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.     

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.     

A thioredoxin fusion protein of VanH, a D-lactate dehydrogenase from Enterococcus faecium: cloning, expression, purification, kinetic analysis, and crystallization.

The gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D-lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition site under a variety of conditions resulted in nonspecific proteolysis and inactivation of the protein. The intact fusion protein was, therefore, used for kinetic studies and crystallization trials. It has been purified to greater than 90% homogeneity by ammonium sulfate precipitation followed by phenyl Sepharose chromatography. Based on k(cat)/KM for pyruvate, it is 20% as active as native VanH. Michaelis constants for NADPH, NADH, and pyruvate, of approximately 3.5 microM, 19.0 microM, and 1.5 mM, respectively, were comparable to those reported for the native VanH (Bugg TDH et al., 1991, Biochemistry 30:10408-10415). Like native VanH, maximum activity of the fusion protein requires the presence of an anion (phosphate or acetate), however, in addition, a strongly reducing environment is needed for optimal efficacy. Competitive inhibition constants for ADP-ribose, NAD+, and oxamate have also been determined. Crystallization by hanging drop vapor diffusion produced two different crystal forms, one hexagonal and the other tetragonal. Flash-frozen crystals of the tetragonal form diffracted to 3.0 A resolution at a synchrotron radiation source.     

Occurrence of multidrug-resistant Enterococcus faecium isolated from environmental samples

Enterococcus species are present in the microbiota of humans and animals and have also been described in the environment. Among the species, Enterococcus faecium is one of the main pathogens associated with nosocomial infections worldwide. Enterococcus faecium isolates resistant to different classes of antimicrobials have been increasingly reported, including multidrug-resistant (MDR) isolates in environmental sources, which is worrying. Therefore, this study aimed to characterize E. faecium isolates obtained from soil and water samples regarding antimicrobial resistance and virulence determinants. A total 40 E. faecium isolates were recovered from 171 environmental samples. All isolates were classified as MDR, highlighting the resistance to the fluoroquinolones class, linezolid and vancomycin. Furthermore, high-level aminoglycoside resistance and high-level ciprofloxacin resistance were detected in some isolates. Several clinically relevant antimicrobial resistance genes were found, including vanC1, ermB, ermC, mefAE, tetM, tetL, ant(6′)-Ia, ant(4′)-Ia, aph(3′)-IIIa and aac(6′)-Ie-aph(2″)-Ia. Three virulence genes were detected among the MDR E. faecium isolates, such as esp, gelE and ace. The results of this study contribute to a better understanding of MDR E. faecium isolates carrying antimicrobial resistance and virulence genes in environmental sources and report for the first time in the world the presence of vanC1-producing E. faecium isolated from soil.     

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.     

Characterization of new insertion-like sequences of Enterococcus hirae and their dissemination among clinical Enterococcus faecium isolates

Sequence analysis of different fragments that hybridized with a 4.5-kb EcoRI fragment originally cloned from Enterococcus hirae ATCC 9790 showed 66% homology to IS-like sequences found in staphylococci and lactococci. We tested several enterococcal ATCC strains and found that only E. hirae ATCC 9790 and Enterococcus faecium ATCC 19434 hybridized with the IS-like sequence. Moreover, we wanted to investigate the dissemination of this new IS among E. faecium strains. We analyzed 131 clinical E. faecium isolated in Italy and the USA for the presence of the IS and we found its presence in more than 63% of the isolates. The hybridization patterns obtained vary considerably between unrelated strains and allow further classification among ribotype-grouped species.     

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.     

重庆市1264株粪肠球菌和屎肠球菌耐药性监测

【摘 要】 目的 了解2011年中国重庆市主要7所教学医院临床分离粪肠球菌和屎肠球菌对各类抗菌药物的耐药性。方法 重庆市主要7所教学医院(6所综合性医院,1所儿童医院)按统一方案、采用统一的材料、方法和判断标准(CLSI 2011年版)进行粪肠球菌和屎肠球菌的耐药性监测。数据用WHONET 5.5软件按照CLSI 2011年版折点进行分析。结果 共分离到非重复粪肠球菌589株、屎肠球菌675株,对利奈唑胺、万古霉素、替考拉宁仍极敏感,耐药率<2%,万古霉素耐药粪肠球菌和屎肠球菌检出率分别为0.3%、0.7%。粪肠球菌对青霉素、氨苄西林、呋喃妥因的耐药率较低,分别为14.8%、8.6%和5.1%,对高浓度庆大霉素的耐药率分别为46.9%;屎肠球菌耐药性明显高于粪肠球菌,对青霉素和氨苄西林耐药率接都在90％左右。儿童和成人耐药率存在一定差别。结论 本市医院肠球菌感染以屎肠球菌为主, 粪肠球菌次之,两者耐药性明显不同, 监测其耐药情况对指导临床用药具有重要意义。     

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.     

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.     

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.     

Drug resistance of Enterococcus faecium clinical isolates and the conjugative transfer of gentamicin and erythromycin resistance traits

Drug resistance and the transferability of resistance were examined in 218 Enterococcus faecium clinical isolates obtained from in-patients of a Japanese university hospital between 1990 and 1999. One hundred and sixty one isolates (73.9%) were drug-resistant and 127 (58.2%) isolates were resistant to two or more drugs. Vancomycin resistant E. faecium (VRE) was not isolated. The transferability of drug-resistance to an E. faecium strain was examined by broth or filter mating. Six (12.5%) of the 48 gentamicin resistance traits, and fifty (50%) of the 101 erythromycin resistance traits were transferred by filter mating. The gentamicin resistance traits of five isolates and the erythromycin resistance traits of four isolates were transferred to the recipient strains by both broth mating and filter mating at a frequency of about 10(-6) and 10(-5) per donor cell, respectively. The five gentamicin resistant strains were shown to harbor pMG1-like plasmids on the basis of their Southern hybridization with pMG1 (65.1 kbp, Gm(r)), which transfers efficiently between enterococci by broth mating. Each of the four erythromycin resistant transconjugants obtained by broth mating harbored a large conjugative plasmid (more than 100 kbp). The plasmids showed no homology with well-characterized enterococcal conjugative plasmids such as pAD1, pPD1, pAM(beta)1, pIP501 and pMG1 by Southern hybridization. Of the erythromycin resistance traits that transferred only by filter mating, it was found that the erythromycin resistance trait was conferred by a 47-kbp transposable element that transferred from the chromosome of the donor strain to different sites within the pheromone responsive plasmid pAD1 (60 kbp) of the recipient strain, suggesting that the erythromycin resistance trait was encoded on a conjugative transposon, which was named Tn950.     

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.