The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

Enterococcus gallinarum meningitis in an immunocompetent host: a case report / Meningite por Enterococcus gallinarum em paciente sem imunodepressão: relato de caso

We describe a rare case of a 53-year-old man with a long history of alcohol abuse, with Enterococcus gallinarum meningitis, an organism that rarely causes human infection and is primarily found in the gastrointestinal tract of poultry. The patient improved with high-dose ampicillin and gentamicin therapy. To our knowledge, this is the first Brazilian reported case of E. gallinarum meningitis and probably the first case described in an immunocompetent host.

Descrevemos caso raro de paciente de 53 anos com história de alcoolismo prévio, com meningite por Enterococcus gallinarum, um organismo que raramente causa infecções em humanos e é encontrado principalmente no trato gastrointestinal de aves. O paciente teve melhora importante após início de tratamento intravenoso com ampicilina e gentamicina combinados. Para o nosso conhecimento, este é o primeiro caso relatado de meningite por E. gallinarum no Brasil e possivelmente o primeiro caso descrito em paciente sem imunodepressão.

PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples / PCR-RFLP do 16S DNA ribossomal para confirmar a identificação de Enterococcus gallinarum e Enterococcus casseliflavus isolados de amostras clínicas e alimentares

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5 percent) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

INTRODUÇÃO: O objetivo deste estudo foi confirmar a identificação de amostras clínicas e alimentos de Enterococcus gallinarum e Enterococcus casseliflavus por PCR-RFLP. MÉTODOS: Cinquenta e duas cepas identificadas por exames bioquímicos convencionais foram submetidos a amplificação por PCR e digestão com HinfI. Apenas 20 (38,5 por cento) das 52 amostras apresentaram um padrão de DNA esperado E. gallinarum e E. casseliflavus. RESULTADOS: Analise dos resultados deste estudo demonstraram que, algumas vezes E. gallinarum e E. casseliflavus são erroneamente identificados e confirmaram a potencial aplicação da análise do 16S rDNA para identificação exata destas espécies. CONCLUSÕES: A correta identificação é importante a fim de distinguir entre resistência intrínseca e adquirida à vancomicina.

Genotype identifications of ESBLs and AmpC β-Iactamase of a Enterococcus gallinarum isolated from chloebia gouidiae / 中国兽医学报

The genotypes of extended spectrum β-laetamases(ESBLs) and AmpC β-lactamases produced by Enterococcus gallinarum isolated from chloebia gouldiae were determined to elucidate the evolution mechanism of the resistant genes.The minimum inhibitory concentrations (MICs) of 18 antibacterial drugs against the Enterococcus gallinarum were detected with two dilution method,and the ESBLs and AmpC β-lactamases from the bacterium were amplified by PCR using the primers of TEM,SHV,CTX-M,ACC,CIT,DHA,EBC,FOX and MOX,respectively.The PCR products were cloned and then the cloned fragments were sequenced to identify their genotypes and subtypes.The bacterium was proved to be a ESBL-producing and AmpC β-lactamase-producing bacterium,showing severe resistant to the other drugs,except the third and forth cephalosporins,carbopenems and fosfomycin.Compared with that of AJ847364 (TEM-116),the sequence of the TEM-type was characterized by two nucleotide mutations (512T→A and 695A→C),which led to two mutations of amino acids(17111e→Lys and 232Lys→Thr),showing that the detected TEM-type was a new genotype,the sequence of the AmpC β-lactamase was similar to that of EF078894 (ACT-like type)with a 97% homology.The genotype of ESBLs of Enterococcus gallinarum was a new TEM-type derived from the TEM-type ESBLs of klebstella pneumoniae isolated from the same avian.The genotype of AmpC lactamase was ACT-type,which probably concerned with β-1actam antibiotics used.

Enterococcus gallinarum native valve endocarditis in an immunocompetent patient / 대한내과학회지

Motile Enterococci, including Enterococcus gallinarum and Enterococcus casseliflavus/flavescens are rarely encountered in human clinical specimens. Enterococcus gallinarum is intrinsically resistant to low levels of vancomycin and causes bacteremia or infection among immunosuppressed or chronically ill patients, sometimes through nosocomial acquisition. We report a case of native valve endocarditis caused by Enterococcus gallinarum in an immunocompetent patient without any medical history.

