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.     

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.     

Comparison of Enterococcus faecium and Enterococcus faecalis Strains Isolated from Water and Clinical Samples: Antimicrobial Susceptibility and Genetic Relationships

Enterococci are part of the normal intestinal flora in a large number of mammals, and these microbes are currently used as indicators of fecal contamination in water and food for human consumption. These organisms are considered one of the primary causes of nosocomial and environmental infections due to their ability to survive in the environment and to their intrinsic resistance to antimicrobials. The aims of this study were to determine the biochemical patterns and antimicrobial susceptibilities of Enterococcus faecalis and E. faecium isolates from clinical samples and from water (groundwater, water from the Xochimilco wetland, and treated water from the Mexico City Metropolitan Area) and to determine the genetic relationships among these isolates. A total of 121 enterococcus strains were studied; 31 and 90 strains were isolated from clinical samples and water (groundwater, water from the Xochimilco wetland, and water for agricultural irrigation), respectively. Identification to the species level was performed using a multiplex PCR assay, and antimicrobial profiles were obtained using a commercial kit. Twenty-eight strains were analyzed by pulsed-field gel electrophoresis (PFGE). E. faecium strains isolated from water showed an atypical biochemical pattern. The clinical isolates showed higher resistance to antibiotics than those from water. Both the enterococci isolated from humans, and those isolated from water showed high genetic diversity according to the PFGE analysis, although some strains seemed to be closely related. In conclusion, enterococci isolated from humans and water are genetically different. However, water represents a potential route of transmission to the community and a source of antimicrobial resistance genes that may be readily transmitted to other, different bacterial species.     

Evidence for the Clustering of Antibacterial Resistance Phenotypes of Enterococci Within Integrated Poultry Companies

From July to December 2006, a panel of 401 enterococci was isolated from carcass rinse samples collected in five poultry processing plants in New Zealand. Agar diffusion assays for nine antibacterial drugs were used to obtain a resistance phenotype for each isolate. Hierarchical clustering techniques and diversity indices showed a high diversity of resistance phenotypes within each plant, with populations of Enterococcus faecalis showing greater heterogeneity than Enterococcus faecium. Bayesian modelling identified three clusters of phenotype patterns within the panel: the E. faecium isolates showed a high probability of containing two distinct clusters, whilst the E. faecalis isolates all grouped together to form the third cluster. The validity of these three clusters was examined using pairwise fixation indices and analysis of variance. Comparing the three clusters to the structure of the participating companies showed that resistance phenotypes for E. faecium isolated from processing plants that were geographically separated but were operated by the same integrated poultry company were more similar than E. faecium isolated from unconnected companies. Company-level management factors, such as the routine use of antibacterial drugs and the genetic line of birds reared, mirrored the structure of these clusters, thus indicating that company-level factors were the dominant selective pressures upon resistance phenotypes across all operating units within these integrated poultry companies.     

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. 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.     

Effect of Conventional and Organic Production Practices on the Prevalence and Antimicrobial Resistance of Campylobacter spp. in Poultry

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.     

Investigation of Antimicrobial Resistance in Escherichia coli and Enterococci Isolated from Tibetan Pigs

Objectives

This study investigated the antimicrobial resistance of Escherichia coli and enterococci isolated from free-ranging Tibetan pigs in Tibet, China, and analyzed the influence of free-ranging husbandry on antimicrobial resistance.

Methods

A total of 232 fecal samples were collected from Tibetan pigs, and the disk diffusion method was used to examine their antimicrobial resistance. Broth microdilution and agar dilution methods were used to determine minimum inhibitory concentrations for antimicrobial agents for which disks were not commercially available.

Results

A total of 129 E. coli isolates and 84 Enterococcus isolates were recovered from the fecal samples. All E. coli isolates were susceptible to amoxicillin/clavulanic acid, and 40.4% were resistant to tetracycline. A small number of isolates were resistant to florfenicol (27.9%), ampicillin (27.9%), sulfamethoxazole/trimethoprim (19.4%), nalidixic acid (19.4%), streptomycin (16.2%) and ceftiofur (10.9%), and very low resistance rates to ciprofloxacin (7.8%), gentamicin (6.9%), and spectinomycin (2.3%) were observed in E. coli. All Enterococcus isolates, including E. faecium, E. faecalis, E. hirae, and E. mundtii, were susceptible to amoxicillin/clavulanic acid and vancomycin, but showed high frequencies of resistance to oxacillin (92.8%), clindamycin (82.1%), tetracycline (64.3%), and erythromycin (48.8%). Resistance rates to florfenicol (17.9%), penicillin (6.0%), ciprofloxacin (3.6%), levofloxacin (1.2%), and ampicillin (1.2%) were low. Only one high-level streptomycin resistant E. faecium isolate and one high-level gentamicin resistant E. faecium isolate were observed. Approximately 20% and 70% of E. coli and Enterococcus isolates, respectively, were defined as multidrug-resistant.

Conclusions

In this study, E. coli and Enterococcus isolated from free-ranging Tibetan pigs showed relatively lower resistance rates than those in other areas of China, where more intensive farming practices are used. These results also revealed that free-range husbandry and absence of antibiotic use could decrease the occurrence of antimicrobial resistance to some extent.     

