Comparative analysis of amplified fragment length polymorphism and pulsed field gel electrophoresis in a hospital outbreak and subsequent endemicity of ampicillin-resistant Enterococcus faecium

Reliable molecular methods for determination of relatedness between bacterial isolates have become increasingly important to evaluate outbreaks and endemic situations with nosocomial pathogens. In the present study Simpson's index of diversity with calculated confidence intervals was used to compare amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE) analysis of a hospital outbreak of ampicillin-resistant Enterococcus faecium and subsequent endemicity. The outbreak, in a Norwegian tertiary hospital, of infections caused by these enterococci started in 1995 and increased in 1996 after which the situation turned endemic. The purpose of this study was to compare the two methods in this setting and to determine the length of time during an outbreak that these methods are sufficiently valid to be of value for hospital infection control efforts. One hundred and sixty clinical isolates from urine specimens collected during the period 1995-1999 were included. The findings indicate that PFGE and AFLP are equally discriminative and could in this setting be used for typing purposes over the whole 5-year period.     

A rapid pulsed-field gel electrophoresis method of genotyping Haemophilus parasuis isolates

Aims: To develop a modified pulsed‐field gel electrophoresis (PFGE) method for characterizing Haemophilus parasuis isolates. Methods and Results: A modified PFGE procedure was designed using CpoI to generate restriction maps of H. parasuis genomic DNA. This approach was used to characterize 47 H. parasuis clinical isolates and 15 reference strains. All strains could be typed by this method, and the procedure was completed in 36 h. A total of 39 different PFGE patterns were identified among 47 epidemiologically unrelated clinical isolates. Conclusions: The modified PGFE described in this report efficiently characterized H. parasuis isolates. This method can be adopted for studying the epidemiology of Glässer’s disease outbreaks in addition to differentiating and classifying previously untypeable H. parasuis isolates. Significance and Impact of the Study: The modified PFGE method described is a novel means of characterizing H. parasuis isolates. It is also a highly discriminatory molecular typing method (discriminatory index of 0·98) that can overcome the limitations of serotyping.     

不同地区宋内志贺菌耐药性及脉冲场凝胶电泳分型分析

摘要:【目的】通过对不同地区的宋内志贺菌株进行药物敏感性检测、耐药基因的扩增以及基因分型,了解不同地区宋内志贺菌的耐药情况与流行趋势。【方法】使用微量肉汤稀释法测定了54 株宋内志贺菌对21种药物的敏感性,用PCR方法扩增相关耐药基因,利用脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis,PFGE)技术进行宋内志贺菌的基因分型,最后采用BioNumerics分析软件对所有菌株进行聚类,分析其相似度。【结果】实验菌株对于甲氧苄氨嘧啶/磺胺甲噁唑、四环素、替卡西林/棒酸、氨苄西林、庆大霉素5种抗生素普遍耐药,对亚胺培南、头孢吡肟、左氟沙星、诺氟沙星、阿米卡星5种抗生素全部表现为敏感。共检测出包括blaTEM,blaCTX以及整合子在内的7 种不同的耐药基因。全部实验菌株可分为26个不同的PFGE带型,分型后表现出较高的基因同源性。宋内志贺菌的耐药性、携带基因与带型具有一定地域相关性。【结论】目前各地区流行的宋内志贺菌株对甲氧苄氨嘧啶/磺胺甲噁唑与四环素已经普遍耐药,不同地区出现相同聚类分型的菌株表明存在跨区域流行的宋内志贺菌群。因此,加强对不同地区宋内志贺菌的监控对减少多重耐药菌株的产生具有重要意义。     

Emergence of CC17 Enterococcus faecium: from commensal to hospital-adapted pathogen

For many years, Enterococcus faecium was considered to be a commensal of the digestive tract, which only sporadically caused opportunistic infections in severely ill patients. Over the last two decades, vancomycin-resistant E. faecium (VREF) has emerged worldwide as an important cause of nosocomial infections, especially in immunocompromised patients. The global Vancomycin-resistant enterococci (VRE) epidemic was preceded by the emergence of ampicillin-resistant E. faecium (AREfm) in the United States in the early 1980s, followed by the rapid emergence of VRE in the 1990s. A similar increase of VRE may occur in countries with still low levels of VRE in hospitals (such as The Netherlands), but increasing incidence of AREfm infections. Molecular epidemiological studies of both human- and animal-derived E. faecium isolates using multilocus sequence typing revealed the existence of host-specific genogroups, including a specific genetic lineage designated CC17, associated with hospital-related isolates. These strains were characterized by ampicillin and quinolone resistance. In addition, the majority of these CC17 isolates contain over hundred hospital-clade-specific genes, including mobile elements, phage genes and plasmid sequences, hypothetical and membrane proteins and antibiotic and regulatory genes and a putative pathogenicity island including the esp gene.     

