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.     

Contamination of milk by enterococci and coliforms from bovine faeces

AIM: To determine the contribution of enterococci and coliforms from bovine faeces and teats to contamination of raw milk. Methods: Putative enterococci (n = 301) and coliforms (n = 365) were isolated from bovine faeces (n = 20), cows' teats (n = 20), the raw milk (n = 1) and the milking environment (n = 4) on one farm. The clonal relationships of each bacterial group were investigated using Pulsed-Field Gel Electrophoresis of genomic macrorestriction fragments. Representatives of the different clusters of enterococci were identified by molecular techniques including rep-PCR, SDS protein profiling, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), phenylalanyl-tRNA synthase (pheS) sequence analysis and/or 16S rDNA gene sequencing. Coliforms were identified by API 20E strips. RESULTS: The majority of the bovine faecal enterococcal isolates were identified as a potential new species of Aerococcus (100 isolates); E. faecium (28 isolates), and Aerococcus viridans (28 isolates) were also found. All coliform isolates from the bovine faeces were identified as Escherichia coli. The coliforms present in the milk were Hafnia alvei, Serratia liquefaciens, Yersinia enterocolitica and Enterobacter amnigenus. No E. coli, Enterococcus or Aerococcus from the bovine faeces were found in the milk. A single clone of H. alvei was found in the water, the milking equipment and the milk, suggesting that the water was the source of the organism in the milk. No vancomycin-resistant aerococci or enterococci were found while most of the isolates tested showed the presence of at least one virulence gene. The milk-sock retained strains that adhered to particulate faecal material. Coliforms were present at approx. 2 orders of magnitude greater than enterococci in the bovine faeces. CONCLUSIONS: The results imply that bovine faeces are not an important source of contamination of raw milk with enterococci or coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm those of two previous studies (Gelsomino et al. 2001, Int J Food Microbiol71, 177-188 and Kagkli et al. 2007, Int J Food Microbiol114, 243-251) on two other farms. The three studies show that contamination of milk by enterococci, lactobacilli and coliforms of bovine faecal origin is extremely low. The results also suggest that where raw milk is implicated in food infection, other factors in addition to faecal contamination must be involved.     

Species distribution and antimicrobial resistance of enterococci isolated from surface and ocean water

Aims: The species identification and antimicrobial resistance profiles were determined for enterococci isolated from Southern California surface and ocean waters. Methods and Results: Species identification was determined for 1413 presumptive Enterococcus isolates from urban runoff, bay, ocean and sewage water samples. The most frequently isolated species were Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus mundtii. All five of these species were isolated from ocean and bay water with a frequency ranging from 7% to 36%. Enterococcus casseliflavus was the most frequently isolated species in urban runoff making up 36–65% of isolates while E. faecium was the most frequently isolated species in sewage making up 53–78% of isolates. The similar distribution of species in urban runoff and receiving water suggests that urban runoff may be the source of Enterococcus. No vancomycin or high level gentamycin resistance was detected in E. faecalis and E. faecium isolates. Conclusions: Enterococcus faecalis, E. faecium, E. casseliflavus and E. mundtii are the most commonly isolated Enterococcus species from urban runoff and receiving waters in Southern California. Significance and Impact of the Study: Determination of the Enterococcus species isolated from receiving waters and potential pollution sources may assist in determining the sources of pollution.     

Low prevalence of vancomycin-resistant enterococci in clinical samples from hospitalized patients of the Canary Islands, Spain

Over the last decade vancomycin-resistant enterococci (VRE) have emerged as nosocomial pathogens. The aim of this study was to determine the prevalence of VRE in clinical samples from hospitalized patients in the Canary Islands. From April to November 2000, 437 enterococci were isolated from patients hospitalized at the four main health care centers in those islands. Identification to the species level was performed with the GPS-TA (Vitek 1) or the Wider I system. A PCR assay was used to determine the genotype of glycopeptide resistance (vanA, vanB, vanC1, and vanC2/C3 genes). Only three (0.7%) VRE were detected: one vanA Enterococcus faecalis, and two vanC1 Enterococcus gallinarum. To our knowledge, this is the first VRE study carried out in the Canary Islands hospitals, and the results showed a low prevalence of VRE. Electronic Publication     

Isolation and biochemical characterisation of enterocins produced by enterococci from different sources

