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.     

Mechanisms of antimicrobial resistance and genetic relatedness among enterococci isolated from dogs and cats in the United States

Aims: In this study, mechanisms of antimicrobial resistance and genetic relatedness among resistant enterococci from dogs and cats in the United States were determined. Methods and Results: Enterococci resistant to chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, streptomycin, lincomycin, quinupristin/dalfopristin and tetracycline were screened for the presence of 15 antimicrobial resistance genes. Five tetracycline resistance genes [tet(M), tet(O), tet(L), tet(S) and tet(U)] were detected with tet(M) accounting for approx. 60% (130/216) of tetracycline resistance; erm(B) was also widely distributed among 96% (43/45) of the erythromycin‐resistant enterococci. Five aminoglycoside resistance genes were also detected among the kanamycin‐resistant isolates with the majority of isolates (25/36; 69%) containing aph(3′)‐IIIa. The bifunctional aminoglycoside resistance gene, aac(6′)‐Ie‐aph(2″)‐Ia, was detected in gentamicin‐resistant isolates and ant(6)‐Ia in streptomycin‐resistant isolates. The most common gene combination among enterococci from dogs (n = 11) was erm(B), aac(6′)‐Ie‐aph(2″)‐Ia, aph(3′)‐IIIa, tet(M), while tet(O), tet(L) were most common among cats (n = 18). Using pulsed‐field gel electrophoresis (PFGE), isolates clustered according to enterococcal species, source and antimicrobial gene content and indistinguishable patterns were observed for some isolates from dogs and cats. Conclusion: Enterococci from dogs and cats may be a source of antimicrobial resistance genes. 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. Although host‐specific ecovars of enterococcal species have been described, identical PFGE patterns suggest that enterococcal strains may be exchanged between these two animal species.     

Enumeration and speciation of enterococci found in marine and intertidal sediments and coastal water in southern California

AIMS: To determine the levels and species distribution of enterococci in intertidal and marine sediments and coastal waters at two beaches frequently in violation of bacterial water standards. METHODS AND RESULTS: Faecal indicator bacteria were extracted from sediment and enumerated using membrane filtration. High levels of enterococci were detected in intertidal sediments in a seasonal river and near a storm drain outlet. Low levels were found in marine sediments at 10 m depths and in surf zone sand. Bacterial isolates presumptively identified as Enterococcus on mEI media were speciated. The predominant species found in both water and sediment included Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus mundtii. A number of isolates (11-26%) from regulatory water samples presumptively identified as enterococci on mEI media were subsequently identified as species other than Enterococcus. At both study sites, the distribution of species present in water was comparable with those in sediments and the distribution of species was similar in water samples passing and exceeding bacterial indicator standards. CONCLUSIONS: High levels of Enterococcus in intertidal sediments indicate retention and possible regrowth in this environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Resuspension of enterococci that are persistent in sediments may cause beach water quality failures and calls into question the specificity of this indicator for determining recent faecal contamination.     

The prevalence and antimicrobial resistance phenotypes of Salmonella,Escherichia coli and Enterococcus sp. in surface water

Surface water is prone to bacterial contamination as it receives wastes and pollutants from human and animal sources, and contaminated water may expose local populations to health risks. This review provides a brief overview on the prevalence and antimicrobial resistance (AR) phenotypes of Salmonella, Escherichia coli and Enterococcus, found in natural freshwaters. These bacteria are frequently detected in surface waters, sometimes as etiological agents of waterborne infections, and AR strains are not uncommonly identified in both developed and developing countries. Data relating to Salmonella, E. coli and Enterococcus present in environmental water are lacking, and in order to understand their development and dissemination using the One Health approach, understanding the prevalence, distribution and characteristics of the bacteria present in surface water as well as their potential sources is important. Furthermore, AR bacteria in natural watersheds are not well investigated and their impacts on human health and food safety are not well understood. As surface water is a receptacle for AR bacteria from human and animal sources and a vehicle for their dissemination, this is a crucial data gap in understanding AR and minimizing its spread. For this review, Salmonella, E. coli and Enterococcus were chosen to evaluate the presence of primary pathogens and opportunistic pathogens as well as to monitor AR trends in the environmental water. Studies around the world have demonstrated the widespread distribution of pathogenic and AR bacteria in surface waters of both developing and developed countries, confirming the importance of environmental waters as a reservoir for these bacteria and the need for more attention on the environmental bacteria for emerging AR.     

