Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR,and the VITEK2 system for identification of Acinetobacter clinical isolates

Since accurate identification of species is necessary for proper treatment of Acinetobacter infections, we compared the performances of 4 bacterial identification methods using 167 Acinetobacter clinical isolates to identify the best identification method. To secure more non-baumannii Acinetobacter (NBA) strains as target strains, we first identified Acinetobacter baumannii in a total of 495 Acinetobacter clinical isolates identified using the VITEK 2 system. Because 371 of 495 strains were identified as A. baumannii using gyrB multiplex 1 PCR and bla_OXA51-like PCR, we performed rpoB gene sequencing and 16S rRNA gene sequencing on remaining 124 strains belonging to NBA and 52 strains of A. baumannii. For identification of Acinetobacter at the species level, the accuracy rates of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK 2 were 98.2%, 93.4%, 77.2%, and 35.9%, respectively. The gyrB multiplex PCR seems to be very useful for the detection of ACB complex because its concordance rates to the final identification of strains of ACB complex were 100%. Both the rpoB gene sequencing and the 16S rRNA gene sequencing may be useful in identifying Acinetobacter.     

Identification of oral bacteria by 16S rRNA gene analysis in elderly persons requiring nursing care

After incubation of saliva from 58 semi-bedridden elderly persons, the cultures were identified based on the 16S rRNA gene base sequence to compare the identification by the conventional culture method. As a result, the 16S rRNA gene base sequence of 198 strains identified by the culture method showed 98.5% or more homology in some of the Human Oral Microbiome database, and the identification of bacterial species and genus was possible. When an organism identified by the 16S rRNA gene sequencing method was compared with that by the culture method, the concordance rates were 54.5% at the genus level and 35.9% at the species level. Streptococcus mitis strains most frequently isolated from saliva that were identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method (32/35), and all the 11 Streptococcus salivarius strains identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method. All the strains identified as Streptococcus anginosus group by the culture method and 8 of the 9 strains identified as Prevotella species by the culture method were identified as the same group and genus by the 16S rRNA gene sequencing method. When an oral microbial flora test with saliva samples from elderly persons is performed, the 16S rRNA gene sequence identification enables us to identify major indigenous bacteria and pathogenic bacteria and is considered useful as a means of supplementing the conventional culture method.     

First case report of prosthetic valve endocarditis caused by Mycobacterium wolinskyi

To date, only 26 cases of Mycobacterium wolinskyi infections have been reported in humans. We herein report a first case of prosthetic valve endocarditis due to this organism after cardiovascular surgery. An 82-year-old man presented with repeat episodes of syncope and fever after aortic valve replacement, mitral valve replacement, left atrial appendage closure, and pulmonary vein isolation. Blood cultures maintained in aerobic bottles were repeatedly positive after 90–100 hours, and Gallium scan revealed abnormal accumulations in the sternum and left testis. While colonies formed by culturing the fluid of the parasternal area and blood cultures revealed gram-positive rods, we could not analyze the colony using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). M. wolinskyi was finally identified on 16S rRNA, hsp65, and rpoB gene sequencing. We treated the patient with multiple antimycobacterial drugs, i.e., amikacin, imipenem, and clarithromycin for 6 weeks, which was changed to oral ciprofloxacin and minocycline for 12 months. This case highlights the need to consider rapidly growing mycobacteria, including M. wolinskyi, if chronic fever persists from weeks to months after surgery, the blood culture is positive, and the organism is not identified. In addition, sequencing the 16S rRNA, hsp65, and rpoB genes is essential for diagnosis.     

Utility of high-performance liquid chromatography analysis of mycolic acids and partial 16S rRNA gene sequencing for routine identification of Mycobacterium spp. in a national reference laboratory

High-performance liquid chromatography analysis of mycolic acids and partial gene sequencing for the first 500-bp 5′ end of the 16S rRNA gene were used singularly and in combination to evaluate the final identification of species. Examination of 200 cultures revealed 100 strains of slowly growing mycobacteria (SGM), 91 strains of rapidly growing mycobacteria (RGM), and 9 strains of other genera. SGM were discriminated in complexes with both methods for 56 strains, composed primarily of the Mycobacterium spp.: Mycobacterium avium, Mycobacterium terrae, and Mycobacterium simiae–Mycobacterium lentiflavum. For RGM, 73 strains were associated with complexes designated as Mycobacterium abscessus–Mycobacterium chelonae, Mycobacterium fortuitum–Mycobacterium peregrinum, and Mycobacterium mucogenicum–Mycobacterium phocaicum. Consistent identification of all the isolates differentiated to single species within the Mycobacterium genus was not possible with either test method. Sequencing results often distinguished complexes containing fewer species, and combining the results from each method increased the confidence of identifying the correct species.     

