John B. Barrett

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.

Serum leptin concentrations, luteinizing hormone and growth hormone secretion during feed and metabolic fuel restriction in the prepuberal gilt

Two experiments were conducted to determine 1) the effect of acute feed deprivation on leptin secretion and 2) if the effect of metabolic fuel restriction on LH and GH secretion is associated with changes in serum leptin concentrations. Experiment (EXP) I, seven crossbred prepuberal gilts, 66 +/- 1 kg body weight (BW) and 130 d of age were used. All pigs were fed ad libitum. On the day of the EXP, feed was removed from four of the pigs at 0800 (time = 0) and pigs remained without feed for 28 hr. Blood samples were collected every 10 min from zero to 4 hr = Period (P) 1, 12 to 16 hr = P 2, and 24 to 28 hr = P 3 after feed removal. At hr 28 fasted animals were presented with feed and blood samples collected for an additional 2 hr = P 4. EXP II, gilts, averaging 140 d of age (n = 15) and which had been ovariectomized, were individually penned in an environmentally controlled building and exposed to a constant ambient temperature of 22 C and 12:12 hr light: dark photoperiod. Pigs were fed daily at 0700 hr. Gilts were randomly assigned to the following treatments: saline (S, n = 7), 100 (n = 4), or 300 (n = 4) mg/kg BW of 2-deoxy-D-glucose (2DG), a competitive inhibitor of glycolysis, in saline iv. Blood samples were collected every 15 min for 2 hr before and 5 hr after treatment. Blood samples from EXP I and II were assayed for LH, GH and leptin by RIA. Selected samples were quantified for glucose, insulin and free fatty acids (FFA). In EXP I, fasting reduced (P < 0.04) leptin pulse frequency by P 3. Plasma glucose concentrations were reduced (P < 0.02) throughout the fast compared to fed animals, where as serum insulin concentrations did not decrease (P < 0.02) until P 3. Serum FFA concentrations increased (P < 0.02) by P 2 and remained elevated. Subcutaneous back fat thickness was similar among pigs. Serum IGF-I concentration decreased (P < 0.01) by P 2 in fasted animals compared to fed animals and remained lower through periods 3 and 4. Serum LH and GH concentrations were not effected by fast. Realimentation resulted in a marked increase in serum glucose (P < 0.02), insulin (P < 0.02), serum GH (P < 0.01) concentrations and leptin pulse frequency (P < 0.01). EXP II treatment did not alter serum insulin levels but increased (P < 0.01) plasma glucose concentrations in the 300 mg 2DG group. Serum leptin concentrations were 4.0 +/- 0.1, 2.8 +/- 0.2, and 4.9 +/- 0.2 ng/ml for S, 100 and 300 mg 2DG pigs respectively, prior to treatment and remained unchanged following treatment. Serum IGF-I concentrations were not effected by treatment. The 300 mg dose of 2DG increased (P < 0.0001) mean GH concentrations (2.0 +/- 0.2 ng/ml) compared to S (0.8 +/- 0.2 ng/ml) and 100 mg 2DG (0.7 +/- 0.2 ng/ml). Frequency and amplitude of GH pulses were unaffected. However, number of LH pulses/5 hr were decreased (P < 0.01) by the 300 mg dose of 2DG (1.8 +/- 0.5) compared to S (4.0 +/- 0.4) and the 100 mg dose of 2DG (4.5 +/- 0.5). Mean serum LH concentrations and amplitude of LH pulses were unaffected. These results suggest that acute effects of energy deprivation on LH and GH secretion are independent of changes in serum leptin concentrations.

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.

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.

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.

