|Year : 2014 | Volume
| Issue : 4 | Page : 462-465
Vancomycin resistance due to VanA in an Aerococcus viridans isolate
WQ Zhou1, DM Niu2, ZZ Zhang1, MZ Ning1, H Shen1, K Zhang1
1 Department of Laboratory Medicine , Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, China
2 Department of Clinical Laboratory , Nanjing Jinling Hospital, Nanjing University Medical School, Nanjing, Jiangsu, China
|Date of Submission||21-Oct-2013|
|Date of Acceptance||03-Feb-2014|
|Date of Web Publication||4-Oct-2014|
Department of Laboratory Medicine , Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Zhou W Q, Niu D M, Zhang Z Z, Ning M Z, Shen H, Zhang K. Vancomycin resistance due to VanA in an Aerococcus viridans isolate
. Indian J Med Microbiol 2014;32:462-5
|How to cite this URL:|
Zhou W Q, Niu D M, Zhang Z Z, Ning M Z, Shen H, Zhang K. Vancomycin resistance due to VanA in an Aerococcus viridans isolate
. Indian J Med Microbiol [serial online] 2014 [cited 2019 Oct 16];32:462-5. Available from: http://www.ijmm.org/text.asp?2014/32/4/462/142238
Wanqing Zhou and Dongmei Niu contributed equally to this paper.
The genus Aerococcus was created by Williams et al., to accommodate some Gram-positive, microaerophilic, catalase-negative organisms that were clearly distinguishable from streptococci, which were widely distributed in air, soil, milk, etc. It was described as a probable opportunistic pathogen and can be isolated from a variety of clinical specimens including blood cultures of patients with sub-bacterial endocarditis, urine cultures of patients suffered with urinary tract infections, synovial fluid cultures of septic arthritis, blood cultures of patients with bacteraemia, and cerebrospinal fluid (CSF) cultures of patients with meningitis.  Before 1992, only one species, Aerococcus viridans, was included in this genus. However, up to date, another five species including A. urinae, A. christensenii, A. sanguicola, A. urinaeequi and A. urinaehominis have been described, which could be identified by partial sequencing of the 16S rRNA gene. , Accurate species-level identification of isolates from relevant clinical specimens such as blood and abscess material is important in understanding the pathogenic mechanisms of the particular species. Since phenotypic test systems do not always allowed an accurate identification of this group bacteria, additional detection of 16S rRNA gene and cell matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF-MS) analysis are needed besides classical biochemical tests. , The present study was designed to identify an isolate of vancomycin resistance Aerococcus viridans from peritoneal-related ascites samples using MALDI-TOF-MS and partial sequencing of the 16S rRNA gene.
A 37-year-old female with 3 days of fever and bellyache was admitted to Nanjing Drum Tower Hospital on 03 August 2013, who had a medical history of chronic kidney disease, chronic nephritis and hypertensive. She received a peritoneal-related catheter surgery on 21 May 2008 and then followed by regular peritoneal dialysis. After admission to our hospital, dialysate was collected for routine and culture tests. The dialysate was turbid with a leucocyte count of 8.6 × 10 9 /L and most was neutrophils. Laboratory test revealed an elevated leucocyte count (10.7 × 10 9 /L with 89.8% neutrophils) and serum c reaction protein (121.7 mg/L). Empirical initial therapy included ceftizoxime sodium and teicoplanin were administered for the infection symptoms. The ascites, which was collected on 06 August 2013, was injected into blood culture bottle (bioMérieux, France), and later transferred to a sheep blood agar (bioMérieux, France). Cultures on sheep blood agar grew small alpha-haemolytic colonies of weakly positive catalase, non-motile, Gram-positive cocci. The strain was then characterised biochemically by using API 20 Strep and Vitek-2 Compact GP systems according to the manufacturer's instructions (bioMérieux, France). Conventional physiological tests were also conducted as described by Facklam and Elliott.  Anti-microbial susceptibility testing was performed using the Kirby-Bauer disc diffusion method on Mueller-Hinton (MH) agar with sheep blood including ceftriaxone, cefepime, erythromycin, levofloxacin, ofloxacin, chloromycetin, teicoplanin, clindamycin, vancomycin and linezolid, and minimal inhibitory concentrations (MICs) were determined by methods described by the Clinical and Laboratory Standards Institute (CLSI, 2010) for testing streptococci other than Streptococcus pneumoniae, including penicillin, quinupristin/dalfopristin, vancomycin and linezolid. The antimicrobial susceptibility testing (AST) results showed that this isolate was susceptible to linezolid and chloromycetin, but showed extent resistant to other drugs detected. After given ceftizoxime sodium via intravenous and teicoplanin by dialysate for 5 days, the patient has a clear dialysate tested and a relief of abdominal pain, which showed an effective anti-infection treatment. The patient was then followed by negative culture results for dialysate and blood. Patient responded to such combination therapy and was discharged on post-admission day 14.
