Analysis of peptidoglycan precursors in vancomycin-resistant enterococci.

Analysis by high-pressure liquid chromatography of the cytoplasmic peptidoglycan precursors of a high- and a low-level vancomycin-resistant Enterococcus spp. was performed before and after induction of resistance. This analysis showed a decrease of the D-Ala-D-Ala and UDP-MurNac-pentapeptide pools, an increase of the UDP-MurNac-tripeptide pool, and the appearance of new UDP-MurNac-containing material. These results lead us to suggest that the vancomycin-induced carboxypeptidase activity cleaves the D-Ala-D-Ala (L. Gutmann, D. Billot-Klein, S. Al-Obeid, I. Klare, S. Francoul, E. Collatz, and J. van Heijenoort, Antimicrob. Agents Chemother. 36:77-80, 1992), which in turn would prevent formation of the normal UDP-MurNac-pentapeptide and thereby of the vancomycin target. The novel UDP-MurNac-containing material is thought to correspond to peptidoglycan precursors which might be synthesized by an alternate pathway (T. D. H. Bugg, G. D. Wright, S. Dutka-Malen, M. Arthur, P. Courvalin, and C. T. Walsh, Biochemistry 30:10408-10415, 1991) and which would be unable to bind vancomycin in glycopeptide-resistant enterococci.     

Inducible carboxypeptidase activity in vancomycin-resistant enterococci.

Vancomycin was found to coinduce DD-carboxypeptidase activity, together with resistance, in eight low- or high-level glycopeptide-resistant strains of enterococci. The constitutively resistant mutant (MT10) of a low-level-resistant strain of Enterococcus faecium (D366) spontaneously expressed a level of carboxypeptidase similar to that of the induced strain D366. Pentapeptide, UDP-MurNac-pentapeptide, as well as D-alanyl-D-alanine were in vitro substrates for the carboxypeptidase which was not inhibited by penicillin. The level of vancomycin resistance correlated roughly with the level of carboxypeptidase activity. We infer from these results that the carboxypeptidase is one component of the glycopeptide resistance mechanism.     

Altered peptidoglycan composition in vancomycin-resistant Enterococcus faecalis.

The muropeptide compositions of isogenic vancomycin-resistant and -susceptible Enterococcus faecalis strains were analyzed by reverse-phase high-performance liquid chromatography combined with amino acid analysis and fast atom bombardment mass spectrometry. Peptidoglycan of the susceptible strain contained pentapeptides as stem peptides, whereas peptidoglycan of the isogenic resistant strain was composed of muropeptides with tetrapeptide stem peptides. Despite the synthesis of lactate-terminating peptidoglycan precursors, no lactate-containing muropeptides were detected in peptidoglycan of the resistant strain. These findings indicate that either lactate-terminating precursors are not incorporated into peptidoglycan of the resistant strain or that the lactate residues are removed from peptidoglycan during synthesis.     

Evidence for biliary excretion of vancomycin into stool during intravenous therapy: potential implications for rectal colonization with vancomycin-resistant enterococci

Sixty-three stool samples and five bile samples were prospectively collected from 33 patients receiving intravenous vancomycin therapy and were quantitatively analyzed for vancomycin by a competitive immunoassay. Vancomycin was excreted via bile into the stools of almost all patients at concentrations of 3.3 to 94.8 microg/ml after >/=5 days of a therapy of 1 g every 12 h.     

Clinical impact of vancomycin-resistant enterococci

In humans, vancomycin-resistant enterococci (VRE) most commonly result in intestinal colonization, which does not result in symptoms, may persist for a long time and serves as a reservoir for transmission of VRE to other patients. Certain VRE-colonized patients are at risk of infection, including haematology and oncology patients, patients in intensive care units and recipients of solid (especially abdominal) organ transplants. Controlling the spread of VRE colonization and preventing colonized patients from becoming infected are important aims. Ramoplanin is a member of a new class of antimicrobial agents; it may have a role in preventing infection in patients colonized with VRE.     

Presence of vancomycin-resistant enterococci in farm and pet animals.