High-resolution melt analysis for species identification of coagulase-negative staphylococci derived from bovine milk

Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens isolated from bovine milk. In this study, we report a rapid assay for species identification of CNS using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time polymerase chain reaction amplification of 16S rRNA gene fragment, spanning the variable region V1 and V2, was performed with a resulting amplicon of 215 bp. A library of distinct melt curves of reference strains of 13 common CNS species was created using HRMA. Sequencing of 16S rRNA and rpoB genes, and, when needed, tuf gene, of 100 CNS isolates obtained from Canadian Bovine Mastitis Research Network was done to determine their species identity, allowing for subsequent evaluation of the performance of HRMA for field isolates of bovine CNS. A combination of HRMA and sequencing revealed that Staphylococcus chromogenes, S. xylosus, S. simulans, and S. sciuri had multiple genotypes, complicating their resolution by HRMA. As the 3 genotypes of S. chromogenes had distinct melt curves, the 3 distinct genotypes were employed as reference strains in a blinded trial of 156 CNS isolates to identify S. chromogenes. HRMA correctly identified all S. chromogenes isolates which were later confirmed by sequencing. Staphylococcus chromogenes (68%) was most frequently found among the CNS isolates, followed by S. haemolyticus (10%) and S. xylosus (6%). The present study revealed that HRMA of 16S rRNA gene (V1–V2) could be used as a rapid, efficient, low-cost, and minimally cumbersome technique for S. chromogenes identification, the most common CNS derived from bovine milk.     

不同分子生物学方法快速鉴别非结核分枝杆菌的应用评价

目的 评价3种分子生物学方法快速鉴定非结核分枝杆菌的优缺点.方法 收集41株临床分离的非结核分枝杆菌,以16S rRNA基因测序方法为标准,同时以hsp65基因测序方法及PCR-RFLP方法鉴定菌株,与16S rRNA基因测序结果进行比较.结果 41株非结核分枝杆菌16SrRNA基因测序结果:9株龟分枝杆菌复合群,7株偶发分枝杆菌,7株胞内分枝杆菌,3株鸟分枝杆菌,3株堪萨斯分枝杆菌复合群,3株耻垢分枝杆菌,3株土分枝杆菌,2株草分枝杆菌,2株无色分枝杆菌,1株瘰疬分枝杆菌,1株M.arupense.与16S rRNA基因测序相比较,hs65 PCR-RFLP能鉴定9株龟分枝杆菌复合群至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌;1株偶发分枝杆菌及1株无色分枝杆菌与其不符;其余菌株鉴定结果一致,符合率为95.1%(39/41).hsp65基因测序结果显示,1株爱尔兰分枝杆菌与16S rRNA测序结果不符,其余菌株鉴定结果与其一致,符合率为97.6%(40/41),并且能进一步将9株龟分枝杆菌复合群鉴定至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌.结论 3种方法均能快速鉴定非结核分枝杆菌.与16S rRNA基因测序相比,hsp65基因测序及hsp65 PCR-RFLP更容易鉴定临床最常见非结核分枝杆菌(如堪萨斯分枝杆菌和脓肿分枝杆菌),可在临床推广使用.     

A case of Bordetella trematum and Kerstersia gyiorum infections in a patient with congestive dermatitis

Bordetella trematum and Kerstersia gyiorum are rare gram-negative bacilli that are not frequently detected in human infections. In this report, we describe a case of a 48-year-old man who presented to our hospital with an infected wound on his leg. Discharges from the cracks of the granulation were collected and evaluated in our microbiology laboratory. Gram staining of the specimen showed polymorphonuclear leukocytes and abundant gram-negative bacilli. Three types of colonies were isolated on blood agar and were identified as B. trematum and Alcaligenes faecalis using VITEK MS. Moreover, K. gyiorum and B. trematum were identified and confirmed via 16S ribosomal RNA (rRNA) gene sequencing. The patient successfully recovered following application of meropenem antibacterial therapy and surgical debridement. This is the first reported case of complex wound infection caused by both B. trematum and K. gyiorum. Identification of B. trematum has recently been made possible by routine bacterial identification using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, K. gyiorum isolation is still rare, and species identification requires 16S rRNA sequencing. Thus, this case highlighted the importance of using multiple methods, such as MALDI-TOF MS and 16S rRNA gene sequencing, for identification of rarely isolated species from clinical specimens.     

