Multiple-Antibiotic Resistance of Enterococcus spp. Isolated from Commercial Poultry Production Environments

The potential impact of food animals in the production environment on the bacterial population as a result of antimicrobial drug use for growth enhancement continues to be a cause for concern. Enterococci from 82 farms within a poultry production region on the eastern seaboard were isolated to establish a baseline of susceptibility profiles for a number of antimicrobials used in production as well as clinical environments. Of the 541 isolates recovered, Enterococcus faecalis (53%) and E. faecium (31%) were the predominant species, while multiresistant antimicrobial phenotypes were observed among all species. The prevalence of resistance among isolates of E. faecalis was comparatively higher among lincosamide, macrolide, and tetracycline antimicrobials, while isolates of E. faecium were observed to be more frequently resistant to fluoroquinolones and penicillins. Notably, 63% of the E. faecium isolates were resistant to the streptogramin quinupristin-dalfopristin, while high-level gentamicin resistance was observed only among the E. faecalis population, of which 7% of the isolates were resistant. The primary observations are that enterococci can be frequently isolated from the poultry production environment and can be multiresistant to antimicrobials used in human medicine. The high frequency with which resistant enterococci are isolated from this environment suggests that these organisms might be useful as sentinels to monitor the development of resistance resulting from the usage of antimicrobial agents in animal production.     

Genetic Variability of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates from Humans,Chickens, and Pigs in Malaysia

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.     

Persistence of Vancomycin-Resistant Enterococci in New Zealand Broilers after Discontinuation of Avoparcin Use

Large amounts of tylosin, zinc-bacitracin, and avilamycin are currently used as prophylactics in New Zealand broiler production. Avoparcin was also used from 1977 to 2000. A total of 382 enterococci were isolated from 213 fecal samples (147 individual poultry farms) using enrichment broths plated on m-Enterococcus agar lacking antimicrobials. These isolates were then examined to determine the prevalence of antimicrobial resistance. Of the 382 isolates, 5.8% (22 isolates) were resistant to vancomycin, and 64.7% were resistant to erythromycin. The bacitracin MIC was ≥256 μg/ml for 98.7% of isolates, and the avilamycin MIC was ≥8 μg/ml for 14.9% of isolates. No resistance to ampicillin or gentamicin was detected. Of the 22 vancomycin-resistant enterococci (VRE) isolates, 18 (81.8%) were Enterococcus faecalis, 3 were Enterococcus faecium, and 1 was Enterococcus durans. However, when the 213 fecal enrichment broths were plated on m-Enterococcus agar containing vancomycin, 86 VRE were recovered; 66% of these isolates were E. faecium and the remainder were E. faecalis. Vancomycin-resistant E. faecium isolates were found to have heterogenous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, whereas the PFGE patterns of vancomycin-resistant E. faecalis isolates were identical or closely related, suggesting that this VRE clone is widespread throughout New Zealand. These data demonstrate that vancomycin-resistant E. faecalis persists in the absence and presence of vancomycin-selective pressure, thus explaining the dominance of this VRE clone even in the absence of avoparcin.     

A Comparison of BOX-PCR and Pulsed-Field Gel Electrophoresis to Determine Genetic Relatedness of Enterococci from Different Environments

Genetic relatedness of enterococci from poultry litter to enterococci from nearby surface water and groundwater in the Lower Fraser Valley regions of British Columbia, Canada was determined. A new automated BOX-PCR and Pulsed-Field Gel Electrophoresis (PFGE) were used to subtype enterococcal isolates from broiler and layer litter and surface and groundwater. All surface water samples (n = 12) were positive for enterococci, as were 11% (3/28) of groundwater samples. Enterococcus faecium (n = 90) was isolated from all sources, while Enterococcus faecalis (n = 59) was isolated from all sources except layer litter. The majority of E. faecalis originated from broiler litter (28/59; 47.5%) while the majority of E. faecium were isolated from layer litter (29/90; 32.2%). E. faecalis grouped primarily by source using BOX-PCR. Isolates from water samples were dispersed more frequently among PFGE groups containing isolates from poultry litter. E. faecium strains were genetically diverse as overall clustering was independent of source by both molecular methods. Subgroups of E. faecium isolates based upon source (layer litter) were present in BOX-PCR groups. Three individual E. faecalis groups and two individual E. faecium groups were 100% similar using BOX-PCR; only one instance of 100% similarity among isolates using PFGE was observed. Although enterococci from litter and water sources were grouped together using BOX-PCR and PFGE, isolates originating from water could not be definitively identified as originating from poultry litter. Automation of BOX-PCR amplicon separation and visualization increased the reproducibility and standardization of subtyping using this procedure.     

