Vancomycin-Resistant Enterococci from Humans and Retail Chickens in Taiwan with Unique VanB Phenotype-vanA Genotype Incongruence

Vancomycin resistant enterococci (VRE) with VanB phenotype-vanA genotype incongruence were found in all 39 VRE isolated from chicken carcasses and four human VRE isolates in Taiwan. Three identical mutations in the vanS gene were found in the VanB phenotype-vanA genotype VRE sequenced. This finding indicates possible transmission of glycopeptide resistance among different hosts.     

Comparison of Tn1546-like elements in vancomycin-resistant Staphylococcus aureus isolates from Michigan and Pennsylvania

In 2002, the first two clinical isolates of vancomycin-resistant Staphylococcus aureus (VRSA) containing vanA were recovered in Michigan and Pennsylvania. Tn1546, a mobile genetic element that encodes high-level vancomycin resistance in enterococci, was present in both isolates. With PCR and DNA sequence analysis, we compared the Tn1546 elements from each isolate to the prototype Tn1546 element. The Michigan VRSA element was identical to the prototype Tn1546 element. The Pennsylvania VRSA element showed three distinct modifications: a deletion of nucleotides 1 to 3098 at the 5' end, which eliminated the orf1 region; an 809-bp IS1216V-like element inserted before nucleotide 3099 of Tn1546; and an inverted 1,499-bp IS1251-like element inserted into the vanSH intergenic region. These differences in the Tn1546-like elements indicate that the first two VRSA isolates were the result of independent genetic events.     

Diversity of Tn1546 elements in clinical isolates of glycopeptide-resistant enterococci from Scottish hospitals

The Tn1546-related elements of 48 Van glycopetide-resistant enterococci were compared. Ten distinct Tn1546 types were identified with variation primarily due to IS1542 and IS1216V-like insertions. Clonal isolates frequently differed in their Tn1546 type, indicating instability of Tn1546-related elements. A putative hybrid promoter was identified, generated upstream of vanR by the insertion of IS1542. The presence of this hybrid promoter was associated with constitutive expression of the van genes and elevated teicoplanin resistance.     

Epidemiology of Vancomycin-Resistant Enterococci in Canadian Hospitals (CANWARD Study, 2007 to 2013)

Of 1,927 Enterococcus species isolates collected across Canada from 2007 to 2013, 80 (4.2%) were identified as vancomycin-resistant enterococci (VRE). VRE infections during this time tripled in Canadian hospitals, from 1.8% to 6.0% (P = 0.03). All VRE were Enterococcus faecium, with 90% possessing vanA. The prevalence of vanB decreased from 37.5% in 2007 to 0% in 2013 (P < 0.05). The VRE were multidrug resistant, but 70.6%, 86.3%, and 100% were susceptible to doxycycline, linezolid, and daptomycin, respectively.     

Diversity of VanA Glycopeptide Resistance Elements in Enterococci from Humans and Nonhuman Sources

Elements mediating VanA glycopeptide resistance in 106 diverse enterococci from humans and nonhuman sources were compared with the prototype VanA transposon, Tn1546, in Enterococcus faecium BM4147. The isolates included 64 from individual patients at 15 hospitals in the United Kingdom (isolated between 1987 and 1996) and 42 from nonhuman sources in the United Kingdom (27 from raw meat, 7 from animal feces, and 8 from sewage). VanA elements were assigned to 24 groups (designated groups A to X) with primers that amplified 10 overlapping fragments of Tn1546. Ten groups of elements were found only in human enterococci, eight groups of elements were unique to nonhuman strains, and six groups of elements were common in enterococci from all sources. Elements indistinguishable from Tn1546 (group A) were observed more frequently in enterococci from nonhuman sources (34 versus 9%) but were identified in enterococci that caused outbreaks in hospital patients between 1987 and 1995. The most common group found in human enterococci (group H; 33%) was rarely observed in enterococci from other sources (5%). Group H elements differed from Tn1546 in three regions and included a novel insertion sequence, designated IS1542, between orf2 and vanR. The VanA elements of 14 other groups had a similar insertion at this position and/or distinct insertions at other positions. We conclude that VanA elements in enterococci are heterogeneous, although all show regions of homology with Tn1546. Furthermore, the elements most common among the human and nonhuman enterococci studied were different. This approach may be useful for monitoring the evolution of VanA resistance and may also be applicable in local “snapshot” epidemiological studies. However, as transposition events involving insertion sequences accounted for the differences observed between several groups, the stability of the elements must be assessed before their true epidemiological significance can be determined.     