Comparison of Tn1546-like Elements in vanA Genotype Enterococcus gallinarum and E. faecalis or E. faecium / 대한임상미생물학회지

BACKGROUND: Enterococcus gallinarum, an organism often found in the intestine, is intrinsically resistant to low level vancomycin, but some of them are highly resistant to vancomycin due to acquisition of vanA gene. We occasionally detected both vanA carrying E. gallinarum and E. faecalis or E. faecium in the same patients, suggesting transfer of the resistance gene from the latter. In this study, the structures of Tn1546-like elements in E. gallinarum, and E. faecalis or E. faecium from the same patients were compared to determine the clinical significance of the vanA genotype E. gallinarum isolates. METHODS: Six pairs of vanA genotype E. gallinarum and E. faecalis or E. faecium were isolated at a tertiary- care hospital in Korea during 2000 to 2004. Species identification was performed by conventional methods. and the vancomycin-resistance genotypes were determined by PCR. For structural analysis of Tn1546-like elements, overlapping PCR amplification and sequencing of the internal regions were performed. RESULTS: All isolates were positive for vanA genes by PCR. The analysis of Tn1546-like elements showed structurally related three types of distribution of IS elements integrated: Type I had insertion of an IS1542 in the orf2-vanR intergenic region, and an IS1216V in the vanX-vanY intergenic region. Type II was similar with Type I but accompanied with the partial and complete deletions of orf1 and orf2. Type III had an IS1216V and an IS1542 in the orf2-vanR intergenic region, and an IS1216V in the vanX-vanY intergenic region. Two of the six vanA clusters in E. gallinarum isolates had structures identical to those in E. faecalis or E. faecium strains isolated from the same patients. However, in some isolate pairs, the origin of Tn1546 cannot be determined, because the structures were not identical, and colonization of multiple clones was supposed. CONCLUSION: vanA clusters in some E. gallinarum, and in E. faecalis or E. faecium isolates from the same patients had an identical structure, indicating their transfer from the latter. It is assumed that vanA type E. gallinarum can act as a reservoir of vanA gene for interspecies spread.

Análisis clínico-epidemiológico de la portación intestinal de enterococos resistentes a vancomicina en una unidad de terapia intensiva / Clinical and epidemiologic analysis of intestinal tract colonization with vancomycin-resistant enterococci in an intensive care unit

En un período de cinco meses y 25 días se investigó la portación intestinal de enterococos resistentes a vancomicina (EVR). Se estudiaron 124 pacientes (73%) de 171 admitidos en la unidad de terapia intensiva (UTI), 35 de los cuales (28%) resultaron ser portadores. Los aislamientos de EVR (n=35) fueron identificados como Enterococcus faecium (n=18), Enterococcus gallinarum (n=16) y Enterococcus raffinosus (n=1). Todos los aislamientos estudiados fueron resistentes a vancomicina (VAN) (CIM90= 512 µg/ml) y teicoplanina (CIM90= 32 µg/ml) y portaban el gen vanA. Los estudios de tipificación molecular mostraron un alto grado de homología entre los aislamientos de E. faecium (un clon dominante) y E. gallinarum (dos tipos clonales), sugiriendo su diseminación a modo de brote. No se encontraron diferencias significativas con la edad y el sexo de los pacientes no portadores (p>0,05), pero si con el tiempo de hospitalización y el uso de esquemas antibióticos de amplio espectro (p<0,05), estando estos dos factores asociados al estado de portación. Se deduce de este estudio, la importancia de maximizar las medidas de prevención y control de las infecciones nosocomiales, analizar los esquemas empíricos empleados, tratar de disminuir el tiempo de hospitalización y continuar con los estudios de vigilancia para evaluar la eficacia de las acciones implementadas.

Intestinal tract colonization with vancomycin resistant enterococci (VRE) was studied during five months and 25 days. Out of 171 patients hospitalized in the intensive care unit, 124 (73%) were included in this study. Thirty five of them (28%) were recognized as colonized with VRE. VRE isolates (n = 35) were identified as Enterococcus faecium (n=18), Enterococcus gallinarum (n=16), and Enterococcus raffinosus (n=1). All of them were resistant to vancomycin (MIC90= 512 µg/ml) and to teicoplanin (MIC90= 32 µg/ml), having the vanA gene. By means of molecular methods a high homology was found among E. faecium and E. gallinarum isolates, respectively, suggesting their spread as a kind of outbreak. No significant differences in age or sex were found among colonized and non-colonized patients (p>0.05). On the other hand, the hospitalization time and the use of broad-spectrum antibiotics were associated with colonization. From this study we highlight the importance of enhancing all measures of control and prevention of hospital infections, carefully analyzing the empiric antimicrobial schemes, trying to reduce the hospital stage, and following the surveillance to evaluate the efficacy of such procedures.