Occurrence of multidrug-resistant Enterococcus faecium isolated from environmental samples

Enterococcus species are present in the microbiota of humans and animals and have also been described in the environment. Among the species, Enterococcus faecium is one of the main pathogens associated with nosocomial infections worldwide. Enterococcus faecium isolates resistant to different classes of antimicrobials have been increasingly reported, including multidrug-resistant (MDR) isolates in environmental sources, which is worrying. Therefore, this study aimed to characterize E. faecium isolates obtained from soil and water samples regarding antimicrobial resistance and virulence determinants. A total 40 E. faecium isolates were recovered from 171 environmental samples. All isolates were classified as MDR, highlighting the resistance to the fluoroquinolones class, linezolid and vancomycin. Furthermore, high-level aminoglycoside resistance and high-level ciprofloxacin resistance were detected in some isolates. Several clinically relevant antimicrobial resistance genes were found, including vanC1, ermB, ermC, mefAE, tetM, tetL, ant(6′)-Ia, ant(4′)-Ia, aph(3′)-IIIa and aac(6′)-Ie-aph(2″)-Ia. Three virulence genes were detected among the MDR E. faecium isolates, such as esp, gelE and ace. The results of this study contribute to a better understanding of MDR E. faecium isolates carrying antimicrobial resistance and virulence genes in environmental sources and report for the first time in the world the presence of vanC1-producing E. faecium isolated from soil.     

Antimicrobial Resistance of Enterococcus Species Isolated from Produce

The purpose of this study was to characterize the antibiotic resistance profiles of Enterococcus species isolated from fresh produce harvested in the southwestern United States. Among the 185 Enterococcus isolates obtained, 97 (52%) were Enterococcus faecium, 38 (21%) were Enterococcus faecalis, and 50 (27%) were other Enterococcus species. Of human clinical importance, E. faecium strains had a much higher prevalence of resistance to ciprofloxacin, tetracycline, and nitrofurantoin than E. faecalis. E. faecalis strains had a low prevalence of resistance to antibiotics used to treat E. faecalis infections of both clinical and of agricultural relevance, excluding its intrinsic resistance patterns. Thirty-four percent of the isolates had multiple-drug-resistance patterns, excluding intrinsic resistance. Data on the prevalence and types of antibiotic resistance in Enterococcus species isolated from fresh produce may be used to describe baseline antibiotic susceptibility profiles associated with Enterococcus spp. isolated from the environment. The data collected may also help elucidate the role of foods in the transmission of antibiotic-resistant strains to human populations.     

Characterization of vanA-type Enterococcus faecium isolates from urban and hospital wastewater and pigs

Aims:  In this study we analysed urban, hospital wastewater and pig faeces samples to investigate the presence of vancomycin-resistant Enterococcus faecium strains (VREF) and to determine potential links among the strains originating from the above sources and VREF strains causing clinical infections.
Methods and Results:  Urban, hospital wastewater and pig faeces exhibited high VREF prevalence of 52%, 87% and 85%, respectively. Pulsed field gel electrophoresis (PFGE) clustering of VREF genotypes as well as discriminant analysis of antibiotic resistance patterns of VREF strains revealed their source specificity while strains isolated from hospitalized humans were genetically distinct.
Conclusions:  PFGE genotypes and antimicrobial resistance patterns in VREF isolates are distinguishable by each sample origin. The observed high genetic diversity of VREF suggests horizontal transfer of genetic elements among VREF. Phenotypic and genotypic data indicate that VREF isolates of hospital-treated wastewater might pass to the urban wastewater system.
Significance and Impact of the Study:  This study provides information to understand the origin and the mechanism of circulation of vancomycin resistance in food animals and wastewater treatment plants for minimizing the risk of transmission of VRE in human population.     

