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.     

Buruli ulcer caused by Mycobacterium ulcerans subsp. shinshuense: A case report

Buruli ulcer is the third most common mycobacterial infection worldwide and is mainly diagnosed in tropical regions. Globally, this progressive disease is caused by Mycobacterium ulcerans; however, Mycobacterium ulcerans subsp. shinshuense, an Asian variant, has been exclusively identified in Japan. Because of insufficient clinical cases, the clinical features of M. ulcerans subsp. shinshuense–associated Buruli ulcer remain unclear. A 70-year-old Japanese woman presented with erythema on her left backhand. The skin lesion deteriorated without an apparent etiology of inflammation, and she was referred to our hospital 3 months after disease onset. A biopsy specimen was incubated in 2% Ogawa medium at 30 °C. After 66 days, we detected small yellow-pigmented colonies, suggesting scotochromogens. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI Biotyper; Bruker Daltonics, Billerica, MA, USA) indicated that the organism was Mycobacterium pseudoshottsii or Mycobacterium marinum. However, additional PCR testing for the insertion sequence 2404 (IS2404) was positive, suggesting that the pathogen was either M. ulcerans or M. ulcerans subsp. shinshuense. Further examination by 16S rRNA sequencing analysis, focusing on nucleotide positions 492, 1247, 1288, and 1449–1451, we finally identified the organism as M. ulcerans subsp. shinshuense. The patient was successfully treated with 12 weeks of clarithromycin and levofloxacin treatment. Mass spectrometry is the latest microbial diagnostic method; however, it cannot be used to identify M. ulcerans subsp. shinshuense. To accurately detect this enigmatic pathogen and uncover its epidemiology and clinical characteristics in Japan, more accumulation of clinical cases with accurate identification of the causative pathogen is essential.     

Soft tissue infection caused by Mycolicibacter kumamotonensis

Mycolicibacter kumamotonensis (M. kumamotonensis), formerly Mycobacterium kumamotonense, is a nontuberculous mycobacteria species, which was first separated from Mycobacterium terrae complex in 2006. Reports about infections caused by M. kumamotonensis are extremely rare, with most of them being lung infection. Here, we report the case of a 68-year-old man with a hobby of gardening who developed swelling in his right middle finger. He underwent surgical debridement at a previous hospital and was diagnosed with nontuberculous mycobacteria infection based on positive findings of acid-fast staining of pus obtained from the surgical specimen. He was treated with rifampicin, ethambutol, and clarithromycin, but the swelling worsened. Therefore, he was referred to our hospital for further examination and treatment. We performed a second debridement and added isoniazid to the treatment regimen, but the swelling continued to worsen. We then administered levofloxacin, but his condition did not change. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and DNA sequencing analysis confirmed M. kumamotonensis as the causative bacterium. Since the finger swelling did not improve, the patient underwent a third debridement and amikacin was added to the treatment regimen. Finally, the infection was controlled. He completed amikacin therapy and will continue treatment with the other five antibiotics for a total of 24 months. To the best of our knowledge, this is the first report of a patient with M. kumamotonensis soft tissue infection. We consider this case might provide important insights into the diagnosis and treatment of soft tissue infections caused by M. kumamotonensis.     

First case of a bloodstream infection caused by the genus Brachybacterium

An 83-year-old previously self-sufficient man was referred to our hospital for a fever, severe tenderness over the lumbar spine, and elevated C-reactive protein levels. Computed tomography revealed fluid collection in the intervertebral space of L3/4. Gram-positive, short rod-shaped bacteria were isolated from two sets of blood cultures. A 16S rRNA sequence analysis of an isolate showed a similarity of 98.1% to the nearest type strain Brachybacterium squillarum JCM 16464^T. Biochemical characteristics of the presently isolated strain differed from those of the most closely related species of the genus Brachybacterium. The patient was successfully discharged on day 73 of admission with antimicrobial therapies and showed no recurrence during outpatient visits. Brachybacterium spp. have mainly been isolated from the environment, and human Brachybacterium infections have rarely been documented to date. To our knowledge, this is the first clinical isolation of Brachybacterium sp. as a causative pathogen of bloodstream infection.     