Enterococcus faecium strains with vanA-mediated glycopeptide resistance were isolated by enrichment culture from the intestines and feces of several animal species, mainly horses and dogs (8% positive), chickens (7% positive), and pigs (6% positive). Other vanA-positive enterococcal strains were identified as E. durans in gallinaceous birds, E. faecalis in a horse, and E. gallinarum in a pheasant. Samples from pigeons, cage birds, and ruminants were negative. It was concluded that vancomycin resistance is widespread among isolates from farm and pet animals.     

多重PCR检测万古霉素耐药的肠球菌

目的检测2005～2007年澳大利亚墨尔本地区万古霉素耐药的肠球菌基因。方法用多重PCR检测肠球菌4种耐药基因(vanA,vanB,vanC1和vanC2),鉴定4种肠球菌E.faecalis(粪肠球菌),E.faecium(屎肠球菌),E.gallinarum(母鸡肠球菌)和E.casseliflavus(产黄肠球菌)。结果vanA基因扩增阳性可见732bp条带;vanB基因为635bp条带;vanC1基因为822bp条带;vanC2/3基因为439bp条带。粪肠球菌基因扩增见941bpDNA条带,屎肠球菌为550bpDNA条带。3年回顾性研究中,最常见的万古霉素耐药基因是vanB型(84.9%),菌种是屎肠球菌(76.3%)。2007年还出现2株同时含有vanA和vanB耐药基因的屎肠球菌菌株。结论万古霉素耐药的肠球菌菌株逐年增多,多重PCR检测万古霉素耐药的肠球菌,简便迅速,有利于准确及时地发出报告。     

A PCR assay for rapid detection of vancomycin-resistant enterococci

Since the first report of a vancomycin-resistant enterococcal clinical isolate, these Gram-positive bacteria have emerged as important nosocomial pathogens. Several glycopeptide resistance phenotypes can be distinguished on the basis of the level and inducibility of resistance to vancomycin and teicoplanin. In the present study, we developed a multiplex PCR, which allows the simultaneous identification of enterococci at the genus level and detection of the most frequent glycopeptide resistance genotypes. Five primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 and tuf were used in one reaction tube with bacterial DNA extracted from three to five colonies. This PCR method is suitable for the rapid detection of vancomycin-resistant enterococci.     

Selective media for detecting gastrointestinal carriage of vancomycin-resistant enterococci

Nosocomial infection with vancomycin-resistant enterococci (VRE) has become a significant problem. Effective institution of infection control measures depends on rapid identification of carriage of the organism, especially in asymptomatic individuals. We compared two selective media for use in screening for the presence of VRE and found that an agar medium containing bile esculin azide supplemented with 8 μg/ml of vancomycin was a useful and cost-effective means for primary screening for asymptomatic gastrointestinal carriage of VRE.     

In-vitro activity of fosfomycin against vancomycin-resistant enterococci

The effect of fosfomycin against 69 vancomycin-resistant isolates of Enterococcus faecium (VanA), five of E. faecium (VanB), 11 of Enterococcus faecalis (VanA), three of E. faecalis (VanB), 10 of Enterococcus gallinarum (VanC1) and two of Enterococcus casseliflavus (VanC2) and glycopeptide-sensitive E. faecium (n = 8) and E. faecalis (n = 10) was tested in vitro. Fosfomycin inhibited 97%, 94% and 96% of the vancomycin-resistant strains, according to results of agar dilution, broth microdilution, and a disc diffusion method (DIN 58940). The disc diffusion test by the NCCLS method does not include fosfomycin; using breakpoints suggested by Andrews et al. (< or = 11 mm, resistant; > or = 18 mm, susceptible), 5% of the vancomycin-resistant strains tested would have been considered fosfomycin resistant. Minimal inhibitory concentrations of most vancomycin-resistant isolates were in the intermediate sensitivity range, yielding an MIC50 of 32 mg/L and an MIC90 of 64 mg/L. Moreover the majority of inhibitory zone sizes by the disc diffusion method (DIN 58940) corresponded to intermediate susceptibility. These results suggest that fosfomycin at a high dosage and possibly used in combination with other drugs could be a potentially useful drug for the treatment of infections caused by vancomycin-resistant enterococci.     