Development of a DNA Microarray to Detect Antimicrobial Resistance Genes Identified in the National Center for Biotechnology Information Database

To understand the mechanisms and epidemiology of antimicrobial resistance (AR), the genetic elements responsible must be identified. Due to the myriad of possible genes, a high-density genotyping technique is needed for initial screening. To achieve this, AR genes in the National Center for Biotechnology Information GenBank database were identified by their annotations and compiled into a nonredundant list of 775 genes. A DNA microarray was constructed of 70mer oligonucelotide probes designed to detect these genes encoding resistances to aminoglycosides, beta-lactams, chloramphenicols, glycopeptides, heavy metals, lincosamides, macrolides, metronidazoles, polyketides, quaternary ammonium compounds, streptogramins, sulfonamides, tetracyclines, and trimethoprims as well as resistance transfer genes. The microarray was validated with two fully sequenced control strains of Salmonella enterica: Typhimurium LT2 (sensitive) and Typhi CT18 (multidrug resistance [MDR]). All resistance genes encoded on the MDR plasmid, pHCM1, harbored by CT18 were detected in that strain, whereas no resistance genes were detected in LT2. The microarray was also tested with a variety of bacteria, including MDR Salmonella enterica serovars, Escherichia coli, Campylobacter spp., Enterococcus spp., methicillin-resistant Staphylococcus aureus, Listeria spp., and Clostridium difficile. The results presented here demonstrate that a microarray can be designed to detect virtually all AR genes found in the National Center for Biotechnology Information database, thus reducing the subsequent assays necessary to identify specific resistance gene alleles.

Genetic Relatedness of High-Level Aminoglycoside-Resistant Enterococci Isolated from Poultry Carcasses

Abstract Approximately 46% (75/162) or poultry enterococci collected between 1999 and 2000 exhibited high-level resistance to gentamicin (minimum inhibitory concentration [MIC] ≥ 500 μg/ml), kanamycin (MIC ≥ 500 μg/ml), or streptomycin (MIC ≥ 1000 μg/ml). Forty-one percent of the isolates were resistant to kanamycin (n = 67), whereas 23% and 19% were resistant to gentamicin (n = 37) and streptomycin (n = 31), respectively. The predominant species identified was Enterococcus faecium (n = 105), followed by Enterococcus faecalis (n = 40) and Enterococcus durans (n = 8). Using polymerase chain reaction, the isolates were examined for the presence of 10 aminoglycoside resistance genes [ant(6)-Ia, ant(9)-Ia, ant(4′)-Ia, aph(3′)-IIIa, aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id, aac(6′)-Ie-aph(2″)-Ia, and aac(6′)-Ii]. Five aminoglycoside resistance genes were detected, most frequently aac(6′)-Ii and ant(6)-Ia from E. faecium. Seven E. faecalis isolates resistant to gentamicin, kanamycin, or streptomycin were negative for all genes tested, indicating that additional resistance genes may exist. Phylogenetic analysis revealed that the isolates were genetically different with little clonality. These data indicate that enterococci from poultry are diverse and contain potentially unidentified aminoglycoside resistance genes.

Carriage of methicillin‐resistant staphylococci by healthy companion animals in the US

Antimicrobial‐resistant staphylococci have been associated with wounded or ill companion animals, but little is known about the prevalence of resistant staphylococci among healthy animals. In this study, 276 healthy dogs and cats from veterinary clinics were tested for the presence of antimicrobial‐resistant Staphylococcus spp. Isolates were tested for antimicrobial susceptibility and the presence of select resistance genes, and typed using Pulsed‐Field Gel Electrophoresis (PFGE). Staphylococcus aureus and Staphylococcus pseudintermedius were also characterized using multilocus sequence typing (MLST), spa typing and SCCmec typing. Approximately 5% (14/276) of the animals were positive by enrichment for five species of staphylococci [Staph. aureus (n = 11), Staph. pseudintermedius (n = 4), Staphylococcus sciuri (n = 6), Staphylococcus simulans (n = 1) and Staphylococcus warneri (n = 1)]. Seventy‐eight per cent (18/23) of staphylococci were resistant to oxacillin and also multidrug resistant (resistance to ≥ 2 antimicrobials). All Staph. aureus isolates were mecA+ and blaZ+, SCCmec type II, spa type t002, ST5 and clonal using PFGE. Staphylococcus pseudintermedius were SCCmec type IV or V, spa type t06 and ST170; two of the isolates were pvl+. These results suggest that healthy companion animals may be a reservoir of multidrug‐resistant staphylococci, which may be transferred to owners and others who handle companion animals.