Molecular determination of van genes among clinical isolates of enterococci at a hospital setting

Vancomycin-resistant enterococci (VRE) poses a formidable challenge to public health due to its inherent resistance to multiple antibiotics coupled with the ability to transfer genetic determinants to dangerous pathogens like Methicillin-resistant Staphylococcus aureus (MRSA). The purpose of this study was to investigate the incidence of vancomycin resistance in enterococci among clinical isolates at a tertiary care military hospital in the eastern region of Saudi Arabia and to detect van genes using multiplex-PCR. Overall, 246 isolates of enterococci were collected from various clinical specimens. The isolates were identified, and antimicrobial susceptibility testing was done using the Vitek 2 system. Multiplex PCR was performed on the VRE isolates, thus identified to determine the van genes harbored. A total of 15 VRE were identified, of which 14 (93.3%) were Enterococcus faecium, and 1(6.7%) was Enterococcus casseliflavus with intrinsic vanC resistance. Of the 14 vancomycin-resistant Enterococcus faecium, 8 (57.1%) harbored vanB genes, while 6 (42.8%) harbored vanA genes. All the VRE were susceptible to linezolid and tigecycline. Our study detected a low prevalence (6.1%) of VRE among clinical isolates of enterococci and that the vanB gene predominates in such strains. Susceptibility profiles indicated that linezolid and tigecycline are still effective against these multidrug-resistant pathogens. Pus specimens yielded the highest percentage (53.3%) of isolates from which VRE was obtained, and this finding is novel among studies done in Saudi Arabia.     

Multilocus sequence typing and antimicrobial resistance in Enterococcus faecium isolates from fresh produce

The purpose of the present study was to determine the relatedness of Enterococcus faecium isolates from fresh produce to E. faecium strains from other sources by using multi-locus sequence typing (MLST) and to determine the antimicrobial resistance of the isolates. MLST analysis of 22 E. faecium isolates from fresh produce revealed 7 different sequence types (ST 22, ST 26, ST 43, ST 46, ST 55, ST 94 and ST 296). Most isolates belonged to ST 296 (40.9 %), followed by ST 94 (27.3 %). All isolates were sensitive to vancomycin and to imipenem, and only one was resistant to ampicillin (MIC 32 mg/l). However, all were resistant to cefotaxime and ceftazidine. E. faecium isolates from fresh produce were inhibited by quaternary compounds (benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide), biguanides (chlorhexidine), polyguanides [poly-(hexamethylene guanidinium) hydrochloride], bisphenols (triclosan, hexachlorophene) and biocidal solutions of P3 oxonia and P3 topax 66. Didecyldimethylammonium bromide and triclosan were the least effective biocides in growth inhibition, while hexadecylpyridinium chloride was the most effective. Results from MLST typing and antibiotic resistance suggest that the studied E. faecium isolates from fresh produce are not related to the clinically-relevant clonal complex CC17.     

Prevalence of antimicrobial resistance and molecular characterization of tetracycline resistance mediated by tet(M) and tet(L) genes in Enterococcus spp. isolated from food in Southern Brazil

Enterococcus spp. are opportunistic pathogens that are widely distributed in the natural environment. Two remarkable characteristics of enterococci is their intrinsic resistance against several of the antimicrobial agents routinely prescribed in the treatment of Gram-positive cocci, and their enormous capacity to acquire different genetic markers by conjugation. The aim of this study was to evaluate the prevalence of antimicrobial resistance and the frequency of tet(M) and tet(L) genes in 112 Enterococcus spp. strains isolated from food. Fifty-two strains (64%) of Enterococcus faecalis, 10 (55%) of Enterococcus faecium, 2 (66%) of Enterococcus casseliflavus and 3 (42%) of Enterococcus gallinarum showed multidrug resistance. Tet(M) gene associated with or without the tet(L) gene was the most prevalent genotype found in food. Nine erythromycin-resistant and tetracycline-susceptible enterococci strains harbor silencing tet(M) or tet(L) genes were present in our investigation. In conclusion, antibiotic-resistant enterococci current in food may act as a reservoir of resistant strains creating a potential route of genes transference by horizontal gene transfer.     