Influence of avilamycin administration and its subsequent withdrawal on emergence and disappearance of antimicrobial resistance in enterococci in the intestine of broiler chickens

AIMS: To investigate the influence of avilamycin (AVM) administration and its subsequent withdrawal on the emergence and disappearance of AVM-resistant enterococci in the intestine of broiler chickens. METHODS AND RESULTS: Five chicks each of C, L and H groups were given the basal diet, the basal diet supplemented with 5 g AVM/ton and the basal diet supplemented with 50 g AVM/ton, respectively. The AVM-resistant Enterococcus faecalis population did not emerge during 30 days of the AVM administration period, whereas the AVM-resistant Enterococcus faecium with a minimum inhibitory concentration of >512 microg ml(-1) in the faeces of chicks of the L and H groups emerged on 3 and 1 days after the AVM administration, respectively. Thereafter, the AVM-resistant Ent. faecium population density in both L and H groups maintained high levels during the AVM administration period. Twenty days after the AVM withdrawal, the AVM-resistant Ent. faecium population disappeared from the intestines of both four of five chicks of L group and three of five chicks of H group. The AVM-resistant Ent. faecium population density in one chick from each of the groups, L and H, did not change before and after the AVM removal. CONCLUSIONS: The AVM-resistant Ent. faecium emerged during the AVM administration, and disappeared from the intestine of most chicks after the AVM withdrawal. However, the AVM-resistant Ent. faecium persisted in some chicks 20 days after AVM withdrawal. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that introducing an AVM withdrawal period could minimize the risk of AVM-resistant Ent. faecium becoming carcass contaminants, and that prudent antibiotic use alone is not sufficient to stem emergence of the AVM-resistant Ent. faecium.     

Persistence and diversity of faecal coliform and enterococci populations in faecally polluted waters

Aim: To assess the persistence and diversity of faecal bacterial populations (faecal coliforms and enterococci) that have recently been included in microbial source tracking (MST) predictive models. Methods and Results: The analysed bacterial populations included members of the enterococci group (ENT) [Enterococcus faecium (FM), Enterococcus faecalis (FS) and Enterococcus hirae (HIR)] and the faecal coliform group (FC) [diverse Escherichia coli phenotypes (ECP) and cellobiose‐negative faecal coliforms (CNFC)]. The inactivation of these distinct groups was monitored over time on‐site in river by biochemical fingerprinting, and diversity indices were calculated. Among the different analysed species belonging to the ENT group, HIR persisted longer and was able to replicate in the environment at a higher rate. On the other hand, ECP and NCFC showed a similar persistence throughout the different seasons. The diversity index (Di) for FC increased substantially in the summer after 96 h to a maximum value of 0·96. On the other hand, the Di for ENT diminished over the same period to a value of 0·86, suggesting a different persistence for the different species integrating this group. Conclusions: The persistence of ECP, CNFC, FM and FS in the aquatic environment is high, particularly for the members of the FC and in the summer season. On the contrary, HIR is able to replicate in the environment at a high rate even in winter, and therefore, its inclusion in MST predictive models is discouraged. Significance and Impact of the Study: ECP, CNFC, FMFS and HIR have been proposed as additional variables in MST predictive models. However, the different persistence of HIR compared with the other variables should be taken into account for the development of such models.     

Chemiluminescence of enterococci isolates from freshwater

All Enterococcus spp., isolated from environmental water samples (n=81), emitted a high chemiluminescence signal in the presence of luminol (10(-2) M). Kinetic studies of chemiluminescence show a close correlation between chemiluminescence and growth curves during the exponential phase, with a maximum chemiluminescence reached just before bacterial growth entered in the stationary phase. On the other hand, genera closely related to Enterococcus such as Streptococcus or Lactococcus produced a very weak chemiluminescent signal. Chemiluminescence of enterococci could therefore offer a rapid test, in aiding the identification of the genus Enterococcus and in the survey of the microbiological quality of water supplies.     

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed

AIMS: The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. METHODS AND RESULTS: Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. CONCLUSIONS: None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.     

临床分离肠球菌的分布特点及耐药性变迁

目的分析临床分离肠球菌的分布特点及耐药性变迁,为临床合理应用抗生素提供依据。方法从中国医科大学附属盛京医院门诊和住院病人采集血液、尿液、手术切口分泌物、腹腔引流液等标本,应用法国生物梅里埃API细菌鉴定系统进行细菌的分离和种属鉴定,用K-B纸片琼脂扩散方法对不同肠球菌做药物敏感试验。结果肠球菌属的数量占全部分离到细菌种类的第6位。其中屎肠球菌的构成比,2002年为30.9%,2006年上升到71.6%,超过粪肠球菌的比例。肠球菌主要分布于ICU、普通外科、呼吸内科、肾内科、干诊、感染科和新生儿科病房。粪肠球菌对青霉素、氨苄西林和呋喃妥因的耐药率分别从2002年的53.6%、38.5%和30.4%下降到2006年的4.5%、0和4.5%,屎肠球菌对氯霉素的耐药率从2002年的37.1%下降到2006年的14.3%,两种菌对万古霉素和替考拉宁的敏感性均高,但临床出现耐万古霉素和耐替考拉宁的肠球菌。结论中国医科大学附属盛京医院临床肠球菌出现新的分布特点,屎肠球菌比例有增加趋势。调查肠球菌尤其是屎肠球菌感染的危险因素和采取相应的防治措施是当前研究的重要课题。     

