Effect of banning vancomycin analogue avoparcin on vancomycin-resistant enterococci in chicken farms in Taiwan

Avoparcin, a vancomycin analogue, was banned as a feed additive in Taiwan in 2000. A nationwide surveillance was conducted to study the prevalence of vancomycin-resistant enterococci (VRE) on chicken farms between 2000 and 2003. Among the 1021 E. faecalis and 967 E. faecium isolates studied, resistance to tetracycline, erythromycin, high-level aminoglycosides, ciprofloxacin and chloramphenicol either increased or remained high except vancomycin. The proportion of VRE decreased, between 2000 and 2003, from 13.7% (22/161) to 3.7% (11/299) for E. faecalis, and 3.4% (4/119) to 0% (0/300) for E. faecium. Only 8.8% (7/80) of the chicken farms surveyed harboured VRE in 2003 compared with 25% (15/60) in 2000. All VRE were resistant to tetracycline and erythromycin. All VRE possess the vanA gene but nearly all (79 of 83 isolates) were susceptible to teicoplanin, indicating VanB phenotype. Some clones were detected from different farms in various regions over the years. We conclude that the frequency of VRE in chicken farms decreased in association with a ban on avoparcin; and the continued presence of VRE may be due to the ability of some strains to persist in the farms, transfer of vancomycin resistance determinants or co-selection by the continued use of other antibiotics.     

Conjugal transfer of the vancomycin resistance determinant vanB between enterococci involves the movement of large genetic elements from chromosome to chromosome

Abstract Resistance to various levels of vancomycin and susceptibility to teicoplanin in enterococci (VanB phenotype) is mediated by the vanB gene cluster. VanB-type resistance was transferred by intra- and inter-specific conjugation between different strains of Enterococcus . Analysis of S⨍i I-digested genomic DNA by zero integrated-field electrophoresis followed by Southern hybridization revealed vanB -containing chromosomal insertions of approximately 90–250 kb in the transconjugants. Thus, transfer of VanB-type resistance is associated with the movement of large genetic elements from chromosome to chromosome.     

肠球菌万古霉素耐药基因簇遗传特性

万古霉素耐药肠球菌自20世纪80年代后期被发现以来,已逐渐发展成为重要的医院感染病原菌。此类耐药肠球菌携带的万古霉素耐药基因簇编码产物可催化合成与万古霉素、替考拉宁等糖肽类抗生素亲和力极低的细胞壁前体导致耐药。目前已在肠球菌中发现的万古霉素耐药基因簇根据基因序列及构成不同分为9个型别;依据它们编码的连接酶合成产物不同又可分为D-Ala:D-Lac连接酶基因簇(VanA、VanB、VanD及VanM型)和D-Ala:D-Ser连接酶基因簇(VanC、VanE、VanG、VanL和VanN型)。这些耐药基因簇介导的耐药水平及其传播模式各有特点。文章综述了肠球菌中万古霉素耐药基因簇的类型、基因构成及传播特性。     

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopeptide resistance by conjugative transfer of VanA elements.     

Isolation of Tn1546-like elements in vancomycin-resistant Enterococcus faecium isolated from wood frogs: an emerging risk for zoonotic bacterial infections to humans

Aim: Isolation and characterization of vancomycin‐resistant enterococci (VRE), mainly Enterococcus faecium, from the faecal pellet of wood frogs (Rana sylvatica). Methods and Results: The frog VRE isolates were tested for their susceptibility to various antibiotics and were found resistant to ampicillin (Am), chloramphenicol (Cm), erythromycin (Em), gentamicin (Gm), tetracycline (Tc), teicoplanin (Tp) and vancomycin (Vn). The linkage of multiple antibiotic resistances to Em, Tc, Tp and Vn was observed in 84% of resistant Ent. faecium. Inducible antibiotic resistance (MIC ≥ 512 μg ml^?1) to Vn was also detected in these isolates. PCR analysis revealed the presence of vanA in all strains, and none of the strains were positive for vanB, indicating the existence of vanA phenotype. Furthermore, the PCR–RFLP analysis of the frog vanA amplicon with PstI, BamHI and SphI generated identical restriction patterns similar to Tn1546‐like elements found in human VRE isolates. DNA homoduplex analysis also confirmed that vanA from the frog VRE has DNA sequence homology with the vanA of Tn1546‐like elements of human and animal isolates. Blastx analysis of frog vanA sequence showed similarities with protein sequences generated from protein database of Vn‐resistant Ent. faecium, Baccilus circulans, Paenibacillus apiarius and Oerskovia turbata isolates. Horizontal transfer of Vn resistance was not detected in frog isolates as revealed by filter mating conjugal experiment. Conclusions: In summary, our results demonstrated that wood frogs carry Vn‐resistant bacteria, and resistance genes (vanA) are located on Tn1546‐like elements. Significance and Impact of the Study: This study highlights a previously less recognized role of amphibians as sentinels for multidrug‐resistant bacteria and alerts the public health workers for an emerging risk of zoonotic bacterial infections to humans.     

