.g., alleles of aacC, aadA, aadB, ant, aphA, and StrAB), β-lactams (e.g., blaCMY−2, TEM−1, PSE−1), chloramphenicol (e.g., floR, cmlA, cat1, cat2), folate pathway inhibitors (e.g., alleles of sul and dfr), and tetracycline [e.g., alleles of tet(A), (B), (C), (D), (G), and tetR]. In the U.S., multi-drug resistance (MDR) mechanisms in Salmonella animal isolates were associated with integrons, or mobile genetic elements (MGEs) such as IncA/C plasmids which can be transferred among bacteria. It is thought that AR Salmonella originates in food animals and is transmitted through food to humans. However, some AR Salmonella isolated from humans in the U.S. have different AR elements than those isolated from food animals, suggesting a different etiology for some AR human infections. The AR mechanisms identified in isolates from outside the U.S. are also predominantly different. For example the extended spectrum β-lactamases (ESBLs) are found in human and animal isolates globally; however, in the U.S., ESBLs thus far have only been found in human and not food animal isolates. Commensal bacteria in animals including Escherichia coli and Enterococcus spp. may be reservoirs for AR mechanisms. Many of the AR genes and MGEs found in E. coli isolated from U.S. animals are similar to those found in Salmonella. Enterococcus spp. isolated from animals frequently carry MGEs with AR genes, including resistances to aminoglycosides (e.g., alleles of aac, ant, and aph), macrolides [e.g., erm(A), erm(B), and msrC], and tetracyclines [e.g., tet(K), (L), (M), (O), (S)]. Continuing investigations are required to help understand and mitigate the impact of AR bacteria on human and animal health.

Prevalence and Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Retail Meat and Humans in Georgia

ABSTRACT There is increasing interest in the presence of Staphylococcus aureus, specifically methicillin-resistant S. aureus (MRSA), on retail meat products. In this study, staphylococci were isolated from retail pork and retail beef in Georgia, and MRSA from the products was compared to human MRSA from the same geographic area using broth microdilution antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 45% (45/100) of pork products and 63% (63/100) of beef products; mecA was detected in S. aureus from both pork (3/100; 3%) and beef (4/100; 4%). Fifty percent (50/100) of human S. aureus also contained mecA. Multidrug resistance was detected among MRSA from all sources. All MRSA (n = 57) was SCCmec type IV, and nine different spa types were present among the isolates (t002, t008, t012, t024, t179, t337, t548, t681, and t1062). Four sequence types (ST5, ST8, ST9, and ST30) were detected using MLST; the majority of MRSA isolates belonged to ST8, followed by ST5. One retail beef MRSA isolate belonged to ST8, while the remaining three were ST5. In retail pork MRSA, ST5, ST9, and ST30 were observed. The majority of human MRSA isolates belonged to ST8. Thirty-seven MRSA isolates, one of which was a retail beef MRSA isolate, were pvl +. Using PFGE, MLST, and spa typing, three retail beef MRSA isolates were found to be identical in PFGE pattern, ST, and spa type to two human clonal MRSA isolates (USA100 and USA300). One additional retail beef MRSA isolate had a PFGE pattern similar to that of a human MRSA isolate, whereas none of the retail pork MRSA isolates had PFGE patterns similar to those of human MRSA isolates. These data suggest that the retail beef samples were contaminated by a human source, possibly during processing of the meat, and may present a source of MRSA for consumers and others who handle raw meat.

Epidemiology and Genotypic Characteristics of Methicillin-Resistant Staphylococcus aureus Strains of Porcine Origin