Two new rapid PCR-based methods for identification of Acinetobacter baumannii isolated from clinical samples

The accurate identification of Acinetobacter spp. is challenging due to their high phenotypic and biochemical similarities. Because clinical relevance and antibiotic susceptibility are significantly different among different genomic species of Acinetobacter, the exact identification of A. baumannii is necessary and it can help us prevent inappropriate antibiotic use and inferior clinical care. This project employed a sequence-specific PCR assay for the rpoB region in A. baumannii to distinguish it from non-Acinetobacter baumannii Acinetobacter species. Moreover, a duplex PCR assay was used to detect bla_OXA-51-like and gluconolactonase genes as a second identification method. In this study, 210 isolates of Acinetobacter spp. were considered and identified by PCR-sequencing of rpoB gene as a reference test. PCR-sequencing of rpoB revealed that 179 isolates were A. baumannii and 31 were non- A. baumannii Acinetobacter strains. PCR amplification targeting the rpoB gene as the first method, detected 182 isolates of A. baumannii, while duplex PCR assay confirmed 163 isolates as A. baumannii. Data analysis indicated that the sensitivities of sequence-specific PCR of the rpoB gene and duplex PCR assay were 100% and 91.06%, respectively, while specificities were 91.18% and 100%, respectively. Given the data, it was revealed that these two methods showed a reasonable potential for the accurate identification of A. baumannnii from non- A. baumannii species. Sequence-specific PCR assay for the rpoB gene and duplex PCR assay for bla_OXA-51-like and gluconolactonase genes are rapid, reliable and cost-effective methods which can be used in clinical laboratories for the accurate identification of A. baumannii.     

肠球菌菌种鉴定方法的探索

目的 选择一种能将肠球菌属鉴定到种水平的快速准确的方法.方法 分别使用生化鉴定方法、16SrRNA基因测序技术、rpoA基因测序技术、基因组杂交技术(DNA-DNA hybridization,DDH)和平均核苷酸相似度(Average nucleotide identity,ANI)对12株肠球菌进行种水平鉴定,并比...     

Rapid identification of strains belonging to the Mycobacterium abscessus group through erm(41) gene pyrosequencing

Mycobacterium abscessus and Mycobacterium massiliense lung infections have different clarithromycin susceptibilities, making proper identification important; however, standard multi-gene sequencing in clinical laboratories is laborious and time consuming. We developed a pyrosequencing-based method for rapid identification of strains belonging to the M. abscessus group by targeting erm(41). We examined 55 isolates from new pulmonary M. abscessus infections and identified 28 M. abscessus, 25 M. massiliense, and 2 Mycobacterium bolletii isolates. Multi-gene sequencing of 16S rRNA, hsp65, rpoB, and the 16S–23S ITS region was concordant with the results of erm(41) pyrosequencing; thus, the M. abscessus group can be identified by single-nucleotide polymorphisms in erm(41). The method also enables rapid identification of polymorphic, inducible clarithromycin-resistant sequevars (T28 or C28). Pyrosequencing of erm(41) is a rapid, reliable, high-throughput alternative method for identifying and characterizing M. abscessus species. Further testing of a diverse collection of isolates is necessary to demonstrate the discriminatory power of erm(41) sequencing to differentiating species with this highly divergent group.     

Filifactor alocis brain abscess identified by 16S ribosomal RNA gene sequencing: A case report

We report a clinical case of Filifactor alocis brain abscess in an 85-year-old man who had decayed teeth 1 week prior. In this case, the abscess was surgically drained after empirical antibiotics had been initiated. Although the causative organism could not be identified by culture, F. alocis was detected via 16S ribosomal RNA (16S rRNA) gene sequencing of the pus isolated from the abscess. The patient recovered without serious sequelae after surgical drainage and prolonged antibiotic treatment, including metronidazole, ceftriaxone and meropenem for 8 weeks. The findings in this case emphasize that 16S rRNA gene sequencing allows bacterial diagnosis of brain abscess when phenotypic identification fails, such as in cases where patients are undergoing antimicrobial treatment at the time of sampling or where patients are infected with fastidious organisms.     