Frequent occurrence of multidrug‐resistant CC17 Enterococcus faecium among clinical isolates in Sweden

Aims: To screen for the globally spread cluster of Enterococcus faecium, clonal complex 17 (CC17) and characterize the genetic profile of Swedish clinical Ent. faecium isolates. Methods: A total of 203 consecutive isolates collected from 2004 to 2007 from patients with bacteraemia in Sweden. All isolates were genotyped using multiple‐locus variable‐number tandem repeat analysis (MLVA) and 20 isolates representing different MLVA types (MT) were chosen for multilocus sequence typing (MLST). Minimal inhibitory concentrations against clinically relevant antibiotics were determined with agar dilution. Presence of the virulence genes esp and hyl was investigated using PCR. Results: A total of 65% (n = 109) of all isolates belonged to MT‐1, and the second most common MLVA type was MT‐159 (13%, n = 21). MLST analysis confirmed the presence of CC17 during the entire study period. The number of isolates resistant to gentamicin and vancomycin, as well as the presence of hyl, increased significantly during the investigation period. Conclusions: The present study demonstrates that nosocomial infections caused by Ent. faecium CC17 are commonly occurring in Sweden. Significance and Impact of the Study: This is the first report of CC17 Ent. faecium in Sweden. The increase of antibiotic resistance and virulence indicates that these strains are further adapting to the hospital environment.     

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.     

400株肠球菌临床耐药性分析

了解2001~2003年肠球菌对常用抗菌药物的耐药性,为临床治疗肠球菌感染提供参考。采用Kirby-Bauer纸片扩散法进行药敏试验。结果显示:2001~2003年肠球菌属的耐药率呈上升趋势。万古霉素和替考拉宁依然是敏感性最高的抗生素。青霉素、氨苄西林和呋喃妥因对粪肠球菌具有较好的抗菌活性,敏感率分别为62.5%、67.1%和92.4%。屎肠球菌对除糖肽类抗生素以外的所有临床常用抗菌药物显示高水平耐药。可见,动态监测肠球菌的耐药状况对指导临床治疗具有重要意义。     

Cross‐transmission of clinical Enterococcus faecium in relation to esp and antibiotic resistance

Aims: To investigate clonality among clinical Enterococcus faecium isolates and normal intestinal microflora isolates as well as cross‐transmission between patients in relation to the presence of the esp gene and antibiotic resistance. Methods and Results: Blood‐culture isolates (n = 101) deriving from tertiary, secondary and primary hospitals were analysed. Antibiotic susceptibility was investigated. Polymerase chain reaction and pulsed‐field gel electrophoresis were used for detection of esp and genotyping, respectively. Nearly half (43%) of the patients included were involved in a cross‐transmission event with Ent. faecium. These strains disseminated both within and between all hospitals. The antibiotic resistance and presence of esp were highest in isolates from the tertiary hospital. Isolates harbouring esp showed less genetic diversity compared with esp negative ones. Conclusions: Cross‐transmission with Ent. faecium between patients was readily detected, indicating that hospital‐adapted clones circulate within and between hospitals. Acquired characteristics, such as antibiotic resistance and esp, seem to accumulate in the isolates disseminating in the tertiary hospital. Significance and Impact of the Study: It is important to characterize Ent. faecium isolates causing infections and to determine the extent of dissemination in order to prevent further spread of these pathogens.     

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.     

屎肠球菌的临床分布与耐药性分析

目的了解医院屎肠球菌的临床分布和耐药情况,为临床抗感染的预防与治疗提供参考。方法回顾性分析1999年1月至2011年12月临床标本中分离的1161株屎肠球菌;用WHONET5．6软件分析耐药率变迁。结果临床分离的1161株屎肠球菌,在同期分离的1944株肠球菌属中占59．72％。主要分离自尿液和血液,分别占40．91％和26．87％;主要分离自外科病区、内科病区、ICU和儿科病区的菌株,分别占29．37％、25．15％、13．95％和13．53％;屎肠球菌对多种抗菌药物耐药,对万古霉素、替考拉宁和利奈唑胺的耐药率较低,分别为1．04％、0．94％和1．85％。结论屎肠球菌在临床的分离率逐年增加,已成为医院内感染的主要病原菌之一,其多药耐药和高耐药现象相当严重,目前万古霉素、替考拉宁和利奈唑胺仍然是治疗肠球菌属引起感染的有效药物。     

DNA profiling of Stenotrophomonas maltophilia by PCR targeted to its species-specific repetitive palindromic sequences