ABSTRACT The main goal of this study was to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), particularly livestock-associated MRSA (LA-MRSA) in pigs and pork. The genotypic relatedness of isolates on the farm, at slaughter, and at the retail level was assessed. Paired nasal and perianal swab samples were collected from 10 cohorts of market-age pigs (24 pigs per cohort) and carcasses at slaughterhouse, and pork samples were collected at retail. Staphylococci were isolated using selective enrichment method. Isolates were tested for antimicrobial resistance by broth microdilution. Duplex PCR was used to confirm MRSA using species-specific (nuc) and methicillin resistance (mecA) genes. The clonal relatedness of isolates was determined using pulsed-field gel electrophoresis (PFGE), Staphylococcus protein A (spa) typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec element (SCCmec) typing. MRSA was detected in 5 of the 10 cohorts (50%), with the prevalence ranging from 0% to 12.5% per cohort. Of all the pigs sampled on the farm before they went to market, 3% (7/240) were MRSA positive. A higher prevalence of MRSA was detected at holding pens at the slaughterhouse (11% [27/240]). MRSA was also detected in 2% (4/235) of the carcasses and 4% (5/135) of the retail pork. While the isolates appear predominantly to be highly clonal, PFGE had a relatively higher discriminatory power (discriminatory index [DI] = 0.624). Four genotypic clusters were identified by PFGE; of the four clusters, clonal type B was predominant across the farm-to-retail continuum. MLST findings revealed that sequence type 5 (ST5) was the most predominant subtype (32/50). The livestock-associated MRSA (clonal complex 398 [CC398] or sequence type 398 [ST398]) was the second common type (12/50) and was detected at all stages from farm to retail. Nine of the 50 (18%) MRSA isolates belonged to spa type 539/t034 that were of ST398 based on MLST. The results of this study confirm that MRSA, including LA-MRSA, is common in herds of swine in Ohio and hereby shown to persist in the farm to processing and retail continuum.

Prevalence, species distribution and antimicrobial resistance of enterococci isolated from dogs and cats in the United States.

Aims:  The contribution of dogs and cats as reservoirs of antimicrobial resistant enterococci remains largely undefined. This is increasingly important considering the possibility of transfer of bacteria from companion animals to the human host. In this study, dogs and cats from veterinary clinics were screened for the presence of enterococci.

Prevalence and antimicrobial resistance of enterococci isolated from retail fruits, vegetables, and meats

Although enterococci are considered opportunistic pathogens, they can be reservoirs of antimicrobial resistance. Antimicrobial resistance is increasingly important because of foodborne illnesses from meat and infections from produce. From 2000 through 2001, food items (vegetables, fruits, and meats) were obtained from grocery store chains in northern Georgia and cultured for the presence of enterococci; 47.7% (189 of 396) of these samples were positive for enterococci. For the fruits and vegetables, enterococci were cultured most often from tomatoes (9 of 27 samples, 33%) and radishes (10 of 11 samples, 91%), respectively. Among the meat items tested, enterococci were isolated from 95% (21 of 22) of the chicken samples, 73% (16 of 22) of the beef samples, 95% (20 of 21) of the turkey samples, and 68% (15 of 22) of the pork samples. The predominant species identified was Enterococcus faecalis (n = 80) from meat and Enterococcus casseliflavus (n = 66) from fruits and vegetables. Although high numbers of isolates were resistant to lincomycin (176 of 185 isolates, 95.1%) and bacitracin (150 of 185 isolates, 81.1%), very few isolates were resistant to salinomycin (2 isolates, 1.1%), penicillin (3 isolates, 1.6%), or nitrofurantoin (9 isolates, 4.9%). None of the isolates were resistant to linezolid or vancomycin. These data suggest that foods commonly purchased from grocery stores are a source of enterococci; however, overall resistance to antimicrobials is relatively low.

Salmonella Enteritidis in Meat, Poultry, and Pasteurized Egg Products Regulated by the U.S. Food Safety and Inspection Service, 1998 through 2003

The U.S. Food Safety and Inspection Service (FSIS) tests for Salmonella in meat, poultry, and egg products through three regulatory testing programs: the Pathogen Reduction-Hazard Analysis and Critical Control Point (PR-HACCP) program, the ready-to-eat program for meat and poultry products, and the pasteurized egg products program. From 1998 through 2003, 293,938 samples collected for these testing programs were analyzed for the presence of Salmonella enterica serotypes. Of these samples, 12,699 (4.3%) were positive for Salmonella, and 167 (1.3%) of the positive samples (0.06% of all samples) contained Salmonella Enteritidis. The highest incidence of Salmonella Enteritidis was observed in ground chicken PR-HACCP samples (8 of 1,722 samples, 0.46%), and the lowest was found in steer-heifer PR-HACCP samples (0 of 12,835 samples). Salmonella Enteritidis isolates were characterized by phage type, pulsed-field gel electrophoretic pattern, and antimicrobial susceptibility. Phage typing of 94 Salmonella Enteritidis isolates identified PT13 (39 isolates) and PT8 (36 isolates) as the most common types. One isolate from a ready-to-eat ham product was characterized as PT4. Electrophoretic analysis of 148 Salmonella Enteritidis isolates indicated genetic diversity among the isolates, with 28 unique XbaI electrophoretic patterns identified. Of these 148 isolates, 136 (92%) were susceptible to each of 16 antimicrobials tested. Two isolates were resistant to ampicillin alone, and 10 isolates were resistant to two or more antimicrobials. Isolation of Salmonella Enteritidis from FSIS-regulated products emphasizes the need for continued consumer education on proper food handling and cooking practices and continued work to decrease the prevalence of Salmonella in meat, poultry, and pasteurized egg products.