An Enterococcus gallinarum Strain Carrying Both vanA and vanC1 Genes / 대한임상병리학회지

Enterococcus gallinarum carrying both vanA and vanC1 genes were detected from a surveillance culture from a patient staying at the surgical intensive care unit for a few years. E. gallinarum, SI04, was highly resistant to vancomycin (MIC of >or=256ng/mL) and teicoplanin (MIC of >or=256ng/mL). Multiplex PCR for vanA, vanB, vanC1 and vanC2/3 genes revealed SI04 to be positive for both vanA and vanC1 genes. This finding supports the fact that genotyping is needed to classify vancomycin-resistant enterococci (VRE). This is the first report on VanC VRE accompanying vanA gene in Korea.

Susceptibility of Fosfomycin against Vancomycin Resistant Enterococci / 감염

BACKGROUND: Vancomycin-resistant enterococci (VRE) were first recovered from clinical isolates in Korea in 1992, and the incidence has been steadily increasing. Alternatives to vancomycin are few because VRE are frequently resistant to commonly used antimicrobial agents. The present study was designed to assess the in-vitro activity of fosfomycin to clinical isolates of VRE. METHODS: For 199 VRE isolates from 1995 to 2000, and 91 enterococcal isolates that were consecutively isolated during the January of 2001 at Wonju Christian Hospital, fosfomycin (200 microgram) disk diffusion test was done by NCCLS method. The number of enterococcal isolates tested for fosfomycin were as follows:58 E. faecalis (42 vancomycin susceptible isolates, 16 vancomycin resistant isolates, and 1 vancomycin intermediate resistance isolate); 210 E. faecium (185 vancomycin resistant and 25 vancomycin susceptible isolates); 15 E. gallinarum, and 6 E. casseliflavus isolates. RESULTS: Among the VRE isolates, the resistance rates of fosfomycin according to enterococcal species were 6.3% in E. faecalis, 4.9% in E. faecium, 0% in E. casseliflavus, and 16.7% in E. gallinarum. CONCLUSION: Fosfomycin could be a potentially useful drug for the treatment of infections caused by VRE.

Biochemical Tests for Differential Identification of Enterococci with VanC phenotype / 대한임상미생물학회지

BACKGROUND: Pigment production and acidification of ribose are most frequently used biochemical tests for the differentiation of three enterococcal species carrying vanC genes such as Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens. However, pigment production may occasionally be negative in E. casseliflavus, and some of E. casseliflavus may be negative or delayed reaction with ribose fermentation test. So, we performed this study to find out biochemical tests capable of distinguishing the strains possessing vanC genotypes. METHOD: A total of 17 enterococci composed of 14 clinical isolates with motility or pigment positive strains and three ATCC strains(E. gallinarum ATCC 49573, E. casseliflavus ATCC 25788, and E. flavescens ATCC 49997) Were tested by multiplex PCR of the vanC genes(vanC-1, vanC-2 and vanC-3)and various biochemical tests. RESULTS: Among the 17 isolates including three ATCC control strains, four were genotyped as VanC-1, 11 were VanC-2, one were vanC-2/3, and any of vanC genes were not detected in one clinical isolate, respectively, Among the enterococci with vanC genotype, acid production from alphaD-cyclodextrin and hippurate hydrolysis were positive only in VanC-1 gneotype(E. gallinarum), acid production from glycerol and methyl-alpha-D-mannopyranoside were positive only in vanC-2 genotype(E. casseliflavus), and acid production from rhamnose and pigment production were negative only in VanC-1 genotype. Acid production from alphaD-cyclodextrin was negative only in vanC-2 genotype. The positive rate of ribose fermentation of VanC-1, VanC-2, and VanC-2/3(E. flavescens) genotype were 100%, 82%, and 0%, respectively. CONCLUSION: Acid production from rhamnose, alphaD-cyclodextrin, betaD-cyclodextrin, glycerol and methly-alphaD-mannopyranoside, pigment production, and hippurate hydrolysis test were useful biochemical tests for differentitating E. gallinarum form E. casseliflavus. The production of acid from alphaD-cyclodextrin, glycerol, methyl-alpha-D-mannopyranoside and were suitable biochemical tests for differentiating E. casseliflavus from E. flavescens.