The strain identity was further confirmed by the MALDI-TOF-MS and 16S rRNA gene sequencing  and with a final identification of A. viridans strain, named A. viridans strain AV208. MALDI-TOF-MS analyses were performed according to the protocol of VITEK MS v2.0 system (bioMérieux, France). In brief, a portion of a colony was smeared onto a target plate and then immediately covered with 1 μl α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution. After drying, the target plate was loaded into the VITEK MS instrument. The spectra were recorded in the linear mode in a mass range of 2-20 kDa and subsequently analysed by using a methodology based on the comparison of the characteristics of the spectrum obtained with the typical spectrum of each claimed species. The main spectrum of the A. viridans strain AV208 analysed in the present study was matched to A. viridans in the VITEK MS MS-ID database (version 2.0). The strain identity was further confirmed by the 16S rRNA gene sequencing.  DNA extraction was performed with the QIAamp DNA mini kit (QIAGEN AG, Switzerland) according to the manufacturer's instructions. A tree constructed by the neighbour-joining method, showing the phylogenetic position of the AV208 with respect to some related Gram-positive bacteria, is shown in [Figure 1]. The primers for amplification of the VanA, VanB, VanC1 and VanC2/C3 genes were designed as previously described.  The amplified products were purified by using Qiagen DNA purification kit (QIAGEN AG, Switzerland) and subjected to sequencing. The sequence similarity was determined by using the BLAST program from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST). A. viridans strain AV208 was positive for VanA only.
|Figure 1: Un-rooted tree showing the phylogenetic relationships of A. viridans AV208 and some related Gram-positive bacteria. The tree, constructed by the neighbour-joining method, was based on a comparison of approximately 1320 nucleotides. Bootstrap values, each expressed as a percentage of 500 replications, are given at the branching points. Bar represents 0.5% sequence divergence|
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Traditional bacterial identification has been performed by conventional phenotypic methods. However, in some cases, only classical phenotypic tests could not form a final identification. , Therefore, more attention should be paid on the identification of such strains in conventional works. For this, a combination of genes and protein detection may achieve a final identification. , This AV208 strain we isolated was identified as A. viridans through multi key reactions, including facultatively anaerobic, oxidase negative, catalase weakly positive, alpha-haemolytic when grown on blood agar, growth in 65 g/L NaCl broth but not on MacConkey plates, as well as with the commercially available API system (20 Strep, bioMérieux, France) and Vitek system (GP card, bioMérieux, France). To check the accuracy of the identification, this AV208 strain was further subjected to MALDI-TOF-MS analysis and 16S rRNA sequencing.