Antimicrobial Resistance in Escherichia coli Isolates from Swine and Wild Small Mammals in the Proximity of Swine Farms and in Natural Environments in Ontario, Canada

Wild animals not normally exposed to antimicrobial agents can acquire antimicrobial agent-resistant bacteria through contact with humans and domestic animals and through the environment. In this study we assessed the frequency of antimicrobial resistance in generic Escherichia coli isolates from wild small mammals (mice, voles, and shrews) and the effect of their habitat (farm or natural area) on antimicrobial resistance. Additionally, we compared the types and frequency of antimicrobial resistance in E. coli isolates from swine on the same farms from which wild small mammals were collected. Animals residing in the vicinity of farms were five times more likely to carry E. coli isolates with tetracycline resistance determinants than animals living in natural areas; resistance to tetracycline was also the most frequently observed resistance in isolates recovered from swine (83%). Our results suggest that E. coli isolates from wild small mammals living on farms have higher rates of resistance and are more frequently multiresistant than E. coli isolates from environments, such as natural areas, that are less impacted by human and agricultural activities. No Salmonella isolates were recovered from any of the wild small mammal feces. This study suggests that close proximity to food animal agriculture increases the likelihood that E. coli isolates from wild animals are resistant to some antimicrobials, possibly due to exposure to resistant E. coli isolates from livestock, to the resistance genes of these isolates, or to antimicrobials through contact with animal feed.     

Prevalence, distribution and antibiotic resistance pattern among enterococci species in two traditional fermented dairy foods

The prevalence, distributions and antibiotic resistance pattern among enterococci species were determined. A total of 30 samples of Nigerian traditional fermented dairy food were positive to presence of enterococci, with viable counts of 4.17 log CFU/g in nunu, lower than 4.55 log CFU/g observed in wara samples. Twenty-five representative strains were characterized by a combination of phenotypic and genomic typing based on 16S rRNA gene and multi-locus sequencing analysis (MLSA) of RNA polymerase A (rpoA) and phenylanaline synthase (pheS) genes sequencing; these strains were identified as Enterococcus faecium (84 %) and Enterococcus faecalis (16 %). All the 95 enterococci isolated from wara and nunu samples were alpha haemolytic with multi-drug resistance to 10 antimicrobials regardless of class. Four strains were sensitive to chloramphenicol (30 μg) while 33.7 % of the total isolates were resistant to vancomycin from 5 μg. This information will enhance understanding of Enterococcus drug resistance and distribution in traditional fermented foods to support safety and guarantee quality of traditional foods in West Africa.     

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.     

Antibiotic Resistance of Enterococci in American Bison (Bison bison) from a Nature Preserve Compared to That of Enterococci in Pastured Cattle

Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by Enterococcus casseliflavus in bison (62.4%), while Enterococcus hirae was the most common isolate from cattle (39.7%). Enterococcus faecalis was the second most common species isolated from bison (16%). In cattle, E. faecalis and Enterococcus faecium were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by tetO and ermB, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10⁻⁶ per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.     

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.     

Phenotypic and genotypic characterization of bacteriocins in clinical <Emphasis Type="Italic">enterococcal</Emphasis> isolates of Tunisia

The presence of bacteriocin structural genes (entA, entB, entP, entQ, entAS-48, entL50A/B, bac31, and cylL) encoding different bacteriocins (enterocin A, enterocin B, enterocin P, enterocin Q, enterocin AS-48, enterocin L50A/B, bacteriocin 31 and cytolysin L, respectively), and the production of bacteriocin activity were analysed in 139 E. faecalis and 41 E. faecium clinical isolates of Tunisia. Forty-eight of 139 E. faecalis isolates (34%) and 7 of 41 of E. faecium isolates (17%) were bacteriocin producers. Sixty-two per cent of the bacteriocin-producing enterococci showed inhibitory activity against L. monocytogenes. Different combinations of entA, entB, entP, and entL50A/B genes were detected among the seven bacteriocin-producer E. faecium isolates, and more that one gene were identified in all the isolates. The entA gene was associated in most of the cases with entB gene in E. faecium isolates. Cyl _LS were the unique genes detected among E. faecalis (in 24 of 48 bacteriocin-producer isolates, 50%). A β-hemolytic activity was demonstrated in 19 of the 24 cyl _LS -positive E. faecalis isolates (79%), this activity being negative in the remaining five isolates. The presence of different bacteriocin structural genes and the production of antimicrobial activities seems to be a common trait of clinical enterococci.     