Risk factors for vancomycin-resistant enterococci colonisation in critically ill patients

Vancomycin-resistant enterococci (VRE) are important hospital pathogens and have become increasingly common in patients admitted to the intensive care unit (ICU). To determine the incidence and the risk factors associated with VRE colonisation among ICU patients, active surveillance cultures for VRE faecal carriages were carried out in patients admitted to the ICU of the University Hospital of Uberlandia, Minas Gerais, Brazil. Risk factors were assessed using a case-control study. Seventy-seven patients (23.1%) were found to be colonised with vanC VRE and only one patient (0.3%) was colonised with vanA VRE. Independent risk factors for VRE colonisation included nephropathy [odds ratio (OR) = 13.6, p < 0.001], prior antibiotic use (OR = 5.5, p < 0.03) and carbapenem use (OR = 17.3, p < 0.001). Our results showed a higher frequency (23.1%) of Enterococcus gallinarum and Enterococcus casseliflavus, species that are intrinsically resistant to low levels of vancomycin (vanC), without an associated infection, associated with prior antibiotic use, carbapenem use and nephropathy as comorbidity. This study is the first to demonstrate the risk factors associated with vanC VRE colonisation in ICU hospitalised patients. Although vanA and vanB enterococci are of great importance, the epidemiology of vanC VRE needs to be better understood. Even though the clinical relevance of vanC VRE is uncertain, these species are opportunistic pathogens and vanC VRE-colonised patients are a potential epidemiologic reservoir of resistance genes.     

Time-kill study and synergistic activity of cell-wall inhibitor antibiotics in combination with gentamicin against Enterococcus faecalis and Enterococcus faecium

The synergy between gentamicin and vancomycin, teicoplanin, ampicillin and linezolid was studied by time-kill method. Two clinical vancomycin resistant enterococci (VRE) and two vancomycin susceptible enterococci (VSE) isolates were used. Different concentrations of antibiotics were combined. Two VSE strains and the control strain exhibited synergism with the combination of gentamicin, vancomycin, teicoplanin, ampicillin and linezolid. Two VRE strains exhibited synergism with the combination of gentamicin and ampicillin. Synergy between gentamicin and vancomycin, teicoplanin and linezolid was not observed against these isolates. The VRE isolates were positive for vanA, aac (6')-Ie aph (2") and aph (3')-IIIa genes and their vancomycin, teicoplanin and gentamicin MICs were 512 μg/ml, 512 μg/ml and >4000 μg/ml, respectively. In order to treat serious enterococcal infections, further clinical evaluation is needed to examine the in vitro combined effects of gentamicin and vancomycin, teicoplanin and linezolid.     

Presence of vancomycin and ampicillin-resistant Enterococcus faecium of epidemic clonal complex-17 in wastewaters from the south coast of England

The occurrence and diversity of vancomycin-resistant enterococci (VRE) in wastewaters from the Brighton and Hove area of south-east England were investigated. VRE were recovered from 71% of raw urban wastewater samples, 22% of treated urban wastewater samples, 15% of hospital wastewater sample and 33% of farm wastewater samples. Two hundred and eighty-eight isolates were typed and identified and the minimum inhibitory concentrations (MICs) to six antibiotics were determined for selected VRE. Vancomycin-resistant Enterococcus faecium (VREF) strains with a vancomycin MIC of more than 32 μg ml⁻¹ were examined by polymerase chain reaction for the vanA , vanB and esp genes. Twenty-three VREF with the vanA or vanB gene were further analysed by multilocus sequence typing which revealed that a cluster of VREF from both hospital and urban wastewaters belonged to the high-risk, epidemic, clonal complex-17 (CC17). Vancomycin-resistant Enterococcus faecium belonging to the CC17 group contained the purK-1 allele, were resistant to ampicillin and frequently ciprofloxacin, and usually contained the esp gene. To the authors' knowledge, this is the first report of CC17 strains isolated from urban wastewaters in the UK, and indicates that certain clones carrying antibiotic resistance or virulence traits indicative of the hospital environment can be detected in the urban wastewater system.     