AIMS: Comparison of enterocins produced by six Enterococcus faecium strains and one Ent. faecalis strain isolated from different origin with regard to their microbiological and biochemical characteristics in view of their technological potential and practical use. METHODS AND RESULTS: The seven enterococci were sensitive to the glycopeptide antibiotics vancomycin and teicoplanin and did not show haemolytic activity. The absence of the glycopeptide-resistant genotypes and the genes involved in the production of the lantibiotic cytolysin was confirmed by PCR. The enterocins were active towards Listeria innocua and other lactic acid bacteria. Their temperature stability was dependent on the pH and their activity was higher at acidic pH. A bactericidal and bacteriolytic effect was shown. PCR analyses revealed that the gene of enterocin A was present in the genome of Ent. faecium CCM 4231, Ent. faecium 306 I.2.20 and Ent. faecalis Y; both enterocin A and B genes were present in the genome of Ent. faecium LMG 11423T, Ent. faecium RZS C5 and Ent. faecium RZS C13. Enterocin P was detected in the genome of Ent. faecium RZS C5 and Ent. faecium RZS C13. No signal was found for Ent. faecium SF 68. Enterocins from Ent. faecium RZS C5, Ent. faecium RZS C13 and Ent. faecium SF 68 were purified to homogeneity. CONCLUSIONS: Ent. faecium RZS C5 and Ent. faecium RZS C13 produced an enterocin with a molecular mass of 5460 and 5477 Da, respectively, which was in the range of that of enterocin B. The amino acid sequence analysis of the enterocin from Ent. faecium RZS C13 revealed 24 N-terminal residues, which were identical to those of enterocin B. The enterocin from Ent. faecium SF 68 had a molecular mass of 4488 Da, which did not correspond to any enterocin known so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The number of characterized enterocins is increasing. As this type of work is tedious and time-consuming, it may be interesting to include PCR as a first step to know if the Enterococcus strain in study produces either a known or a new enterocin. Also, it is important to check the absence of cytolysin and resistance to vancomycin for a further application of the Enterococcus strain in food or health applications.     

Differentiation of enterococci from other group D streptococci by means of a specific monoclonal antibody

We report on the specificity of a monoclonal antibody which reacts with autoclaved extracts of four species of enterococci but does not react to the same extent with similar extracts from two non-enterococcal group D streptococci. The monoclonal antibody also reacts specifically with purified lipoteichoic acid from Streptococcus faecalis but not significantly with purified lipoteichoic acid from the non-enterococcal species Streptococcus bovis and Streptococcus equinus. The specific antigen detected with this antibody could correlate with the definition of the enterococcus subgroup of the streptococci which would provide further evidence that this sub-group is taxonomically distinct from the other group D streptococci.     

Characterization of faecal enterococci from rabbits for the selection of probiotic strains

AIMS: To characterize the facultative anaerobic intestinal microbiota of healthy rabbits, especially enterococci, for the selection of potential probiotic strains. METHODS AND RESULTS: Phenotypic and molecular methods were used to identify enterococcal isolates. Results obtained indicated that enterococcal microbiota widely varied among individuals both in size and in composition. Antibacterial and haemolytic activities, and resistance to acid and bile salts were determined. A small group of strains produced bacteriocins active against listeriae and indigenous clostridia and therefore they were selected as potential probiotics. One such strain, 8G, was assayed for colonization capacity. Results obtained suggested that the fate of the introduced strain depended on the composition of the enterococcal indigenous microbiota. CONCLUSIONS: Enterococcus faecalis and Ent. faecium are the predominant enterococcal species in the gut of rabbits. Other species of lactic acid bacteria were not recovered. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal intestinal microbiota of healthy rabbits has been characterized in detail. Monitoring the fate of an introduced probiotic in vivo is required in order to evaluate potential probiotic strains.     

Chemiluminescence of irradiated animal blood plasma

Spontaneous and induced chemiluminescence of rat blood plasma following irradiation of the animals with fast neurons was studied. Dynamics of the luminescence reflected the degree of radiation injury and an oscillatory response of blood chemiluminescent effect was observed.     

Glycopeptide-resistance transferability from vancomycin-resistant enterococci of human and animal source to Listeria spp

AIMS: The glycopeptide-resistance transferability from vancomycin-resistant enterococci (VRE) of clinical and animal origin to different species of Listeria was investigated. METHODS AND RESULTS: Of 36 matings, performed on membrane filter, the glycopeptide resistance was successfully transferred in six attempts, five with donors of animal origin and only one with donors from clinical source. The acquired glycopeptide resistance in Listeria transconjugants was confirmed by the presence of the conjugative plasmid band and by the amplification of the 732-bp fragment of vanA gene in transferred plasmids. CONCLUSIONS: Despite the lower number of bacteria used in this study, the source of enterococci influenced the outcome of mating. Moreover transferred VanA plasmid induced a different expression in Listeria transconjugants, suggesting that gene expression might be influenced by species affiliation of recipients. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data strengthen the opinion that enterococci are an important source of resistance genes for Listeria via the transfer of movable genetic elements. As these strains are commonly found in the same habitats, a horizontal spread of glycopeptide resistance in Listeria spp. could be possible.     