Dogs Are a Reservoir of Ampicillin-Resistant Enterococcus faecium Lineages Associated with Human Infections

Ampicillin resistance is a marker for hospital-associated Enterococcus faecium. Feces from 208 dogs were selectively screened for the occurrence of ampicillin-resistant E. faecium (AREF). AREF was detected in 42 (23%) of 183 dogs screened in a cross-sectional study in the United Kingdom and in 19 (76%) of 25 dogs studied longitudinally in Denmark. AREF carriage was intermittent in all dogs studied longitudinally. Multilocus sequence typing of 63 canine AREF isolates revealed the presence of 13 distinct sequence types. Approximately 76% of the isolates belonged to hospital-adapted clonal complex 17 (CC17), including those of sequence types ST-78 and ST-192, which are widespread in European and Asian hospitals. Longitudinal screening of 18 healthy humans living in contact with 13 of the dogs under study resulted in the identification of a single, intermittent CC17 carrier. This person carried one of the sequence types (ST-78) recovered from his dog. Based on PCR and Southern hybridization analyses, the putative virulence gene cluster from orf903 to orf907 was widespread in canine AREF isolates (present in 97%), whereas orf2351 (present in 26% of isolates) and orf2430 (present in 31%) were strongly associated with CC17-related sequence types (P < 0.05). Surprisingly, esp and hyl were not detected in any of the isolates. The antimicrobial resistance profiles of canine AREF isolates generally differed from those previously described for clinical human isolates. The results indicate that dogs are frequent carriers of CC17-related lineages and may play a role in the spread of this nosocomial pathogen. The distinctive virulence and antimicrobial resistance profiles observed among canine AREF isolates raise interesting questions about the origin and evolution of the strains causing human infections.Enterococci are opportunistic pathogens and form part of the normal gastrointestinal flora in humans and animals. Over the last two decades, nosocomial infections caused by enterococci have emerged and their incidence has increased rapidly, first in the United States and recently in Europe (25, 26, 29). Although Enterococcus faecalis is the causative agent in most enterococcal infections, a shift toward infections caused by multidrug-resistant E. faecium has been noted in the last years, and presently, up to one-third of enterococcal infections in some countries are attributed to this species (17). This shift may be explained by changes in the patterns of antimicrobial usage, which may have resulted in the emergence of a distinct genogroup of hospital-associated ampicillin-resistant E. faecium (AREF) strains, currently labeled clonal complex 17 (CC17) (33). CC17 isolates are characterized by resistance to ampicillin and fluoroquinolones, as well as by the presence in most isolates of putative virulence genes encoding the enterococcal surface protein (esp) and hyaluronidase (hyl) and five recently described open reading frames (ORFs; orf903, orf904.5, orf906.7, orf2351, and orf2430) encoding LPXTG surface proteins, which are found less frequently among other E. faecium lineages (15, 20, 27).Based on the results of multilocus sequence typing (MLST) (28) and amplified fragment length polymorphism analysis (34), E. faecium isolates of animal origin seem to be host specific and generally unrelated to human lineages of clinical importance. Prior to this study, AREF CC17 strains have been isolated only sporadically from animals, including pigs (2, 10) and more recently dogs (8). Following these unexpected findings, the present study was designed to investigate the prevalence and shedding patterns of AREF in dogs. A cross-sectional study and two longitudinal studies involving a total of 208 dogs and 479 canine fecal samples were conducted in the United Kingdom and in Denmark, respectively. Canine isolates were characterized by MLST, antimicrobial susceptibility testing, and putative virulence gene profiling to assess the genetic relationship between human and canine AREF strains.     

Changes in Antimicrobial Susceptibility of Native Enterococcus faecium in Chickens Fed Virginiamycin

The extent of transfer of antimicrobial resistance from agricultural environments to humans is controversial. To assess the potential hazard posed by streptogramin use in food animals, this study evaluated the effect of virginiamycin exposure on antimicrobial resistance in Enterococcus faecium recovered from treated broilers. Four consecutive broiler feeding trials were conducted using animals raised on common litter. In the first three trials, one group of birds was fed virginiamycin continuously in feed at 20 g/ton, and a second group served as the nontreated control. In the fourth trial, antimicrobial-free feed was given to both groups. Fecal samples were cultured 1 day after chickens hatched and then at 1, 3, 5, and 7 weeks of age. Isolates from each time point were tested for susceptibility to a panel of different antimicrobials. Quinupristin/dalfopristin-resistant E. faecium appeared after 5 weeks of treatment in trial 1 and within 7 days of trials 2 to 4. Following removal of virginiamycin in trial 4, no resistant isolates were detected after 5 weeks. PCR failed to detect vat, vgb, or erm(B) in any of the streptogramin-resistant E. faecium isolates, whereas the msr(C) gene was detected in 97% of resistant isolates. In an experimental setting using broiler chickens, continuous virginiamycin exposure was required to maintain a stable streptogramin-resistant population of E. faecium in the animals. The bases of resistance could not be explained by known genetic determinants.     