Determination of antibiotic resistance and resistance plasmids of clinical Enterococcus species

To determine the antibiotic resistance pattern and resistance plasmids, we studied 23 antibiotic-resistant clinical isolates of Enterococcus spp. which caused infection in Bayindir-Ankara Hospital, Turkey. Biochemical and physiological identification tests were applied by the Vitek system and compared with the results of protein profiles by SDS-PAGE. From 23 isolates, 20 were identified as E. faecalis, 2 as E. faecium and 1 as E. gallinarum. Twenty four antibiotics belong to 10 different groups were used in susceptibility tests. Multiple antibiotic resistance was determined in 10 of 23 Enterococcus spp. Overall resistance to the used antibiotics was 47.3% and low level resistance was 16.6%. Among the isolates tested, 8.7% demonstrated high level gentamicin resistance, 17.4% demonstrated high level streptomycin resistance, and 43.5% demonstrated penicillin resistance. High level vancomycin resistant Enterococcus spp. rate was 34.8%, and 60.9% exhibited low level resistance to vancomycin and teicoplanin. They contain plasmids which varied in numbers between 1 and 11 and the plasmid sizes ranged from 2.08 to 56.15 kb. In curing experiments with acriflavine, two different plasmids were shown in different molecular sizes of 33.49 and 13.6 kb while the first determined glycopeptide and penicillin resistance, the second one determined either glycopeptide or penicillin resistance in two different E. faecalis strains. On the other hand, a 22.58 kb plasmid, determining kanamycin resistance, was detected in an E. faecium strain. After the curing experiments, an elimination of 37.17 and 44.47 kDa protein bands was shown in E. faecium EFA1 and E. faecalis EFA13 in SDS-PAGE, respectively. This survey indicates the increase of antibiotic-resistant enterococci, especially to vancomycin in our hospital isolates.     

Vancomycin-Resistant Enterococci in a Chinese Hospital

In investigating the prevalence of vancomycin-resistant enterococci (VREs) in a hospital in China, 338 enterococci were detected by using multiplex polymerase chain reaction. Eleven VREs were found, including one VanA E. faecalis, one VanB E. faecium, three VanB E. faecalis, five VanC₁ E. gallinarum, and one VanC₂ E. flavescens. VITEK 2, microbroth dilution, and E-test were used to determine the susceptibilities of the VREs to certain antimicrobial agents; multiple-drug resistance of the 11 VREs was distinct. Compared with phenotypic methods, multiplex PCR rapidly detected 11 VREs and provided van genotype information. This is the first study that sequenced van genes of VRE isolates in China to date and found type VanB in China. Because of the relatively low isolating rate, early detection of VRE is vital.     

Nosocomial infection caused by vancomycin‐susceptible multidrug‐resistant Enterococcus faecalis over a long period in a university hospital in Japan

Compared with other developed countries, vancomycin‐resistant enterococci (VRE) are not widespread in clinical environments in Japan. There have been no VRE outbreaks and only a few VRE strains have sporadically been isolated in our university hospital in Gunma, Japan. To examine the drug susceptibility of Enterococcus faecalis and nosocomial infection caused by non‐VRE strains, a retrospective surveillance was conducted in our university hospital. Molecular epidemiological analyses were performed on 1711 E. faecalis clinical isolates collected in our hospital over a 6‐year period [1998–2003]. Of these isolates, 1241 (72.5%) were antibiotic resistant and 881 (51.5%) were resistant to two or more drugs. The incidence of multidrug resistant E. faecalis (MDR‐Ef) isolates in the intensive care unit increased after enlargement and restructuring of the hospital. The major group of MDR‐Ef strains consisted of 209 isolates (12.2%) resistant to the five drug combination tetracycline/erythromycin/kanamycin/streptomycin/gentamicin. Pulsed‐field gel electrophoresis analysis of the major MDR‐Ef isolates showed that nosocomial infections have been caused by MDR‐Ef over a long period (more than 3 years). Multilocus sequence typing showed that these strains were mainly grouped into ST16 (CC58) or ST64 (CC8). Mating experiments suggested that the drug resistances were encoded on two conjugative transposons (integrative conjugative elements), one encoded tetracycline‐resistance and the other erythromycin/kanamycin/streptomycin/gentamicin‐resistance. To our knowledge, this is the first report of nosocomial infection caused by vancomycin‐susceptible MDR‐Ef strains over a long period in Japan.     

Phenotypic and genetic characterization of vancomycin-resistant enterococci from hospitalized humans and from poultry in Korea

Vancomycin resistant enterococci (VRE) isolates from humans (23 isolates) and poultry (20 isolates) were characterized by antibiotic susceptibility, vancomycin resistance transferability, pulsed-field gel electrophoresis (PFGE), and structural analysis of Tn1546-like elements. VRE isolates from humans and poultry showed different resistance patterns, transferability, and transfer rate. In addition to these phenotypic differences between humans and poultry VRE, PFGE and the structure of Tn1546-like elements were also distinct. Most poultry isolates (16/20) were identical to the prototype vanA transposon, Tn1546, while most human isolates (21/23) had multiple integrations of insertion sequence. The transmission of VRE and vancomycin resistance determinant between humans and poultry could not be demonstrated in this study.     