HPV感染及高级别CIN患者阴道微生态研究

目的分析人乳头瘤病毒16、18(HPV16、18)宫颈感染及高级别宫颈上皮内瘤变(CIN)患者阴道微生态特征。方法以3例湿疣样变或CIN1级HPV16和/或18感染患者为低级别CIN组,以5例高级别CIN病变HPV16和/或18感染患者为高级别CIN组,以3例HPV阴性阴道炎患者为对照组,采用16S rRNA基因扩增技术分析各研究组患者阴道微生物菌群组成,采用分层聚类法分析并比较各研究组阴道微生物菌群分布特征。结果高级别CIN组阴道微生物细菌种类多样性及复杂性较低级别CIN组、对照组更为明显;从对照组、低级别CIN组至高级别CIN组,卷曲乳杆菌和穹隆乳杆菌丰度呈下降趋势,惰性乳杆菌丰度呈增加趋势;定植菌犬布鲁氏菌丰度呈下降趋势,致病菌戴阿李斯特琥珀酸纤毛杆菌、阴道加德纳菌和短普雷沃菌丰度呈上升趋势。结论高级别CIN患者阴道菌种结构复杂性、多样性明显。阴道乳杆菌种类和丰度对阴道健康起重要作用,惰性乳杆菌则具有相反作用。犬布鲁氏菌、戴阿李斯特琥珀酸纤毛杆菌、阴道加德纳菌和短普雷沃菌长期存在于阴道,与HPV持续感染、进展至高级别CIN病变密切相关。     

Surgical site abscess caused by Lactobacillus fermentum identified by 16S ribosomal RNA gene sequencing

We report the first case of surgical site abscess caused by Lactobacillus fermentum from a 53-year-old woman with squamous cell carcinoma of the esophagus after transthoracic esophagectomy and neoadjuvant chemoirradiation. 16S rRNA gene sequencing is a useful tool to better characterize the epidemiology and clinical significance of L. fermentum.     

医院内深部真菌感染的临床分布及药敏分析

目的了解临床无菌部位真菌感染的菌种分布及耐药情况,为临床病原学诊断和合理使用抗真菌药物提供依据。方法采用法国生物梅里埃公司生产的ATB微生物鉴定仪、ID32C真菌鉴定板、ATB Fungus3药敏板进行真菌鉴定和药敏试验。结果临床113株真菌标本中以白色假丝酵母菌为主。真菌对各类抗菌药的敏感率分别为：氟康唑86.67%,伊曲康唑76.77%,伏立康唑88.89%,5-氟胞嘧啶95.45%,两性霉素B100%;耐药率分别为：氟康唑10.48%,伊曲康15.15%,伏立康唑8.08%,5-氟胞嘧啶0.91%。结论临床无菌部位真菌感染仍以白色假丝酵母菌为主,药敏显示两性霉素B及5-氟胞嘧对真菌抗菌活性相对较高,而真菌对氟康唑及伊曲康唑耐药性有所增加。     

中国麻风分枝杆菌种型分布与特征

目的了解中国目前传播的麻风分枝杆菌(M.leprae)菌种及单核苷酸多态性(SNPs)型分布与特征。方法对来自中国22个省市171例麻风患者皮损组织样本,采用巢式PCR扩增麻风分枝杆菌特异16S rRNA保守片段,并对PCR阳性产物直接测序,经BLAST进行序列比对;采用PCR产物限制性酶切基因多态性方法对麻风分枝杆菌进行SNP分型。结果 171例标本扩增片段与来自巴西的麻风分枝杆菌Br4923相似性达99%,均为M.leprae,未检出M.lepromatosis。在85例SNP分型标本中,SNP3型、SNP1型和SNP2型分别占78.8%(67/85)、20%(17/85)和1.2%(1/85),未检出SNP4型。130例16S rRNA序列存在C251T位碱基变异,不同临床型别标本中16S rRNA序列突变及SNP型别分布差异无统计学意义;16S rRNA基因C251T突变与麻风分枝杆菌菌株的SNP分型有一定的关联,存在突变的菌株多为SNP3型,少见SNP1型,未见SNP2型;不同地区的SNP型别分布之间差异有统计学意义,内陆地区的SNP3型菌株分布率97.1%(34/35)显著高于沿海66%(33/50)(χ~2=11.96,P0.01)。不同地区的16S rRNA序列突变率差异也有统计学意义,内陆地区的16S rRNA突变率94.8%(92/97)显著高于沿海51.4%(38/74)(χ~2=43.56,P0.01)。结论麻风分枝杆菌16S rRNA基因序列突变C251T与SNP分型有关,可提示不同基因型麻风分枝杆菌的地理分布。未发现麻风分枝杆菌M.lepromatosis菌种。     