Prevalence, Persistence, and Molecular Characterization of Glycopeptide-Resistant Enterococci in Norwegian Poultry and Poultry Farmers 3 to 8 Years after the Ban on Avoparcin

Environmental reservoirs of glycopeptide-resistant enterococci (GRE) in Norway have been linked to former growth promoting use of the glycopeptide avoparcin in poultry production. We have examined the prevalence of fecal GRE in poultry and poultry farmers 3 to 8 years after the Norwegian avoparcin ban in 1995 and performed molecular analyses of the GRE population. Fecal samples from poultry farmers and their flocks on 29 previously avoparcin-exposed farms were collected on five occasions during the study period (1998 to 2003). All flocks (100%) were GRE positive in 1998. Throughout the study period, 78.5% of the poultry samples were GRE positive. Glycopeptide-resistant Enterococcus faecium (GREF) was isolated from 27.6% of the farmer samples in 1998 and from 27.8% of the samples collected between 1998 and 2003. The prevalence of fecal GRE in poultry declined significantly during the study period, but prevalence in samples from the farmers did not decline. PCR analysis revealed a specific Tn1546-plasmid junction fragment in 93.9% of E. faecium isolates. A putative postsegregation killing (PSK) system linked to Tn1546 was detected in 97.1% of the isolates examined. Multilocus sequence typing of glycopeptide-susceptible (n = 10) and -resistant (n = 10) E. faecium isolates from humans (n = 10) and poultry (n = 10) on two farms displayed 17 different sequence types. The study confirms the continuing persistence of a widespread common plasmid-mediated vanA-pRE25-PSK element within a heterogeneous GRE population on Norwegian poultry farms 8 years after the avoparcin ban. Moreover, it suggests an important role of PSK systems in the maintenance of antimicrobial resistance determinants in reservoirs without apparent antimicrobial selection.     

Effect of conventional and organic production practices on the prevalence and antimicrobial resistance of Campylobacter spp. in poultry

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.     

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.     

Stephania suberosa Forman extract synergistically inhibits ampicillin- and vancomycin-resistant Enterococcus faecium

Increasing antibiotic resistance in enterococci is among the most serious public health problems worldwide. The new naturally occurring antibacterial agents were explored. This study, therefore, investigated the antibacterial potential of Stephania suberosa extract (SSE) and its synergism with ampicillin (AMP) or vancomycin (VAN) against AMP- and VAN-resistant Enterococcus faecium. Disc diffusion assay revealed that SSE inhibited E. faecium DMST 12829, 12852, 12970, and a reference strain of Enterococcus faecalis ATCC 29,212 in a dose-dependent manner. The minimum inhibitory concentration (MIC) of SSE against all E. faecium isolates was 0.5 mg/mL. E. faecium DMST 12,829 and 12,852 were highly resistant to AMP, as indicated by high MIC values, and E. faecium DMST 12,829 and 12,970 were resistant to VAN. Enterococcus spp. were killed by SSE at the minimum bactericidal concentrations (MBCs) ranging from 0.5 to 4 mg/mL. Checkerboard determination showed that SSE plus AMP and SSE plus VAN combinations exhibited synergistic interaction against E. faecium isolates. The killing curve assay of E. faecium isolates confirmed the antibacterial and synergistic activities of combined agents by dramatically reducing the viable counts compared to a single agent. Scanning electron microscope elucidated the cell damage and abnormal cell division. Enterococcal proteases were also inhibited by SSE. These findings support that SSE could reverse the activity of AMP and VAN. Moreover, it can synergistically inhibit AMP- and VAN-resistant E. faecium. Our combined agents could be attractive candidates for developing new combinatorial agents to resurrect the efficacy of antibiotics for treating AMP- and VAN-resistant E. faecium infections.     

Distribution of antimicrobial‐resistant lactic acid bacteria in natural cheese in Japan

To determine and compare the extent of contamination caused by antimicrobial‐resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P = 0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm‐made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm‐made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to > 512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial‐resistant LAB in imported and Japanese farm‐made cheeses on the Japanese market, but not in Japanese commercial cheeses.     

Predominance of vanA Genotype among Vancomycin-Resistant Enterococcus Isolates from Poultry and Swine in Costa Rica

The use of avoparcin as a growth promoter is considered to have selected for vancomycin-resistant enterococci (VRE). In Costa Rica, the use of avoparcin for poultry and swine was intensive until the product was withdrawn from the market in 2000. We evaluated the presence of VRE in poultry, swine, and cattle fecal samples obtained during 1998 and 1999. A total of 185 VRE isolates were recovered from 116 out of 893 samples. Enterococcus faecium was the most frequently isolated species (50.8%), being predominant among poultry (71.6%) and swine (37.7%) isolates, but it was not recovered from the bovine samples. The second-most-frequently-isolated species from poultry and swine, respectively, were E. durans (23.2%) and E. faecalis (21.7%). E. casseliflavus was the only species obtained from bovine samples, but it was not found among the avian isolates. An evident predominance of the vanA determinant among vancomycin-resistant enterococcal species from poultry and swine, but not from cattle, was observed and was similar to the situation in European countries before avoparcin was forbidden. The diversity of the vanA determinant in the isolates was assessed by detection of the IS1251 insertion in the vanSH intergenic region and of the IS1476 insertion in the vanXY intergenic region. However, in none of the 154 vanA⁺ isolates recovered in this study were those insertions detected.     

Ecology of Antibiotic Resistance Genes: Characterization of Enterococci from Houseflies Collected in Food Settings

In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 × 10³ CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.