Molecular diversity and evolutionary relationships of Tn1546-like elements in enterococci from humans and animals

We report on a detailed study on the molecular diversity and evolutionary relationships of Tn1546-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn1546 types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn1546 derivatives. Differences between the Tn1546 types included point mutations in orf1, vanS, vanA, vanX, and vanY. Moreover, insertions of an IS1216V-IS3-like element in orf1, of IS1251 in the vanS-vanH intergenic region, and of IS1216V in the vanX-vanY intergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn1546. Identical Tn1546 types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn1546 to VRE isolates causing infections in Hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn1546 combined with epidemiological data suggest that the DNA polymorphism among Tn1546 variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.     

儿科临床分离肠球菌的耐药性监测及大环内酯类耐药机制研究

目的 研究儿科临床分离肠球菌的流行情况及红霉素耐药株基因ermB、mefA、tetM与转座子整合酶基因int-Tn分布特点.方法 采用KB纸片法对北京、上海、广州和重庆5家儿科专科医院2000-2006年临床分离粪、屎肠球菌进行8种抗菌药敏感性试验,并用琼脂稀释法测定225株红霉素耐药粪、屎肠球菌对大环内酯类及四环素最小抑菌浓度.PCR检测红霉素耐药基因ermB和mefA,四环素耐药基因tetM,以及转座子Tn1545的整合酶基因int-Tn.结果 4地5家儿童医院分离所得肠球菌对红霉素耐药率最高,平均耐药率86.5%;对氨苄西林、高浓度链霉素、高浓度庆大霉素、环丙沙星及利福平的耐药率分别为48.0%、47.6%、60.5%、45.4%、63.6%;未发现对万古霉素耐药的肠球菌.225株红霉素耐药粪、屎肠球菌ermB基因阳性率为70.7%,仅发现1株对mefA阳性的菌株.tetM基因阳性率为75.1%.Tn1545基因阳性株组ermB和tetM的携带率分别为84.8%和83.7%,高于阴性组60.9%和70.0%,且差异均有统计学意义(P均＜0.05).结论 我国儿科临床分离粪、屎肠球菌对多种抗菌药均有较高耐药率,对糖肽类抗菌药均保持较好敏感性,分离株对红霉素和四环素耐药主要机制分别是ermB编码的靶位改变和tetM编码的核糖体保护作用;接合性转座子Tn1545与tetM和ermB存在关系密切,可能是肠球菌多重耐药的重要机制之一.     

Diversity of Tn1546 and its role in the dissemination of vancomycin-resistant enterococci in Portugal

We characterized the molecular diversity of vanA vancomycin-resistant enterococci (VRE; 176 isolates/87 pulsed-field gel electrophoresis types) from different sources and cities in Portugal (1996 to 2004): (i) food animals (FA; n = 38 isolates out of 31 samples), hospitalized humans (HH; n = 101/101), healthy human volunteers (HV; n = 7/4), and environmental sources (n = 30/10). Some strains were isolated from different hosts and persistently recovered for years. Twenty-four Tn1546 variants were identified, all located on plasmids (30 to 250 kb). Some Tn1546 variants were associated with specific sources such as FA (3 types), HH (11 types), or HV (1 type), while others were recovered from isolates of different origins (8 types). Polymorphisms in the central vanRSHA region of Tn1546 were scarcely detected, while alterations upstream of vanR and downstream of vanA were frequently identified involving mutations (vanS and vanX), deletions (vanY), insertions (IS1216V, ISEf1, and IS19; sequences with or without homology with others available in GenBank databases), and different genetic rearrangements. Most Tn1546 variants contained IS1216V (14 types) or ISEf1 (6 types). IS1216V was found alone or associated with an IS3-like element at different orientations and positions in Tn1546 from human, animal, and environmental samples. ISEf1 was located within vanX-vanY region at nucleotide 9044 of Tn1546 variants mostly associated with clinical isolates, suggesting a common genetic platform. IS19 was observed within the vanX-vanY region in one Tn1546 variant from poultry. Recent spread of VRE in Portugal reflects a complex epidemiology involving both clonal spread and plasmid dissemination containing a variety of Tn1546 types. Apparent Tn1546 heterogeneity among enterococci from human, animal, and environmental sources might reflect frequent genetic exchange events and evolution of particular widely disseminated genetic elements.     