Aims: The aim of this study was to develop a simple protocol for a PCR‐based fingerprinting of Stenotrophomonas maltophilia (SmrepPCR) that utilizes primers complementary to repetitive extragenic palindromic elements (REPs) of this micro‐organism. Methods and Results: The relatedness of 34 isolates of environmental and clinical origin was investigated by two SmrepPCRs with two different primers, gyrB sequencing and XbaI macrorestriction followed by pulsed‐field gel electrophoresis. While SmrepPCR (with primer DIR) results matched data obtained from the analysis of gyrB nucleotide sequences and identified several clonal complexes, XbaI macrorestriction showed high level of heterogeneity between isolates. The macrorestriction‐based clustering of isolates did not correspond to both gyrB and DIR‐SmrepPCR grouping. Conclusions: Our results show that SmrepPCR‐inferred relationship of isolates is in a good agreement with sequence‐based methods. The combined information from all methods used suggests that rapid evolution of S. maltophilia genomes might be predominantly due to high rate of rearrangements caused by mobile genetic elements. Significance and Impact of the Study: The presented method is an inexpensive and easy to perform alternative to genotype S. maltophilia isolates and to study their population genetics. SmrepPCR demonstrates the usefulness of species‐specific repetitive elements in genomic analyses.     

Induction of vancomycin resistance in Enterococcus faecium by inhibition of transglycosylation

Vancomycin resistance has recently been recognized among clinical isolates of enterococci. Resistance is inducible, and associated with production of a novel 39 kDa membrane protein. The mechanism by which exposure to vancomycin, which does not penetrate the cell membrane, induces resistance is unknown. In the vancomycin resistant strain Enterococcus faecium 228, resistance was also inducible by moenomycin, suggesting that inhibition of the transglycosylation step in peptidoglycan synthesis may be required for induction of resistance. Cytoplasmic pools of peptidoglycan precursors were increased after exposure to vancomycin or moenomycin, representing a potential means for regulation of induction.     

vanA-mediated high-level glycopeptide resistance in Enterococcus faecium from animal husbandry

Abstract Glycopeptide-resistant Enterococcus faecium strains were isolated from a pig farm and a poultry farm both using avoparcin as a food additive. Such organisms were not isolated in a hen's eggs-producing farm not using avoparcin. Glycopeptide-resistant enterococci were also detected in broiler chicken carcasses that were delivered to a hospital's kitchen. The resistance was determined by the vanA gene as indicated by the detection of the inducible 39-kDa cytoplasmic membrane protein and of a vanA -specific DNA sequence amplified by polymerase chain reaction. Genomic DNA fragment patterns of strains from animal sources were different from each other and also from those of strains isolated in hospitals and from sewage treatment plants. This findings suggest the dissemination of the vanA determinant among different enterococcal strains of distinct ecological origin.     

Comparison of enterococcal populations related to urban and hospital wastewater in various climatic and geographic European regions

AIMS: Scarce knowledge about the distribution of enterococci species in wastewaters limits any statement on their reliability as faecal indicators or the implications of antibiotic resistance transmission by these organisms through the water cycle. Enterococci have been involved in nosocomial infections and the spreading of antibiotic resistance through the food chain. The species distribution of enterococci and the presence of resistant strains to vancomycin and erythromycin were analysed in more than 400 raw and treated urban wastewaters, surface waters receiving these treated wastewaters and hospital wastewaters from three European countries. METHODS AND RESULTS: A total of 9296 strains were isolated and biochemically phenotyped. The species identification was based on the comparison of biochemical profiles with those of more than 20000 enterococci isolates from an international study. The prevalence of enterococcal isolates resistant to erythromycin (ERE) and vancomycin (VRE) was also analysed. ERE strains were present in a high proportion in all the studied samples. VRE strains were also isolated in all studied countries despite the time elapsed since the use of antimicrobial glycopeptides in animal production was banned in the European Union. CONCLUSIONS: Enterococcus faecalis and Ent. faecium were the most abundant species in all the studied wastewaters. All the studied wastewaters demonstrated high diversity and similar population structure and composition. ERE and VRE isolates were detected in most of the wastewaters. SIGNIFICANCE AND IMPACT OF THE STUDY: Urban and hospital wastewaters are useful targets for the evaluation of the prevalence of ERE and VRE isolates in the environment. It appears that these bacteria could pass through wastewater treatment plants and be transferred to surface waters.     

Induction of vancomycin resistance in Enterococcus faecium by non-glycopeptide antibiotics

Abstract Bacitracin and other antibiotics that inhibit late stages in peptidoglycan biosynthesis induce vancomycin resistance in a high-level, inducibly vancomycin-resistant strain of Enterococcus faecium . Exposure to bacitracin led to synthesis of the lactate-containing UDP-MurNAc-pentadepsipeptide precursor required for vancomycin resistance. These findings indicate that inhibition of peptidoglycan biosynthesis can lead to induction of vancomycin resistance and raise the possibility that multiple signals may serve to induce resistance.     