Teicoplanin biosynthesis genes in Actinoplanes teichomyceticus

The genetic determinants for the biosynthesis of the glycopeptide antibiotic teicoplanin were identified. In order to isolate the corresponding gene cluster, oligonucleotides derived from highly conserved motifs in peptide synthetases were used. These synthetic probes, and gene fragments derived from the balhimycin gene cluster of Amycolatopsis mediterranei, led to the identification of the likely teicoplanin gene cluster centered on a region of ca. 110 kb from the genome of Actinoplanes teichomyceticus, the teicoplanin producer. Partial nucleotide sequences identified partial ORFs likely to encode two glycosyltransferases, three P-450 monooxygenases and one ABC transporter. The corresponding genes have been found in other glycopeptide gene clusters. Furthermore, upstream to the peptide synthetase region a segment was identified with a remarkable similarity to the vanHAX operon, conferring resistance to glycopeptides in enterococci. Thus, in contrast to the other glycopeptide producers thus far analyzed, in A. teichomyceticusthe genes for teicoplanin biosynthesis are closely linked to homologs of glycopeptide resistance commonly found in vancomycin-resistant enterococci.     

Occurrence and relatedness of vancomycin-resistant enterococci in animals, humans, and the environment in different European regions

Vancomycin-resistant enterococci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 microg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.     

Chemistry and biology of the ramoplanin family of peptide antibiotics

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis.     

Susceptibility to selected chemotherapeutics of Staphylococcus aureus strains resistant to methycyline isolated from clinical materials in the years 1991-1992 and 1997

The susceptibility to selected chemotherapeutic agents was determined in 100 strains of Staphylococcus aureus methicillin-resistant (MRSA) isolated from clinical materials in 1991-1992 (50 strains) and in 1997 (50 strains). Two methods were used for the determination: disc method and antibiotic dilution in agar. The minimal inhibitory concentration (MIC) was determined for vancomycin, teicoplanin, furazolidone, nitrofurantoin, ofloxacin, gentamicin, netilmicin and trimethoprim. The concentrations of the chemotherapeutics in the substrate ranged from 0.125 to 512 mg/l. The obtained results served for drawing of the following conclusions: all studied MRSA strains isolated in 1991-1992 and in 1997 were sensitive to glycopeptide antibiotics: vancomycin and teicoplanin, to nitrofurans: nitrofurantoin and furazolidone, and to fusidic acid. MRSA strains isolated in 1991-1992 were sensitive to ofloxacin, but in 1997 about 80% of the strains were resistant to that antibiotic, and this resistance was noted in S. aureus strains with homogeneous resistance to methicillin. Increasing frequency of resistance to mupirocin was found, in 1991-1992 4% of the strains were resistant, and in 1997 the resistance of MRSA to that antibiotic was found in 12%. No changes occurred in the sensitivity of staphylococci to trimethoprim/sulfamethoxazole (cotrimoxazole). About 94% of strains in 1991-1992 and 1997 were sensitive to that drug. The sensitivity to cotrimoxazole is connected with one of its components (trimethoprim), with 94% of MRSA strains sensitive to it.     

Occurrence and Relatedness of Vancomycin-Resistant Enterococci in Animals, Humans, and the Environment in Different European Regions

Vancomycin-resistant enterococcci (VRE) in Europe are thought to have emerged partly due to the use of the glycopeptide avoparcin in animal husbandry. We compared the occurrence of VRE in geographical regions of Europe in which until 1997 large amounts of avoparcin were used (Spain, United Kingdom, and Denmark) with the occurrence of VRE in Sweden, where avoparcin was banned in 1986. We also studied the relatedness between VRE strains from different regions and habitats. In total, 2,580 samples were collected from humans, animals, and the environment (soil, sewage, recipient water). VRE resistant to 20 μg/ml vancomycin were identified in 8.2% of the samples and were found most frequently in raw and treated urban sewage samples (means, 71% and 36% of the samples, respectively), pig manure (17%), and hospital sewage (16%). The proportions of VRE-positive sewage samples were similar in Sweden, Spain, and the United Kingdom, whereas pig feces and manure were more often positive in Spain than in Sweden (30% versus 1%). Most VRE were Enterococcus faecium carrying vanA, and computerized biochemical phenotyping of the isolates of different ecological origins showed a high degree of polyclonality. In conclusion, it seems that animal-associated VRE probably reflect the former use of avoparcin in animal production, whereas VRE in human-associated samples may be a result of antibiotic use in hospitals. Since there seems to be a reservoir of the resistance genes in all countries studied, precautions must be taken to limit the use of antibiotics and antibiotic-like feed additives.