Campylobacter showae bacteremia with cholangitis

We report a case of Campylobacter showae bacteremia associated with cholangitis. A 71-year-old man with advanced bile duct cancer was admitted to our hospital because of cholangitis with shock, hypoglycemia, and impaired renal function. After replacement of the biliary drainage tube, pus was drained from the tube. Specimens for blood and bile cultures were obtained, and fluid resuscitation and antimicrobial treatment were then begun. Although anaerobic blood culture yielded small curved gram-negative rods, the isolate could not be identified by conventional identification methods. The isolate was identified as C. showae by 16S rRNA gene sequencing analysis. We consider here the pathogenicity of C. showae and the association of C. showae with cholangitis.     

16SrRNA、16S-23SrRNA基因测序分析检测主要血流感染病原菌比较

目的比较细菌16SrRNA、16S-23SrRNA基因测序分析在血流感染病原菌检测中的作用。方法提取临床上血流感染常见的金黄色葡萄菌、表皮葡萄球菌、大肠埃希菌、粪肠球菌、肺炎链球菌、铜绿假单胞菌、阴沟肠杆菌、鲍曼不动杆菌、洛菲不动杆菌、肺炎克雷伯杆菌、化脓性链球菌、奇异变形杆菌、潘尼变形杆菌、屎肠球菌、粘质沙雷菌、宋内志贺菌、产气肠杆菌、小肠结肠炎耶尔森菌、腐生葡萄球菌基因组DNA,运用16SrRNA、16S-23SrRNA基因进行PCR扩增。扩增产物经测序后在美国国家生物技术中心（NCBI）上进行比对分析,确定菌种。结果在所分析的19种临床血流感染常见细菌中,16SrRNA基因测序分析可将除粘质沙雷菌外的细菌鉴定到种的水平,但无法完全区分近缘种属;16S-23SrRNA成功鉴定17种细菌,除大肠埃希菌、宋内志贺菌外所有细菌均成功鉴定到单一种的水平。结论16S-23SrRNA基因可作为血流感染细菌检测较好的分子靶标。     

Bloodstream infection caused by Campylobacter lari

We describe a case of bloodstream infection (BSI) caused by Campylobacter lari in a 58-year-old man diagnosed with lumbar pyogenic spondylitis. Anaerobic blood cultures, taken on the day of admission and on hospital day 4, were positive after 30 h of incubation, although no bacteria were detected by Gram staining. After subculture on 5 % sheep blood agar for 2 days at 35 °C in a 5 % CO₂ environment, capnophilic, curved, gram-negative bacteria were recovered. The bacteria were identified as C. lari using a combination of phenotypic identification methods and partial 16S rRNA gene sequencing. The BSI was eradicated following combination therapy with intravenous tazobactam/piperacillin, oral erythromycin, and sulfamethoxazole/trimethoprim. These results suggest that accurate identification, to the species level, is important to determine effective treatment of BSI caused by Campylobacter spp. and can help us to understand the epidemiology.     

The usefulness of multiplex PCR for the identification of bacteria in joint infection

Background: The diagnosis of septic arthritis (SA) relies on synovial analysis and conventional culture. But, these methods lack of sensitivity and culture is time consuming to establish a definite diagnosis. This study evaluated a new multiplex PCR assay which entailed screening PCR for Gram typing and identification PCR for species identification using two primer mixes. Methods: A total of 80 synovial fluid samples from patients with suspected SA were collected. Culture, multiplex PCR, and 16S rRNA gene PCR were performed. Results: The analytical sensitivity of multiplex PCR assay was 10¹ CFU/ml for each type of bacteria. There was no cross‐reactivity with common bacterial pathogens. Bacteria were detected in 20, 25, and 26 of 80 samples for culture, multiplex PCR, and 16S rRNA gene PCR, respectively. Nineteen (95%) of 20 culture‐positivesamples and 6 (10%) of 60 culture‐negative samples were positive for the multiplex PCR. Five of six samples which were positive only from multiplex PCR were also positive in 16S rRNA gene PCR. The multiplex PCR showed 2 false‐negative in 27 true‐positive samples but no false‐positive. The sensitivity and specificity of the multiplex PCR were 92.6 and 100%, and the agreement with culture and 16S rRNA gene PCR were 91.3 and 96.3%, respectively. The time to detection for multiplex PCR was a maximum of 6 hr. Conclusion: This multiplex PCR assay offers high sensitivity and improved detection speed relative to culture. The appropriate combination of this new multiplex PCR assay with culture may contribute to the accurate and rapid diagnosis of SA. J. Clin. Lab. Anal. 24:175–181, 2010. © 2010 Wiley‐Liss, Inc.     