用MALDI-TOF MS技术快速鉴定临床分离棒状杆菌属细菌

目的评价基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)快速鉴定临床分离的非白喉棒状杆菌的价值。方法收集本院58株非白喉棒状杆菌,用MALDI-TOF MS和16S rRNA基因测序两种方法进行鉴定和比较。用MALDI-Biotyper软件构建不同棒状杆菌MALDI-TOF MS蛋白系统发育树。结果 58株非白喉棒状杆菌中,54株MALDI-TOF MS与16S rRNA基因测序鉴定结果一致,包括34株纹带棒状杆菌(Corynebacterium straitum)、11株杰氏棒状杆菌(C.jeikeium)、3株C.resistens、2株解葡萄糖苷棒状杆菌(C.glucuronolyticum)、2株黏金色棒状杆菌(C.aurimucosum)和2株无枝菌酸棒状杆菌(C.amycolatum)。4株16S rRNA基因测序无法鉴定到种的菌株中,MALDI-TOF MS鉴定为2株产黏棒状杆菌(C.mucifaciens)、1株C.singulare和1株假白喉棒状杆菌(C.commune)。结论 MALDI-TOF MS可将棒状杆菌属细菌快速、准确地鉴定到种。     

医院感染肠球菌的临床分离及耐药性分析

目的 了解医院感染肠球菌的临床分离及其对常用抗生素的耐药性,指导临床合理用药,提高医院感染的治愈率.方法 对本院感染标本中分离出的136株肠球菌,进行常规分离和纸片扩散法进行药敏试验,并以"WHONET 5.5"软件对试验数据进行分析处理.结果 在136株肠球菌中,以粪肠球菌为主(77.94%),主要引起泌尿系感染.药敏试验结果显示:肠球菌对各种抗生素的耐药性差异有统计学意义,粪肠球菌对抗生素的耐药率最低是万古霉素(1.6%),最高是四环素(79.0%).屎肠球菌对大部分抗生素的耐药率明显高于粪肠球菌.结论 医院感染肠球菌属细菌常发生在免疫功能低下、有严重基础疾病、使用过侵袭性操作的患者, 肺部感染最常见.肠球菌对常用抗生素耐药率较高, 屎肠球菌的耐药性明显高于粪肠球菌.万古霉素敏感性高, 是治疗肠球菌感染的最佳抗生素.     

16S metagenomics for diagnosis of bloodstream infections: opportunities and pitfalls

Introduction: Bacterial bloodstream infections (BSI) form a large public health threat worldwide. Current routine diagnosis is based on blood culture (BC) but this technique suffers from limited sensitivity. Molecular diagnostic tools have been developed for identification of bacteria in the blood of BSI patients. 16S metagenomics is an open-ended technique that can detect simultaneously all bacteria in a given sample based on PCR amplification of the 16S ribosomal RNA gene (rDNA) followed by sequencing of the PCR amplicons and taxonomic labeling of the sequence reads at genus or species level.

Areas covered: Here we review the studies that have used 16S metagenomics for the identification of bacteria in human blood samples. We also discuss the potential added value of 16S metagenomics in the diagnosis of BSI, challenges as well as future directions for implementation in clinical settings.

Expert commentary: 16S metagenomics has the potential to complement conventional BC; however, the technique currently suffers from several technical limitations jeopardizing implementation in routine clinical microbiology laboratories. Further studies are required to assess the cost-efficiency and clinical impact of 16S metagenomics in comparison to BC which remains the gold standard diagnostic method for BSI.     