Chemiluminescence in Dictyostelium discoideum

Abstract Oxygen radicals generated during oxidative metabolism participate in chemical reactions resulting in light emission. Chemiluminescence is used therefore to measure their production. We have shown that starvation and heat shock induce chemiluminescence in Dictyostelium discoideum . The peak light emission was found to occur about 4 h after the onset of starvation. The optimum temperature for chemiluminescence by starving amoebae was about 33°C. The heat shock inducibility of chemiluminescence was maximal at the beginning of development. Our results are consistent with suggestions that the product(s) of perturbed mitochondrial metabolism might be intracellular signal(s) controlling gene expression in stressed cells. They also suggest a role for intracellular stress signal(s) in the initiation of development in Dictyostelium by starvation.     

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.     

神经生长因子的发光免疫分析法

介绍了一种灵敏的神经生长因子化学发光免疫测定方法。小鼠神经生长因子（ｍＮＧＦ）的抗体ＩｇＧ用亲和层析纯化并用吖啶酯标记。该法可用于大鼠组织中ＮＧＦ含量的测定。方法的灵敏度为１０ｐｇ／ｍＬＮＧＦ;批内批间变异系数分别为８．７％及１３．２％;回收率平均为１０３％。ｍＮＧＦ抗体对人的ＮＧＦ也有极强的交叉反应,故本方法也可能用于病人血清或脑脊液中ＮＧＦ含量的测定。     

Analysis of gyrA and parC mutations in enterococci from environmental samples with reduced susceptibility to ciprofloxacin

The quinolone resistance determining regions of gyrA and parC in four species of enterococci from environmental samples with reduced susceptibility to ciprofloxacin were sequenced. The nucleotide sequence variations of parC could be related to the different enterococcal species. Mutations in Enterococcus faecalis and Enterococcus faecium related to reduced susceptibility were identical to mutations detected in E. faecalis and E. faecium of clinical origin. A minimal inhibitory concentration of 8 microg ml(-1) to ciprofloxacin was not associated with any mutations in the gyrA and parC gene of Enterococcus casseliflavus and Enterococcus gallinarum. These two species may be intrinsically less susceptible to ciprofloxacin.     

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.     

Molecular characterization of methanotrophic isolates from freshwater lake sediment

Profiles of dissolved O(2) and methane with increasing depth were generated for Lake Washington sediment, which suggested the zone of methane oxidation is limited to the top 0.8 cm of the sediment. Methane oxidation potentials were measured for 0.5-cm layers down to 1.5 cm and found to be relatively constant at 270 to 350 micromol/liter of sediment/h. Approximately 65% of the methane was oxidized to cell material or metabolites, a signature suggestive of type I methanotrophs. Eleven methanotroph strains were isolated from the lake sediment and analyzed. Five of these strains classed as type I, while six were classed as type II strains by 16S rRNA gene sequence analysis. Southern hybridization analysis with oligonucleotide probes detected, on average, one to two copies of pmoA and one to three copies of 16S rRNA genes. Only one restriction length polymorphism pattern was shown for pmoA genes in each isolate, and in cases where, sequencing was done, the pmoA copies were found to be almost identical. PCR primers were developed for mmoX which amplified 1.2-kb regions from all six strains that tested positive for cytoplasmic soluble methane mono-oxygenase (sMMO) activity. Phylogenetic analysis of the translated PCR products with published mmoX sequences showed that MmoX falls into two distinct clusters, one containing the orthologs from type I strains and another containing the orthologs from type II strains. The presence of sMMO-containing Methylomonas strains in a pristine freshwater lake environment suggests that these methanotrophs are more widespread than has been previously thought.     

Mechanism of Catechin Chemiluminescence in the Presence of Active Oxygen

The photon emission (chemiluminescence; CL) of catechin in the presence of active oxygen species (hydrogen peroxide, hydroxyl radical tert-butyl hydroperoxide and tert-butyl oxyl radical) and acetaldehyde was confirmed to occur non-enzymatically at room temperature in aqueous neutral conditions. The CL intensity [P] in the presence of active oxygen species (X), catalytic species (Y) and receptors (Z) is predicted by [P] = k [X] [Y] [Z]. The calculated photon constants (k) of 8 catechins and gallic acid were 8.23 × 10⁶ M⁻² s⁻¹ counts ((−)-epigallocatechin), 2.78 × 10⁶ ((−)-epigallocatechin gallate), 4.66 × 10⁵ ((−)-gallocatechin gallate), 4.36 × 10⁵ ((−)-gallocatechin), 2.70 × 10⁵ ((−)-epicatechin), 6.44 × 10⁴ ((−)-catechin), 5.85 × 10⁴ ((−)-epicatechin gallate), 4.78 × 10⁴ (gallic acid) and 3.54 × 10⁴ ((−)-catechin gallate), respectively. The system of active oxygen species, catalytic species and receptors is proposed to be a scavenging mechanism for active oxygen species. In the presence of acetaldehyde, (−)-epigallocatechin (maximum k value among catechins tested) reacted with tert-BuOOH to form tert-BuOH as determined by HPLC analysis.