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.

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.

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.

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. Methods and Results:  A total of 420 enterococci were isolated from nasal, teeth, rectal, belly and hindquarters sites of 155 dogs and 121 cats from three clinics in Athens, GA. Eighty per cent (124 out of 155) of the dogs and 60% (72 out of 121) of the cats were positive for enterococci. From the total number of dog samples (n = 275), 32% (n = 87) were from hindquarter, 31% (n = 86) were rectal, and 29% (n = 79) were from the belly area. The majority of isolates originated from rectal samples (53 out of 145; 37%) from cats. The predominant species identified was Enterococcus faecalis (105 out of 155; 68%) from dogs and E. hirae (63 out of 121; 52%) from cats. Significantly more E. faecalis were isolated from rectal samples than any other enterococcal species (P < 0·05) for both dogs and cats suggesting site specific colonization of enterococcal species. The highest levels of resistance were to ciprofloxacin in E. faecium (9 out of 10; 90%), chloramphenicol resistance in E. faecalis (17 out of 20; 85%) and gentamicin resistance in E. faecalis (19 out of 24; 79%) from dog samples and nitrofurantoin resistance in E. faecium (15 out of 19; 79%) from cats. Multi‐drug resistance (MDR) (resistance ≥2 antimicrobials) was observed to as few as two and as many as eight antimicrobials regardless of class. Conclusion:  This study demonstrated that dogs and cats are commonly colonized with antimicrobial resistant enterococci. Significance and Impact of the Study:  Dogs and cats may act as reservoirs of antimicrobial resistance genes that can be transferred from pets to people.

The ability of antimicrobial resistance (AR) to transfer, on mobile genetic elements (MGEs) between bacteria, can cause the rapid establishment of multidrug resistance (MDR) in bacteria from animals, thus creating a foodborne risk to human health. To investigate MDR and its association with plasmids in Salmonella enterica , whole genome sequence (WGS) analysis was performed on 193 S. enterica isolated from sources associated with United States food animals between 1998 and 2011; 119 were resistant to at least one antibiotic tested. Isolates represented 86 serotypes and variants, as well as diverse phenotypic resistance profiles. A total of 923 AR genes and 212 plasmids were identified among the 193 strains. Every isolate contained at least one AR gene. At least one plasmid was detected in 157 isolates. Genes were identified for resistance to aminoglycosides ( n = 472), β-lactams ( n = 84), tetracyclines ( n = 171), sulfonamides ( n = 91), phenicols ( n = 42), trimethoprim ( n = 8), macrolides ( n = 5), fosfomycin ( n = 48), and rifampicin ( n = 2). Plasmid replicon types detected in the isolates were A/C ( n = 32), ColE ( n = 76), F ( n = 43), HI1 ( n = 4), HI2 ( n = 20), I1 ( n = 62), N ( n = 4), Q ( n = 7), and X ( n = 35). Phenotypic resistance correlated with the AR genes identified in 95.4% of cases. Most AR genes were located on plasmids, with many plasmids harboring multiple AR genes. Six antibiotic resistance cassette structures (ARCs) and one pseudo-cassette were identified. ARCs contained between one and five resistance genes (ARC1: sul 2, strAB, tetAR ; ARC2: aac3-iid ; ARC3: aph, sph ; ARC4: cmy-2 ; ARC5: floR ; ARC6: tetB ; pseudo-ARC: aadA, aac3-VIa, sul 1). These ARCs were present in multiple isolates and on plasmids of multiple replicon types. To determine the current distribution and frequency of these ARCs, the public NCBI database was analyzed, including WGS data on isolates collected by the USDA Food Safety and Inspection Service (FSIS) from 2014 to 2018. ARC1, ARC4, and ARC5 were significantly associated with cattle isolates, while ARC6 was significantly associated with chicken isolates. This study revealed that a diverse group of plasmids, carrying AR genes, are responsible for the phenotypic resistance seen in Salmonella isolated from United States food animals. It was also determined that many plasmids carry similar ARCs.

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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 g/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.

Background: Nosocomial salmonellosis is often assumed to occur because infection control and surveillance practices are inadequate, but published evidence is lacking to support the related contention that rigorous application of these practices can impact the severity of outbreaks. Objective: Describe active surveillance, early recognition, and intensive mitigation efforts used in an effort to control an outbreak of nosocomial Salmonella enterica serotype Newport infections without hospital closure. Animals: Large animals hospitalized at a referral hospital. Methods: This prospective outbreak investigation was initiated when Salmonella Newport infections were detected among hospitalized animals by active surveillance. Data were analyzed to identify temporal and spatial patterns for epidemic spread of Salmonella in the hospital. Mitigation efforts were aggressively adjusted in response to surveillance data. Genetic relatedness of isolates was investigated by pulsed‐field gel electrophoresis. Results: Of 145 large animals sampled, 8 (5.6%) were infected with the Salmonella strain associated with this outbreak, and all but 1 shed Salmonella in the absence of or before the onset of disease. This strain was recovered from 14.2% (42/295) of environmental samples (ENV samples), indicating that widespread environmental contamination had occurred. Isolates of Salmonella Newport obtained from infected animals and the environment were genetically indistinguishable, confirming clonal dissemination. Conclusions and Clinical Importance: Active surveillance allowed early detection of nosocomial Salmonella transmission and hospital contamination. Use of aggressive interventions was followed by cessation of transmission. Active surveillance can allow earlier recognition and mitigation compared with programs by only sampling of clinically affected animals.