临床分离肠球菌的耐药性研究

目的了解近3年来粤东地区临床分离的肠球菌的耐药特征,为预防耐万古霉素肠球菌（VRE）的产生和控制VRE播散提供理论依据和实验基础。方法收集临床分离的215株肠球菌,细菌鉴定及药敏试验采用VITEK-60全自动细菌鉴定仪。结果215株肠球菌中,尿标本中分离肠球菌75株（35％）;痰液中分离出34株（16％）。粪肠球菌141株（65．5％）,屎肠球菌74株（34．5％）。粪肠球菌对万古霉素、呋喃妥因、青霉素G和莫西沙星的敏感率较高,70％～100％;对红霉素和四环素的敏感性较差,11％～33％。屎肠球菌对万古霉素敏感性较好为100％,四环素为62％;而对呋喃妥因、高链霉素、青霉素和左氧氟沙星等敏感性较差（5％～47％）。未检出耐万古霉素肠球菌。结论粤东地区近3年来未发现耐万古霉素肠球菌。肠球菌对各种常见抗生素敏感程度呈下降趋势。屎肠球菌较粪肠球菌耐药性更高。应根据细菌学培养结果合理用药,减少耐药菌株的发生率。     

Determination of antimicrobial resistance and virulence gene signatures in surface water isolates of Escherichia coli

Aims: To determine the occurrence of Escherichia coli harbouring virulence markers of shiga‐ or entero‐toxins and resistance to antimicrobials in surface waters. Methods and Results: Surface water samples were collected at six locations of the river Gomti. E. coli isolates (n = 90) were characterized for their pathogenic potential using polymerase chain reaction to detect virulence genes as well as their sensitivity to antimicrobial agents using disc diffusion methods. In this study, 57·8% of E. coli isolates exhibited resistance to three or more antimicrobial agents. Sensitivity to cephotaxime, gentamicin and norfloxacin was observed in 7·8%, 48·9% and 77·8% of isolates, respectively. Both stx1 and stx2 genes were present in 15·6% of isolates while remaining isolates had either stx1 (17·8%) or stx2 (6·7%). The stx1 gene (33·3%) was more prevalent than stx2 (22·2%). The results indicate that the LT1 and ST1 genes were positive in 21·2% of isolates. Conclusions: The presence of multi‐drug resistance and virulence genes in E. coli isolated from surface water being used for domestic and recreational purposes may result in waterborne outbreaks. Significance and Impact of the Study: The data will be useful in monitoring surface waters for forecasting and management of waterborne outbreaks.     

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.     

Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci

Fifty-four Enterococcus faecalis and 20 Enterococcus faecium isolates from clinical and non-human sources in Rome, Italy, were characterized by antibiotic resistance and pulsed field gel electrophoresis (PFGE). Resistance to vancomycin, teicoplanin, ampicillin, and ciprofloxacin was more frequent in E. faecium than in E. faecalis, whereas high-level resistance to aminoglycoside was found primarily in E. faecalis. Multi-resistance was found primarily among clinical isolates, but was also observed among environmental isolates. Common genotypes shared among clinical and environmental isolates were observed, however, the majority of isolates occurred as unique, source-specific clones. Several PFGE types were associated with shared features in their antibiotic resistance patterns; evidences of clonal spread between and within wards were also noted. This is the first report indicating clonal relatedness between human and environmental enterococci isolated in Italy.     

Incidence of antibiotic resistance and sex pheromone response among enterococci isolated from clinical human samples and from municipal waste water

The incidence of resistance to various antibiotics as well as the capacity to elicit aggregation response to sex pheromones have been investigated in strains of Enterococcus faecalis isolated from clinical and municipal waste waters (MWW). While clinical isolates showed a high incidence of antibiotic resistance (87%) and sex pheromone response (33%), these traits appeared with a much lower frequency in MWW isolates (12% and 4% respectively). The simultaneous incidence of both traits was of 52% and 0% for clinical and MWW isolates, respectively. The capacity to elicit a positive pheromone response as well as antibiotic resistance traits seemed to be strongly correlated with the presence of gelatinase activity among clinical isolates. Among MWW isolates, only sex pheromone response seemed to correlate with the presence of gelatinase activity.     

Phenotypic variations of enterococci in surface waters: analysis of biochemical fingerprinting data from multi-catchments

Aims:  The aim of this study was to identify the prevalence of environmentally adapted enterococci strains by analysing biochemical fingerprinting (BF) data of 3952 enterococci isolates collected over 5 years from the six catchments in Southeast Queensland, Australia.
Methods and Results:  A BF method was used to type 3952 enterococci isolates from six catchments. The environmental isolates were compared with a large existing BF library comprised of 5803 enterococci isolates from 10 host groups. Environmental isolates belonged to 801 biochemical phenotypes (BPTs), of which, an average of 29.2% was specific to each catchment. When compared with the BF library, an average of 79·5% BPTs from each catchment was identical to those in the library (i.e. host-origin BPTs). The remaining 20·5% was regarded as non-host origin BPTs, as they were not in the library and constituted only 5·3% of the total isolates tested for each catchment.
Conclusions:  Our data suggest that less than 5% of studied environmental strains was not identical to those in the library and seemed to be of environmental origin. From a microbial source tracking context, such low level of environmentally adapted strains can have a minimal impact on the performance of the library-based methods if a large number of isolates were tested from both the host groups and environmental waters.
Significance and Impact of the Study:  These data shed light on the importance of the size and representativeness of library–based source-tracking methods and their implications for the identification of faecal pollution in environmental waters.