Enterococcus faecalis infection in root canals – host‐derived or exogenous source?

Aims: Enterococcus faecalis is associated with a significant number of refractory endodontic infections. Previous studies report a prevalence of Ent. faecalis ranging from 24% up to 77% in teeth with failed endodontic treatment. The origin of the micro‐organism remains unclear, as enterococci do not belong to the normal oral microflora. The aim of this study was to determine whether these enterococci were of endogenous or exogenous origin. Methods and Results: Fifty consecutive patients with apical periodontitis in need of endodontic orthograde re‐treatment were included. Samples were collected from root canals, saliva and faeces and subjected to microbiological culturing. The genetic relationship between Ent. faecalis from root canals and isolates from the different host sources was determined using pulsed‐field gel electrophoresis. In 16% (8/50) of the patients, enterococci were collected from the root canal samples. The genetic analysis showed that the isolates from the root canals were not related to those from the normal gastrointestinal microflora. None of these patients had enterococci in their saliva samples. Conclusions: Endodontic infections with Ent. faecalis are probably not derived from the patient’s own normal microflora, which indicates that these infections ent. faecalis are of exogenous origin. Significance and Impact of the Study: This is the first study to genetically compare endodontic infectious Ent. faecalis isolates with isolates from the hosts’ own normal microflora.     

Effect of Mexican propolis extracts from Apis mellifera on Candida albicans in vitro growth

Propolis is a resinous substance collected by bees (Apis mellifera) from different trees and bushes. Due to its antifungal, antibacterial, antiviral and antiparasitic properties, it has continued to be very popular throughout the time showing variable activity depending on its geographical origin. In Mexico, information about this product is very limited. The aim of this work was to evaluate the antifungal activity of four propolis ethanolic extracts from three different Mexican states, and four commercial extracts on Candida albicans growth. A reference strain (ATCC 10231) and 36 clinical isolates of C. albicans were used. The Minimal Inhibitory Concentration (MIC) was determined by the dilution on agar method. Growth curves on Sabouraud Dextrose broth with and without different propolis ethanolic extracts concentrations were performed. In addition, whether the effect was fungistatic or fungicide was determined. The propolis ethanolic extract obtained from Cuautitlán Izcalli, State of Mexico, showed the best biological activity, inhibiting 94.4% from the clinical isolates at 0.8 mg/ml; the reference strain was inhibited at 0.6 mg/ml. The propolis effect was fungistatic in low concentrations and fungicide in concentrations higher to MIC. The Mexican propolis ethanolic extract could be further investigated for its alternative use for the treatment of some C. albicans infections.     

Host species-specific metabolic fingerprint database for enterococci and Escherichia coli and its application to identify sources of fecal contamination in surface waters

A metabolic fingerprint database of enterococci and Escherichia coli from 10 host groups of animals was developed to trace the sources of fecal contamination in surface waters. In all, 526 biochemical phenotypes (BPTs) of enterococci and 530 E. coli BPTs were obtained from 4,057 enterococci and 3,728 E. coli isolates tested. Of these, 231 Enterococcus BPTs and 257 E. coli BPTs were found in multiple host groups. The remaining 295 Enterococcus BPTs and 273 E. coli BPTs were unique to individual host groups. The database was used to trace the sources of fecal contamination in a local creek. The mean diversities (Di) of enterococci (Di = 0.76 +/- 0.05) and E. coli (Di = 0.88 +/- 0.04) were high (maximum 1) in water samples, indicating diverse sources of fecal contamination. Overall, 71% of BPTs of enterococci and 67% of E. coli BPTs from water samples were identified as human and animal sources. Altogether, 248 Enterococcus BPTs and 282 E. coli BPTs were found in water samples. Among enterococci, 26 (10%) BPTs were identical to those of humans and 152 BPTs (61%) were identical to those of animals (animal BPTs). Among E. coli isolates, 36 (13%) BPTs were identical to those of humans and 151 (54%) BPTs were identical to those of animals. Of the animal BPTs, 101 (66%) Enterococcus BPTs and 93 (62%) E. coli BPTs were also unique to individual animal groups. On the basis of these unique Enterococcus BPTs, chickens contributed 14% of contamination, followed by humans (10%), dogs (7%), and horses (6%). For E. coli, humans contributed 13% of contamination, followed by ducks (9%), cattle (7%), and chickens (6%). The developed metabolic fingerprint database was able to distinguish between human and animal sources as well as among animal species in the studied catchment.     