应用基质辅助激光解吸电离飞行时间质谱技术鉴定临床常见厌氧菌

目的：应用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）技术对临床分离的厌氧菌进行鉴定,与传统Vitek32 ANI鉴定卡及16 S rRNA基因序列法鉴定进行比较,分析MALDI-TOF MS技术用于鉴定临床常见厌氧菌的可行性。方法收集从临床标本培养分离的厌氧菌56株,运用 MALDI-TOF MS 技术对其进行鉴定,同时运用 Vitke32 ANI 鉴定卡及16 S rRNA基因序列法分别进行鉴定,将三者的结果进行比较。结果56株厌氧菌中有41株采用 MALDI-TOF MS技术鉴定至种的水平（分值大于或等于2．0）,11株鉴定至属的水平（分值为1．7～2．0）,4株无可靠结果（分值小于1．7）。MALDI-TOF MS和16S rRNA基因序列法鉴定结果一致的占94．6％（53/56）,不一致的占5．6％（3/56）。MALDI-TOF MS与Vitke32 ANI 鉴定卡的鉴定结果一致的占80．4％（45/56）,不一致的占19．6％（11/56）。结论 MALDI-TOF MS与16S rRNA基因序列法鉴定的符合率高,可应用于临床常见厌氧菌的鉴定。     

Utilization of elongation factor Tu gene (tuf) sequencing and species-specific PCR (SS-PCR) for the molecular identification of Acetobacter species complex

The aim of this study was to use tuf gene as a molecular target for species discrimination in the Acetobacter genus, as well as to develop species-specific PCR method for direct species identification of Acetobacter aceti. The results showed that most Acetobacter species could be clearly distinguished, and the average sequence similarity for the tuf gene (89.5%) among type strains was significantly lower than that of the 16S rRNA gene sequence (98.0%). A pair of species-specific primers were designed and used to specifically identify A. aceti, but none of the other Acetobacter strains. Our data indicate that the phylogenetic relationships of most strains in the Acetobacter genus can be resolved using tuf gene sequencing, and the novel species-specific primer pair could be used to rapidly and accurately identify the species of A. aceti by the PCR based assay.     

A case of infective endocarditis caused by “Neisseria skkuensis”

“Neisseria skkuensis” is a gram-negative coccus that is endemic in the human oral cavity, with only few reports of infection in humans. Herein, we report a case of a male patient in his sixties presenting with infective endocarditis (IE) caused by “N. skkuensis”. To our knowledge, this is the second case of IE confirmed using 16S rRNA gene to have been caused by “N. skkuensis”. The accurate diagnosis of rare or difficult-to-identify pathogens is a major challenge for clinical microbiological laboratories. Although Neisseria spp. are common in the oral cavity and are often seen in routine tests, identification of their biochemical properties and mass spectrometric analysis are difficult. In this case report, we describe the accurate identification of “N. skkuensis” by 16S rRNA gene sequencing analysis compared to other identification methods. Further cases of “N. skkuensis” are needed to fully evaluate the clinical approach of this detection method.     

基于16S rRNA和gyrB基因的施万菌种水平鉴定分析

目的 构建基于16S rRNA和gyrB基因对施万菌(Shewanella)进行种水平鉴定的方法,比较2个基因的鉴定能力差异.方法 利用DnaSP 6.0软件对施万菌16S rRNA和gyrB基因的信息位点数及其百分比、核苷酸多态性值、平均G+C含量、非同义突变率与同义突变率的比值(Ka/Ks)、Tajima检验进行基...