As shown in numerous studies, MALDI-TOF-MS appears to be a powerful tool for species classification for a broad spectrum Gram-positive and -negative bacterium, , and represents a valid and rapid alternative method for identification and classification of human pathogens in microbiology. VITEK MS allowed the identification of A. viridans strain AV208 in the present study to the species level with a 99.9% confidence value. Based on the 16S rRNA gene phylogenetic analysis, the Aerococcus-related species could be classified as new species, which were isolated from human and animal clinical specimens. ,, Results obtained from the VITEK MS v2.0 were comparable with reference 16S rRNA gene sequencing. These findings demonstrate that the VITEK MS v2.0 system can provide accurate results for the identification of the rare species of A. viridans. VITEK MS system is a new commercial MALDI-TOF-based method with a database of 755 species including 645 bacterial and 110 fungal taxa. This system is simplified for both bacteria and fungi based on a single spot without prior protein extraction step, which is different from the protocol typically used in other MALDI-TOF-MS systems. 
Since the CLSI does not provide any anti-microbial susceptibility criteria for Aerococci, we used the criteria for viridans group streptococci to identify A. viridans in this study. The AST results showed that this isolate was susceptible to linezolid and chloromycetin, but showed extent resistant to other drugs detected, such as vancomycin with the MIC > 32 μg/mL and an inhibition zone of 6 mm for Kirby-Bauer disc diffusion method. As revealed by a cluster of sporadic reports, anti-microbial susceptibility patterns of A. viridans have been rapidly changed. Until the late 1980s, this organism had been reported as susceptible to most commonly used antibiotics, but recent studies have documented that A. viridans showed resistance not only to penicillin but also to chloramphenicol and quinolones.  After that, multi-drug resistance A. viridans isolates were reported that have highly resistance to penicillin, ampicillin, erythromycin, clindamycin, ceftriaxone and gentamicin. , Although β-lactam-resistance A. viridans strains can been isolated from human clinical specimens, the isolates of A. viridans from clinical specimens in animals were susceptible to above anti-microbials. , It may suggest that drug resistance of A. viridans could be induced by selective pressure by prolonged antibiotic use.  The AV208 isolate we isolated in the present study exhibited a multi-drug resistance phenotype. The emergence of resistant A. viridans strains constantly, however, there was no report for vancomycin-resistant strains. Further polymerase chain reaction (PCR) reaction showed this isolate was only positive for VanA gene, which suggested a VanA-mediated drug resistance.
Resistance to vancomycin among enterococci strains is mostly due to production of Van-related genes, and the most common types are VanA and VanB. , The AV208 isolate was manifested in highly resistance to vancomycin and teicoplanin and later defined with the VanA gene type, which is consistent with the classification. Interesting, VanA-tpye gene of the AV208 strain shared 100% identity with VanA in Entercoccus faecalis (Gene Bank accession no. GQ484956) and Enterococcus faecium (Gene Bank accession no. FN424376). The resistance factor of the most common types of VanA and VanB can be transferred to other enterococci or other types of bacteria, especially to the methicillin-resistant Staphylococcus aureus (MRSA), which caused the appearance of vancomycin-resistant S. aureus.  The VanA-tpye gene was often transferred by the transposon Tn1546, which is always located on a transferred plasmid and was highly conserved between different strains. There was a clone dissemination of vancomycin-resistant E. faecium in our hospital (data not provided), and these raise some interesting questions: Where this VanA gene is located on the AV208 isolate and what is the relationship of VanA between these two species bacteria? Is there has any possibility of interspecies transmission of the VanA gene? Therefore, the location of VanA gene in A. viridans and the relationship of VanA gene between E. faecium and A. viridans will be confirmed by further research. The mechanisms of the AV208 strain resistant to other drugs will also be studied in the future.
| ~ Conclusion|| |
In conclusion, to our knowledge, this is the first case of peritoneal dialysis-related infections caused by A. viridans and our study is the first report to show the occurrence of VanA-producing A. viridans strain in the world. The results of the present study show that reliable identification of the clinically isolate of A. viridans can be obtained by use of a combination of colony morphology, haemolysis type, catalase reaction, PCR with specific primer restricted to the 16S rRNA, and the MALDI-TOF-MS, which is a fast method that shows promising results. Further study will be performed on the identification of the genetic environment of this VanA gene in A. viridans AV208 isolate.
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