In vitro antimicrobial activity of a novel propolis formulation (Actichelated propolis)

AIMS: This study compared in vitro activities of Actichelated propolis (a multicomposite material obtained with mechano-chemichal activation) and of a hydroalcoholic extract of propolis. METHODS AND RESULTS: Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), determined by means of microdilution broth method, against five strains of Staphylococcus aureus, Streptococcus pyogenes, Haemophilus influenzae, Enterococcus spp., Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa, showed a greater potency of Actichelated propolis (MIC range: 0.016-4 mg flavonoids ml(-1)) in respect to the hydroalcoholic extract (MIC range: 0.08-21.4 mg flavonoids ml(-1)). Concentrations of Actichelated propolis active against adenovirus, influenza virus, parainfluenza virus and herpes virus type 1 were at least 10 times lower than those of the hydroalcoholic extract. Preincubation of Strep. pyogenes and H. influenzae with subinhibitory concentrations of Actichelated propolis (1/4 and 1/8 x MIC) significantly reduced the number of bacteria that adhered to human buccal cells. CONCLUSIONS: Actichelated propolis has proven to possess antibacterial and antiviral activity higher than a hydroalcoholic extract, being also able to interfere on bacterial adhesion to human oral cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This new formulation of propolis showing better antimicrobial and physical characteristics could improve the application of propolis in respiratory tract infections.     

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 > or =256 microg/ml for 98.7% of isolates, and the avilamycin MIC was > or =8 microg/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.     

Enumeration by a miniaturized method of Escherichia coli, Streptococcus bovis and enterococci as indicators of the origin of faecal pollution of waters

Counts of Escherichia coli, faecal streptococci and enterococci were made on faecal specimens from human and animal origin and urban raw sewage waters, with microtiter plates containing selective substances. Escherichia coli was more numerous than faecal streptococci and enterococci in 80% of the samples regardless of the origin. Consequently the use of the ratio E. coli/faecal streptococci to distinguish human from animal origin of faecal pollution is questionable. Enterococcus faecalis was predominant in human and poultry faeces, Streptococcus bovis was typical of the bovine faeces and to a lesser extent also of pig faeces whereas Enterococcus durans, Ent. hirae and Ent. faecium did not characterize any faecal source. Streptococcus bovis could be distinguished in the microtiter plate by its inability to reduce triphenyl tetrazolium chloride (TTC) in the medium.     

Enumeration by a miniaturized method of Escherichia coli, Streptococcus bovis and enterococci as indicators of the origin of faecal pollution of waters

Counts of Escherichia coli , faecal streptococci and enterococci were made on faecal specimens from human and animal origin and urban raw sewage waters, with microtiter plates containing selective substances. Escherichia coli was more numerous than faecal streptococci and enterococci in 80% of the samples regardless of the origin. Consequently the use of the ratio E. coli /faecal streptococci to distinguish human from animal origin of faecal pollution is questionable. Enterococcus faecalis was predominant in human and poultry faeces, Streptococcus bovis was typical of the bovine faeces and to a lesser extent also of pig faeces whereas Enterococcus durans, Ent. hirae and Ent. faecium did not characterize any faecal source. Streptococcus bovis could be distinguished in the mictrotiter plate by its inability to reduce triphenyl tetrazolium chloride (TTC) in the medium.     

Using DNA microarrays to identify library-independent markers for bacterial source tracking

Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers.     

Chemical compositions and antimicrobial activities of four different Anatolian propolis samples

Propolis means a gum that is gathered by bees from various plants. It is known for its biological properties, having antibacterial, antifungal and healing properties. The aims of this study were to evaluate the antimicrobial activity of four different Anatolian propolis samples on different groups of microorganisms including some oral pathogens and comparison between their chemical compositions. Ethanol extracts of propolis (EEP) were prepared from four different Anatolian propolis samples and examined whether EEP inhibit the growth of the test microorganisms or not. For the antimicrobial activity assays, minimum inhibitory concentrations (MIC) were determined by using macrodilution method. The MIC values of the most effective propolis (TB) were 2 microg/ml for Streptococcus sobrinus and Enterococcus faecalis, 4 microg/ml for Micrococcus luteus, Candida albicans and C. krusei, 8 microg/ml for Streptococcus mutans, Staphylococcus aureus, Staphylococcus epidermidis and Enterobacter aerogenes, 16 microg/ml for Escherichia coli and C. tropicalis and 32 microg/ml for Salmonella typhimurium and Pseudomonas aeruginosa. The chemical compositions of EEP's were determined by high-temperature high-resolution gas chromatography coupled to mass spectrometry. The main compounds of four Anatolian propolis samples were flavonoids such as pinocembrin, pinostropin, isalpinin, pinobanksin, quercetin, naringenin, galangine and chrysin. Although propolis samples were collected from different regions of Anatolia all showed significant antimicrobial activity against the Gram positive bacteria and yeasts. Propolis can prevent dental caries since it demonstrated significant antimicrobial activity against the microorganisms such as Streptococcus mutans, Streptococcus sobrinus and C. albicans, which involves in oral diseases.     