Antimicrobial resistance and virulence of Enterococcus faecalis isolated from retail food.

Although enterococci are considered opportunistic nosocomial pathogens, their contribution to foodborne illnesses via dissemination through retail food remains undefined. In this study, prevalence and association of antimicrobial resistance and virulence factors of 80 Enterococcus faecalis isolates from retail food items were investigated. The highest rates of resistance were observed for lincomycin (73 of 80 isolates, 91%) and bacitracin (57 of 80 isolates, 71%), and lower rates of resistance (< or =40%) were found for chloramphenicol, ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, nitrofurantoin, penicillin, and tylosin. Overall resistance to antimicrobials was low for most isolates tested. Of the virulence factors tested, the majority of isolates were positive for ccf (78 of 80 isolates, 98%), efaAfs (77 of 80, 96%), and cpd (74 of 80, 93%). Isolates also commonly contained cob (72 of 80 isolates, 90%) and gelE (68 of 80, 85%). Very few isolates contained cylMBA (12 of 80 isolates [15%] for cylM and 9 of 80 isolates [11%] for both cylB and cylA) and efaAfm (2 of 80 isolates, 3%). Positive statistical associations (significance level of 0.05) were found between agg and tetracycline resistance, cylM and erythromycin resistance, and gelE and efaAfs and lincomycin resistance. The presence of the cylB and cylA alleles also was positively correlated with bacitracin and tetracycline resistance. Negative correlations were observed between many of the virulence attributes and resistance to ciprofloxacin, erythromycin, flavomycin, gentamicin, kanamycin, and tylosin. These data suggest that both positive and negative associations exist between antimicrobial resistance genes and virulence factors in E. faecalis isolates from foods commonly purchased from grocery stores.

Comparative antimicrobial susceptibility of Listeria monocytogenes, L. innocua, and L. welshimeri.

The current study compared antimicrobial susceptibility of Listeria innocua, L. welshimeri, and L. monocytogenes isolated from various sources. Antimicrobial susceptibility testing was performed using a microbroth procedure with Sensititre minimum inhibitory concentration plates containing 18 antimicrobials. Resistant isolates were analyzed for the presence of antimicrobial resistance genes using PCR. The majority of L. monocytogenes isolates were resistant to oxacillin (99%, 89/90) and ceftriaxone (72%, 65/90), while few isolates were resistant to clindamycin (21%, 19/90) and ciprofloxacin (2%, 2/90). When selected sources of L. monocytogenes are compared, resistance to ceftriaxone, clindamycin, and oxacillin ranged from 27% to 86%, 7% to 43%, and 96% to 100%, respectively. Resistance to ciprofloxacin (6%, 2/34), quinupristin/dalfopristin (7%, 1/14), and tetracycline (7%, 1/15) was observed with L. monocytogenes isolated from food, animal, and environmental sources, respectively. All L. welshimeri isolates (6/6) were resistant to streptomycin, quinupristin/dalfopristin, ciprofloxacin, rifampin, oxacillin, penicillin, and clindamycin, while most isolates (67%, 4/6) were resistant to trimethoprim/sulfamethoxazole. All L. innocua isolates (4/4) were resistant to oxacillin and penicillin, whereas 75% (3/4) of isolates were resistant to tetracycline, ceftriaxone, and clindamycin. Resistant isolates were negative for aadA, strA-B, sul I-II, penA, vat(A-E), vga(A-B), and vgb(A-B). However, tetM was detected among tetracycline-resistant isolates. L. welshimeri was resistant to more of the tested antimicrobials than the other two Listeria species tested, but resistance was not attributed to selected resistance genes. These data demonstrate the variability in resistance among Listeria species. However, the human pathogen L. monocytogenes appears to be the least resistant among the tested species.