In Vitro Activity of LY333328 Against Vancomycin-Resistant Enterococci,Methicillin-Resistant Staphylococcus aureus,and Penicillin-Resistant Streptococcus pneumoniae

We report the activity of LY333328 against 35 clinical isolates of vancomycin-resistant enterococci (including organisms carrying the vanA, vanB, vanC-1, and vanC-2/3 genes, as determined by PCR), 33 clinical isolates of methicillin-resistant S. aureus, and 29 clinical isolates of high-level penicillin-resistant S. pneumoniae. All isolates of vancomycin-resistant enterococci were inhibited by 2 μg/mL LY333328, and 8 μg/mL LY333328 was bactericidal against all isolates tested. All isolates of methicillin-resistant S. aureus were inhibited by 1 μg/mL LY333328, and 4 μg/mL LY333328 was bactericidal against all methicillin-resistant S. aureus isolates tested. All isolates of penicillin-resistant S. pneumoniae were inhibited by <0.125 μg/mL LY333328, and 0.25 μg/mL LY333328 was bactericidal against all S. pneumoniae isolates tested. LY333328 is a promising new glycopeptide antimicrobial agent.     

Resistance to Linezolid: Characterization of Mutations in rRNA and Comparison of Their Occurrences in Vancomycin-Resistant Enterococci

To assess the potential for emergence of resistance during the use of linezolid, we tested 10 clinical isolates of vancomycin-resistant enterococci (VRE) (four Enterococcus faecalis, five Enterococcus faecium, and one Enterococcus gallinarum) as well as a vancomycin-susceptible control (ATCC 29212) strain of E. faecalis. The enterococci were exposed to doubling dilutions of linezolid for 12 passes. After the final passage, the linezolid plate growing VRE contained a higher drug concentration with E. faecalis than with E. faecium. DNA sequencing of the 23S rRNA genes revealed that linezolid resistance in three E. faecalis isolates was associated with a guanine to uracil transversion at bp 2576, while the one E. faecium isolate for which the MIC was 16 microg/ml contained a guanine to adenine transition at bp 2505.     

Loss of vancomycin resistance not completely dependent on the Tn1546 element in Enterococcus faecium isolates

We investigated characteristics of 3 Enterococcus faecium strains (SHY-1, SHY-2, and SHY-3) isolated successively from 1 patient. In vitro susceptibility testing was performed using broth microdilution method. Change of vancomycin MIC was monitored during incubation with vancomycin for SHY-3 strain. Genetic backgrounds were determined both by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). In addition, the genetic variations among Tn1546 element were investigated by polymerase chain reaction (PCR) assay and sequencing. vanA and vanX expression of 3 strains was evaluated using quantitative real-time (qRT)-PCR method. Although all the strains possessed the vanA gene, SHY-3 was susceptible to glycopeptides, while SHY-1 and SHY-2 were resistant to glycopeptides. Judged by MLST and PFGE, 3 strains have the same genetic background. The vancomycin resistance of SHY-3 was not recovered after exposure to vancomycin. The vanA and vanX genes were expressed in strains SHY-1 and SHY-2 but not in strain SHY-3, although the SHY-2 and SHY-3 strains shared the same arrangement of the van gene cluster, a common 88-bp deletion in vanS gene. Our results indicate that vancomycin resistance might not be completely dependent on the Tn1546 element.     