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.     

Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.     

Antimicrobial effect of Korean propolis against the mutans streptococci isolated from Korean

The aim of this study was to determine the optimal concentration of Korean propolis against clinical isolates of mutans streptococci (MS) from Koreans. The antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) and time-kill curves against mutans streptococci. The MIC(90) values of propolis for MS were 35 μg/ml. Propolis had a bacteriostatic effect on Streptococcus mutans ATCC 25175(T) and bactericidal effects on Streptococcus sobrinus ATCC 33478(T) at > 2 × MIC (70 μg/ml). These results suggest that the propolis can be used in the development of oral hygiene products for the prevention of dental caries.     

Comparsion ofin vitro activities of antifungal drugs and propolis against yeasts isolated from patients with superficial mycoses

Thein vitro susceptibilities of propolis and antifungal drugs were determined against some yeasts isolated from patients with superficial mycoses. The agents tested included fluconazole, itraconazole, ketoconazole, terbinafine and propolis. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P. For allCandida albicans isolates from the patients with superficial mycoses, ketoconazole presented higher (P<0.05) efficiency than that of the other antifungal agents tested. The geometric mean MIC values of antifungal drugs and propolis against the yeasts tested ranged from 0.087 to 12.69 μg/mL and 0.4–0.6 μg/mL, respectively. Propolis also showed an important antifungal activity against the yeasts tested, MIC ranges of the propolis were between 0.01–1.65 μg/mL. Based on these results, propolis requires further investigation as a potential agent for the treatment of superficial mycoses.     

Comparative study of in vitro methods to analyse the antifungal activity of propolis against yeasts isolated from patients with superficial mycoses

AIM: To test a total of 15 strains belonging to four species of yeasts by different in vitro methods against propolis and itraconazole (ITC). METHODS AND RESULTS: Three methods were compared for susceptibility testing of yeast isolates to propolis: disc diffusion method, agar dilution method and National Committee for Clinical Laboratory Standards (NCCLS, M27A) broth microdilution method. ITC was selected as the antifungal agent for comparison study. Using the broth microdilution method, the geometric mean for MIC (microg ml(-1)) with regard to all isolates was < or =0.06 for propolis and < or =0.35 for ITC. The broth microdilution and the agar dilution methods were in good agreement (75%) for propolis against yeasts isolated from patients with superficial mycoses. Using the diffusion method, all strains showed a broad zone of inhibition at the first available reading time (24 or 48 h). An increase of MIC values was accompanied by a decrease of growth inhibition zone diameter. A favourable correlation was found between MIC and inhibition zone around the disc for propolis sample and the correlation coefficient was: r = -0.626 (P < 0.01). CONCLUSIONS: This study suggests the potential value of the agar dilution and disc diffusion method as a convenient alternative method for testing of yeasts to propolis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that propolis and ITC were very active against yeasts from patients with superficial mycoses. The other prominent finding in this study is that RPMI 1640 with L-glutamine was the available broth for the in vitro susceptibility testing of yeasts.     

<Emphasis Type="Italic">In vitro</Emphasis> antifungal activity of propolis samples of Czech and Slovak origin

Propolis has been used in traditional folk medicine for ages owing to a number of biological effects. Four propolis samples of Czech and one of Slovak origin were extracted using Soxhlet apparatus and analysed by thin-layer chromatography. Raw propolis samples and their extracts were tested by microdilution broth method to determine minimal inhibitory concentration (MIC) in eight strains of human pathogenic fungi. Raw propolis samples showed a lower in vitro antifungal activity than their extracts. In general, the petroleum ether extracts exhibited the highest in vitro antifungal activity (MIC range of 16–64 μg/ml). The content of flavonoids in the samples varied according to region. The highest amount of flavonoids was found in sample A that originated from Broumov (4%). The most susceptible to the propolis extracts were Trichophyton mentagrophytes and Candida albicans. The propolis samples of Czech and Slovak origin and their extracts showed a considerable in vitro antifungal effect which was associated especially with nonpolar petroleum ether and toluene extracts. There was only a partial correlation between flavonoids content and in vitro antifungal activity.     

Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food

In the present study, a collection of 187 Enterococcus food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species Enterococcus faecalis (n = 33), E. durans (n = 7), E. faecium (n = 3), E. casseliflavus (n = 1), and E. gallinarum (n = 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 microg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the tet(M) (n = 43), tet(L) (n = 16), and tet(S) (n = 1) genes. In 15 isolates, including all of those for which the MIC was 256 micro g/ml, both tet(M) and tet(L) were found. Furthermore, all tet(M)-containing enterococci also harbored a member of the Tn916-Tn1545 conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the erm(B) gene. Filter mating experiments revealed that 10 E. faecalis isolates, 3 E. durans isolates, and 1 E. faecium isolate could transfer either tet(M), tet(L), or both of these genes to E. faecalis recipient strain JH2-2. In most cases in which only tet(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn916-Tn1545 family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable tet(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of > or = 99.6%. Two of these SHGs contained tet(M) mosaic structures previously found in Tn916 elements and on Lactobacillus and Neisseria plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.     

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.     

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.     

Novel gyrase mutations and characterization of ciprofloxacin-resistant clinical strains of Enterococcus faecalis isolated in Poland

The activity of ciprofloxacin, sparfloxacin and moxifloxacin was determined for 205 Enterococcus faecalis isolates from patients of five hospitals (Warsaw, Poland; collected from 2000 to 2002). Ciprofloxacin resistant and intermediate isolates were numerous (53.7%). Among them, highly resistant (MIC > or = 16 mg/l) isolates predominated (98%). Isolates resistant to ciprofloxacin were also resistant to sparfloxacin and moxifloxacin. The parC and gyrA QRDRs (quinolone-resistance-determining region) of 11 isolates with ciprofloxacin MICs from 1 to 256 mg/l were analysed by DNA sequencing. In ParC one kind of amino acid substitution (of Ser-85 to Ile) in 9 E. faecalis strains with MICs from 16 to 256 mg/l was observed. In GyrA Ser-84 was changed to one of four different amino acids: Arg, Ile, Cys or Tyr, however no association between the amino acid type and MIC value was found. The last two substitutions have not been reported to date for E. faecalis. Moreover, our results may suggest that mutations within parC and gyrA are associated with development of a high-level of ciprofloxacin resistance.     

Impact of medicated feed on the development of antimicrobial resistance in bacteria at integrated pig-fish farms in Vietnam

Integrated livestock-fish aquaculture utilizes animal excreta, urine, and feed leftovers as pond fertilizers to enhance the growth of plankton and other microorganisms eaten by the fish. However, antimicrobial-resistant bacteria may be transferred and develop in the pond due to selective pressure from antimicrobials present in animal feed, urine, and feces. In an experimental pig-fish farm located in periurban Hanoi, Vietnam, nine piglets were provided feed containing 5 μg of tetracycline (TET)/kg pig weight/day and 0.45 μg of enrofloxacin (ENR)/kg pig weight/day during the second and fourth (last) months of the experiment. The aim of this study was to determine the association between the provision of pig feed with antimicrobials and the development of antimicrobial resistance, as measured in a total of 520 Escherichia coli and 634 Enterococcus strains isolated from pig manure and water-sediment pond samples. MIC values for nalidixic acid (NAL) and ENR showed that E. coli and Enterococcus spp. overall exhibited significant higher frequencies of resistance toward NAL and ENR during the 2 months when pigs were administered feed with antimicrobials, with frequencies reaching 60 to 80% in both water-sediment and manure samples. TET resistance for both indicators was high (>80%) throughout the study period, which indicates that TET-resistant E. coli and Enterococcus spp. were present in the piglets before the initiation of the experiment. PCR-based identification showed similar relative occurrences of Enterococcus faecium, Enterococcus faecalis, and other Enterococcus spp. in the water-sediment and manure samples, suggesting that Enterococcus spp. isolated in the ponds originated mainly from the pig manure. The development of antimicrobial resistance in integrated animal husbandry-fish farms and possible transfers and the impact of such resistance on food safety and human health should be further assessed.