2000年至2006年屎肠球菌的临床分离与耐药变迁

目的了解本地区屎肠球菌在临床的分离与耐药变迁情况,为临床抗感染的预防与治疗提供帮助。方法用WHONET 5软件统计分析我院2000年至2006年屎肠球菌临床分离株在各病区、样本中的分布与耐药性的变迁情况。结果分离率呈逐年上升趋势,从2000年的0.32%上升到2006年的0.81%;7年中以重症监护病区（ICU）分离菌株最高,占总分离菌株的68.9%,其次为肾内科病区占13.5%;在送检标本中以尿液标本分离菌株数最高,占总分离数的62.3%,其次为痰液（10.2%）。对11种抗生素的耐药性分析显示,屎肠球菌对青霉素G、氨苄西林、庆大霉素500、环丙沙星、左旋氧氟沙星呈较高的耐药率,而对四环素、呋喃妥因、链霉素2000相对较低;更值得我们注意的是对于万古霉素的耐药率呈逐年上升趋势,对于喹奴普汀/达福普汀、力奈唑烷这两类临床还未运用的抗生素已有一定的耐药率。结论屎肠球菌在临床的分离率在逐年增加,已成为医院内感染的主要病原菌之一,该菌呈多重耐药的特性,并呈不断增加趋势,临床抗感染治疗应以分离菌株的体外抗菌药物敏感性为依据。     

Clonal groups of high-level gentamicin-resistant Enterococcus faecium isolated from municipal wastewater and clinical samples in Tehran, Iran

Aims:  Clonality among high-level gentamicin-resistant Enterococcus faecium (HLGR-EF) isolates obtained from clinical and sewage treatment plants (STP) were investigated using PhePlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE).
Methods and Results:  During 1 year study (September 2005–2006), a total of 106 HLGR-EF isolates were collected from clinical ( n  = 48) and STP ( n  = 58) samples in Tehran, Iran. Biochemical fingerprinting of these isolates using the PhP showed the presence of 21 PhP types (diversity index, Di  = 0·97) among the clinical and 21 PhP types ( Di  = 0·91) among the STP isolates. Representative isolates of each PhP type ( n  = 42) were further characterized by the ribotyping method. Sixteen ribotypes were identified among the isolates with five types shared between the clinical and STP isolates. PFGE recognized 24 clonal types among these isolates with three pulsotypes shared between the clinical and STP isolates. Combination of the two techniques (PFGE and ribotyping) resulted in 24 ( Di  = 0·96) and 16 ( Di  = 0·93) types among the strains isolated from clinical and STP samples, respectively.
Conclusions:  We concluded that the combination of PhP typing, ribotyping and PFGE could be extremely discriminatory when examining HLGR-EF isolates.
Significance and Impact of the Study:  The emergence of highly diverse HLGR-EF population in Iran is of serious concern especially because of their multi-resistances.     

Characterization of a bacteriocin produced by Enterococcus faecium GM-1 isolated from an infant

AIM: To partially characterize the bacteriocin produced by the GM-1 strain of Enterococcus faecium, isolated from the faeces of a newborn human infant. METHODS AND RESULTS: The bacteriocin produced by E. faecium GM-1 showed a broad spectrum of activity against indicator strains of Escherichia coli, Staphylococcus aureus, Vibrio spp., Salmonella typhimurium, Listeria monocytogenes, Lactobacillus acidophilus, and Streptococcus thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 degrees C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from E. faecium GM-1 was obtained when the culture conditions were pH 6.0-6.5 and 35-40 degrees C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of E. faecium GM-1 by PCR and direct sequencing methods. CONCLUSION: E. faecium GM-1, which was isolated from the faeces of a newborn baby, produces an enterocin P-like bacteriocin with inhibitory activity against Gram-positive and Gram-negative bacteria, including food-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium GM-1, isolated from infant faeces, produces a new bacteriocin that is similar to enterocin P. This bacteriocin is heat stable and has a broad antibacterial spectrum that includes both Gram-positive and Gram-negative bacteria.     

Adherence to host extracellular matrix and serum components by Enterococcus faecium isolates of diverse origin

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I (CI) as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm -mediated CI adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (Fn) (20% of the 60 isolates), fibrinogen (17%) and laminin (Ln) (13%), while only one or two of the isolates adhered to collagen type V (CV), transferrin or lactoferrin and none to the other host components tested. Adherence to Fn and Ln was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to Fn and Ln, in addition to CI, may have contributed to the emergence and adaptation of E. faecium , in particular CC17, as a nosocomial pathogen.