Effect of Surotomycin,a Novel Cyclic Lipopeptide Antibiotic,on Intestinal Colonization with Vancomycin-Resistant Enterococci and Klebsiella pneumoniae in Mice

Surotomycin (formerly called CB-183,315) is a novel, orally administered cyclic lipopeptide antibacterial in development for the treatment of Clostridium difficile infection (CDI) that has potent activity against vancomycin-resistant enterococci (VRE) but limited activity against Gram-negative bacilli, including Bacteroides spp. We used a mouse model to investigate the impact of surotomycin exposure on the microbiome, and to test the consequences of the disruption on colonization by vancomycin-resistant enterococci (VRE) and extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-KP), in comparison with the effects of oral vancomycin and metronidazole. Mice (8 per group) received saline, vancomycin, metronidazole, or surotomycin through an orogastric tube daily for 5 days and were challenged with 10⁵ CFU of VRE or ESBL-KP administered through an orogastric tube on day 2 of treatment. The concentrations of the pathogens in stool were determined during and after treatment by plating on selective media. A second experiment was conducted to determine if the antibiotics would inhibit established VRE colonization. In comparison to controls, oral vancomycin promoted VRE and ESBL-KP overgrowth in stool (8 log₁₀ to 10 log₁₀ CFU/g; P < 0.001), whereas metronidazole did not (<4 log₁₀ CFU/g; P > 0.5). Surotomycin promoted ESBL-KP overgrowth (>8 log₁₀ CFU/g; P, <0.001 for comparison with saline controls) but not VRE overgrowth. Surotomycin suppressed preexisting VRE colonization, whereas metronidazole and vancomycin did not. These results suggest that treatment of CDI with surotomycin could reduce levels of VRE acquisition and overgrowth from those with agents such as vancomycin and metronidazole. However, surotomycin and vancomycin may promote colonization by antibiotic-resistant Gram-negative bacilli.     

Molecular analysis of Tn1546-like elements in vancomycin-resistant enterococci isolated from patients in Europe shows geographic transposon type clustering

Resistance mechanism relatedness was studied in 18 clinical, European vanA vancomycin-resistant enterococci. Molecular analysis revealed 10 Tn1546-like elements, suggesting two evolutionary lineages. Lineage I dominated the European mainland, and lineage II dominated the United Kingdom and Israel. Geographic clustering reflected different types of meat consumption between countries, since each lineage is associated with colonization of different animals.     

Streptococcus gallolyticus subsp. gallolyticus from Human and Animal Origins: Genetic Diversity,Antimicrobial Susceptibility,and Characterization of a Vancomycin-Resistant Calf Isolate Carrying a vanA-Tn1546-Like Element

The aim of this work was to characterize the antibiotic susceptibility and genetic diversity of 41 Streptococcus gallolyticus subsp. gallolyticus isolates: 18 isolates obtained from animals and 23 human clinical isolates. Antibiotic susceptibility was determined by the semiautomatic Wider system and genetic diversity by pulsed-field gel electrophoresis (PFGE) with SmaI. Animal isolates grouped separately in the PFGE analysis, but no statistical differences in antimicrobial resistance were found between the two groups. The LMG 17956 sequence type 28 (ST28) strain recovered from the feces of a calf exhibited high levels of resistance to vancomycin and teicoplanin (MIC, ≥256 mg/liter). Its glycopeptide resistance mechanism was characterized by Southern blot hybridization and a primer-walking strategy, and finally its genome, determined by whole-genome sequencing, was compared with four closely related S. gallolyticus subsp. gallolyticus genomes. Hybridization experiments demonstrated that a Tn1546-like element was integrated into the bacterial chromosome. In agreement with this finding, whole-genome sequencing confirmed a partial deletion of the vanY-vanZ region and partial duplication of the vanH gene. The comparative genomic analyses revealed that the LMG 17956 ST28 strain had acquired an unusually high number of transposable elements and had experienced extensive chromosomal rearrangements, as well as gene gain and loss events. In conclusion, S. gallolyticus subsp. gallolyticus isolates from animals seem to belong to lineages separate from those infecting humans. In addition, we report a glycopeptide-resistant isolate from a calf carrying a Tn1546-like element integrated into its chromosome.     

Mercury resistance determinants related to Tn21, Tn1696, and Tn5053 in enterobacteria from the preantibiotic era

Three mer transposons from the Murray collection of preantibiotic enterobacteria show >99% sequence identity to current isolates. Tn5073 is most closely related to Tn5036 and Tn1696, and Tn5074 is most closely related to Tn5053. Tn5075 is most closely related to Tn21 but lacks integron In2 and is flanked by insertion elements.