Comparison of four methods, including semi-automated rep-PCR, for the typing of vancomycin-resistant Enterococcus faecium

We have assessed the performance of semi-automated rep-PCR (Diversilab®) and multilocus sequence typing (MLST) in comparison to pulsed-field gel electrophoresis (PFGE) for typing a collection of 29 epidemiologically characterized vancomycin-resistant Enterococcus faecium (VRE). Sixteen strains that harbored the Tn1546 element were typed by PCR mapping. The discriminative power of the typing methods was calculated by the Simpson's index of diversity, and the concordance between methods was evaluated by the Kendall's coefficient of concordance. Semi-automated rep-PCR appeared as discriminative as PFGE and was further compared with PFGE for typing 67 VRE isolated during a hospital outbreak. Rep-PCR appeared to be more discriminative than PFGE for this second set of strains. Reproducibility of DiversiLab® was also tested against 35 selected isolates. Only three showed less than 97% similarity, indicating high reproducibility at this level of discrimination. In conclusion, semi-automated rep-PCR is a useful tool for rapid screening of VRE isolates during an outbreak, although cost of the system may be limiting for routine implementation. PFGE, which remains the reference method, should be used for confirmation and evaluation of the genetic relatedness of epidemic isolates.     

Application of PFGE to source tracking of faecal pollution in coastal recreation area: a case study in Aoshima Beach, Japan

Aims: The development of a microbial source tracking (MST) method is strongly desired to ensure public health and bacteriological safety in coastal recreation areas. We try to specify the source of faecal pollution by applying pulsed‐field gel electrophoresis (PFGE) to the study of the aquatic environment on Aoshima Beach, Japan. Methods and Results: Enterococcus faecium, an enterococcus, was used as a faecal indicator bacterium in this study. Enterococcus faecium strains were isolated and identified from each water sample collected from Aoshima Beach and five rivers (Oyodo, Kiyotake, Kaeda, Chifuku and Tsukunami Rivers) that might be potential sources of faecal pollution. Enterococcus faecium strains collected from water samples were analysed using PFGE. The similarities of all the PFGE types of the Ent. faecium strains were compared using dendrogram analysis. The PFGE types of the strains isolated from Aoshima Beach showed a high similarity to those of the strains isolated from the Oyodo River at a 0·9 similarity level. It was suggested that the Oyodo River is the source of faecal pollution on Aoshima Beach. Conclusions: The PFGE analysis using enterococci is a potential tool for the MST of faecal indicator bacteria that can be applied to the study of the coastal environment. Significance and Impact of the Study: This is one of the studies that PFGE was applied to the coastal environment. The approach using PFGE could estimate the river that is source of faecal pollution in Aoshima Beach. By applying PFGE as a tool of MST method, detailed information of faecal pollution in coastal area can be provided.     

Antimicrobial resistance of Enterococcus sp. isolated from the intestinal tract of patients from a university hospital in Brazil

This study reports the results about antimicrobial resistance of Enterococcus spp. isolated from intestinal tract of patients from a university hospital in Brazil. The identification of strains at species level was performed by conventional biochemical tests, API 20 Strep (bioMérieux), and polymerase chain reaction assay. The species distribution was E. faecium (34%), followed by E. faecalis (33%), E. gallinarum (23.7%), E. casseliflavus (5.2%), E. avium (1%), and E. hirae (1%). Intrinsic resistance to vancomycin characterized by presence of vanC genes was found in E. gallinarum and E. casseliflavus. The high prevalence of VanC phenotype enterococci is very important because these species have been reported as causing a wide variety of infections. Vancomycin-resistant E. faecium or E. faecalis were not found and no one isolate of these species was a beta-lactamase producer. Thirteen clinical isolates of enterococci (13.4%) showed multiresistance patterns, which were defined by resistance to three classes of antibiotics plus resistance to at least one aminoglycoside (gentamicin and/or streptomycin). The resistance to several antimicrobials shown by enterococcal strains obtained in this study is of concern because of the decrease in the therapeutic options for treatment of infections caused by enterococci.     