Risk factors for acquisition of vancomycin-resistant enterococci among hematology-oncology patients

The incidence of VRE has increased dramatically and hematology-oncology patients are at high risk for acquisition of colonization and development of infection. Therefore, we performed a prospective cohort study to determine risk factors for VRE acquisition among hematology-oncology patients. Patients admitted to a single unit at Northwestern Memorial Hospital, which was predominantly comprised of patients with hematologic malignancies and recipients of hematopoietic stem cell transplants, were enrolled. Rectal or perianal swabs were obtained on hospital day 1, 4, 7 and then weekly thereafter. Data were collected by medical record review. We evaluated 155 study patients; 12 patients (7.7%) converted from VRE negative to positive. Among these 12 patients, 3 were positive on prior admissions, and 9 acquired VRE during the study. The median time to acquisition was 9 days. The median length of stay was significantly longer for patients with VRE compared to those who were VRE negative (31 vs. 6 days, P < 0.01). Patients with VRE were significantly more likely than those without VRE to have had an ICU admission within 3 months (P = 0.003), been admitted from an acute care facility (P = 0.001), or to have received amikacin (P = 0.02). Antimicrobials were commonly prescribed to all of the patients as 87% received an antimicrobial prior to their first swab. The crude mortality rate for patients with VRE was 67%. Prolonged length of stay, prior hospitalization, previous ICU admission and receipt of amikacin were risk factors associated with VRE acquisition among hematology-oncology patients. Mortality among these patients was high, due to serious underlying disease.     

万古霉素耐药肠球菌的检测和分子流行病学分析

目的为了解本院万古霉素耐药肠球菌（VRE）的流行情况，对VRE株进行实验室检测和分子流行病学检测分析。方法采用多重PCR及测序检测万古霉素耐药基因van，Etest法测定VRE株对万古霉素、替考拉宁的耐药表型；应用脉冲场凝胶电泳（PFGE）对11株VRE进行检测流行病学分析。结果检出1株VanA、4株VanB、5株vanC1，1株VanC2，对万古霉素、替考拉宁的药敏表型与基因型一致；11株VRE中2号vanB株与5号vanB株显示出相同片段，其他VRE株则分别有多于5个区带不同。结论实验室准确检测VRE对防止VRE感染和流行是非常重要的；PFGE电泳分析结果表明了本院11株VRE医院感染为散发。     

对万古霉素耐药的11株肠球菌的药敏表型及基因检测

目的 检测11株耐万古霉素肠球菌(VRE)的耐药表型、基因型以及多耐基因。方法 采用纸片扩散法、微量稀释法、自动化仪器、浓度梯度法测定11株VRE对万古霉素(Van)、替考拉宁(Tec)的耐药表型，微量稀释法测定11株VRE对10种抗菌药物的最低抑菌浓度；多重聚合酶链反应检测vanA、vanB、vanC1、vanC2并对van基因产物进行测序，聚合酶链反应检测TEM、tetM、ermB、aac(6′)／aph(2″)、ant(6)-Ⅰ、aph(3′)-Ⅲ基因。结果 不同药敏方法测得11株VRE对Van、Tec的药敏结果有差异；11株VRE对红霉素(9／11)、环丙沙星(7／11)、左氧氟沙星(7／11)、利福平(8／11)、氯霉素(7／11)耐药程度较高，高水平庆大霉素耐药株(HLGR)、高水平链霉素耐药株(HLSR)分别占10／11和9／11。11株VRE检出1株VanA、4株VanB、5株VanC1，1株VanC2，基因型和表型一致；耐药基因TEM(8／11)、tetM(6／11)、ermB(7／11)、aac(6′)／印h(2″)(7／11)、ant(6)-Ⅰ(5／11)、aph(3′)-Ⅲ(9／11)检出率高。结论 VRE确证试验以及准确检测其MIC值是筛查VRE不漏诊的可靠方法；van基因的检测是从分子水平确定VRE准确特异的方法；耐药基因的高检出率体现了VRE复杂的耐药机制。