Molecular Analysis of Human, Porcine, and Poultry Enterococcus faecium Isolates and Their erm(B) Genes

Fifty-nine erm(B)-positive Enterococcus faecium strains isolated from pigs, broilers, and humans were typed using multilocus sequence typing (MLST), and the coding sequence of the erm(B) gene was determined. Identical erm(B) gene sequences were detected in genetically unrelated isolates. Furthermore, genetically indistinguishable strains were found to contain different erm(B) alleles. This may suggest that horizontal exchange of the erm(B) gene between animal and human E. faecium strains or the existence of a common reservoir of erm(B) genes might be more important than direct transmission of resistant strains.     

Partial Characterization of Bacteriocins Produced by Two New Enterococcus faecium Isolated from Human Intestine

This study aimed at characterizing two novel bacteriocin-producing enterococcal strains isolated from human intestine. A total of 200 lactic acid bacteria were isolated from a woman stool sample. Two of them were selected for characterization due to their high antimicrobial activity against five strains of Listeria monocytogenes. The selected bacteria were identified as two different strains of Enterococcus faecium and designated MT 104 and MT 162. The bacteriocins produced by MT 104 and MT 162 were stable at different pH ranging from 2 to 11 and were active after different treatments such as heat, enzymes, detergents, and γ-irradiation. The two isolated strains exhibited some probiotic properties such as survival in simulated gastric fluid and intestinal fluid, lack of expression of bile salt hydrolase or hemolytic activity, adhesion to Caco-2 cells efficiently, and sensitivity to clinical antimicrobial agents. Thus, the two isolated strains of E. faecium could become new probiotic bacteria and their bacteriocins could be used for controlling L. monocytogenes in combination with irradiation for food preservation.     

A Comparison of Non-Typhoidal Salmonella from Humans and Food Animals Using Pulsed-Field Gel Electrophoresis and Antimicrobial Susceptibility Patterns

Salmonellosis is one of the most important foodborne diseases affecting humans. To characterize the relationship between Salmonella causing human infections and their food animal reservoirs, we compared pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility patterns of non-typhoidal Salmonella isolated from ill humans in Pennsylvania and from food animals before retail. Human clinical isolates were received from 2005 through 2011 during routine public health operations in Pennsylvania. Isolates from cattle, chickens, swine and turkeys were recovered during the same period from federally inspected slaughter and processing facilities in the northeastern United States. We found that subtyping Salmonella isolates by PFGE revealed differences in antimicrobial susceptibility patterns and, for human Salmonella, differences in sources and invasiveness that were not evident from serotyping alone. Sixteen of the 20 most common human Salmonella PFGE patterns were identified in Salmonella recovered from food animals. The most common human Salmonella PFGE pattern, Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS), was associated with more cases of invasive salmonellosis than all other patterns. In food animals, this pattern was almost exclusively (99%) found in Salmonella recovered from chickens and was present in poultry meat in every year of the study. Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS) was associated with susceptibility to all antimicrobial agents tested in 94.7% of human and 97.2% of food animal Salmonella isolates. In contrast, multidrug resistance (resistance to three or more classes of antimicrobial agents) was observed in five PFGE patterns. Typhimurium patterns JPXX01.0003 (JPXX01.0003 ARS) and JPXX01.0018 (JPXX01.0002 ARS), considered together, were associated with resistance to five or more classes of antimicrobial agents: ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (ACSSuT), in 92% of human and 80% of food animal Salmonella isolates. The information from our study can assist in source attribution, outbreak investigations, and tailoring of interventions to maximize their impact on prevention.     

Persistence of Animal and Human Glycopeptide-Resistant Enterococci on Two Norwegian Poultry Farms Formerly Exposed to Avoparcin Is Associated with a Widespread Plasmid-Mediated vanA Element within a Polyclonal Enterococcus faecium Population

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that “plasmid addiction systems” may contribute to the persistence of GREF in nonselective environments.     

VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants

VanA-type human (n = 69), animal (n = 49), and food (n = 36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n = 4) and M49 (n = 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.