玫瑰单胞菌一株的鉴定及分析

目的 确定临床脓液标本中1株革兰阴性球杆菌K8756的分类学位置.方法 抽取患者深部脓肿物,置于Amies培养基室温保存、运输.脓液标本分区划线接种血平板、巧克力平板,置于35 ℃含5%CO2培养箱.采用API、Vitek2细菌鉴定系统,结合传统形态学检查、手工生化实验对分离株进行鉴定.从纯培养物提取脂肪酸、甲基化,采用气相色谱仪分析脂肪酸成分.PCR扩增16SrRNA基因并测序,对所测得的核酸序列进行同源性比对及系统发育分析.结果 血平板、巧克力平板分离到1株缓慢生长的革兰阴性球杆菌K8756.API 20NE生化编码为1245045(72 h),提示为人苍白杆菌;Vitek2 GN鉴定卡重复实验3次,提示为皮氏罗尔斯顿菌或支气管炎鲍特菌.但基于16SrRNA基因序列的系统发育分析表明,菌株K8756属于玫瑰单胞菌的成员,与该属的合格发表种黏液玫瑰单胞菌MDA5527T核酸匹配度高达100%(菌株K8756的GenBank登录号为GU207841).其菌落形态、表型特征及主要细胞脂肪酸成分亦与黏液玫瑰单胞菌相似.结论 菌株K8756(=GIMCC 1.0030)在分类学上属于黏液玫瑰单胞菌.16S rRNA基因序列分析是鉴定临床疑难菌株及新发现菌株的重要手段.

Abstract：

Objective To identify one runny mucoid-like Gram-negative bacteria with pink pigment isolated from clinical pus sample. Methods The pus sample was aseptically extracted from a deep lesions of one patient, then stored in Amies medium at room temperature for transportation. One sheep blood plate and one chocolate plate were used to detect the possible pathogens from the specimens. After inoculation, the plates were placed in a humidified incubator with 5% CO2 at 35 ℃. To identify the obtained isolates, we used the commercial Vitek2 and API systems, combining some traditional morphological examination and classical biochemical and physiological characteristics. For pure cultures, the cellular fatty acids were extracted, methylated, and determined by gas chromatography method. The 16S rRNA gene was amplified and sequenced by a commercial broad-spectrum PCR primers. The phylogenetic tree based on 16S rRNA gene was constructed by Mega 4.1 software using the neighbour-joining methods with 1 000 bootstrap replications. Results One runny mucoid-like Gram-negative bacterium, named K8756, was isolated both on sheep blood and chocolate plates after 72 h incubation. The API 20NE profile was 1245045 after a 3-day culture, which would be identified as Ochrobactrum anthropi with a good confidence of 98% probability. It was identified as Ralstonia pickettii and Bordetella bronchiseptica by VITEK 2 GN kits. However, further comparative 16S rRNA gene sequences showed that strain K8756 was closely related to the valid published Roseomonas mucosa MDA 5527 with 100% identity. Colonial morphologic features, phenotypic characteristics and major cellular fatty acid composition were also with high similarity to Roseomonas mucosa. Conclusions Strain K8756( = GIMCC 1.0030 ) is identified as Roseomonas mucosa by the polyphasic phenotypic and genotypic characteristics. The comparative analysis based on 16S rRNA gene sequences is a useful method for identifying the problematic and newly named bacteria.     

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.     

Catheter-related bloodstream infection by Microbacterium paraoxydans in a pediatric patient with B-cell precursor acute lymphocytic leukemia: A case report and review of literature on Microbacterium bacteremia

Microbacterium species are coryneform gram-positive rods that are widely distributed in the environment and have been recently recognized as rare pathogens in humans. However, information about the epidemiologic and clinical characteristics of Microbacterium species is scarce. We herein reported an 11-year-old girl with acute leukemia who was found to have catheter-related bloodstream infection in her neutropenic phase. Gram-positive bacilli repeatedly grew on the blood cultures and were later confirmed by 16S rRNA analysis as Microbacterium paraoxydans. A literature review found available clinical courses in 21 cases (7 pediatric cases) of Microbacterium spp. bacteremia. Our case and those in literature suggested that Microbacterium spp. bacteremia often occurs in patients with indwelling central venous catheters; the literature review further suggested that removal of central venous catheters is required in most cases and that 16S rRNA sequence was useful in identifying in detail the species of Microbacterium.     

Bacterial pericarditis caused by Lactobacillus iners in an infant

We report the case of a 6-month-old male infant with bacterial pericarditis due to Lactobacillus iners. Although the culture of pericardial fluid was negative, L. iners was identified by 16S rRNA gene amplification by polymerase chain reaction and a subsequent sequence analysis. This weakly pathogenic bacterium could develop a severe infection in infants.     

The first case report of pediatric acute appendicitis caused by “Candidatus Actinobaculum timonae”

A 14-year-old boy presented to the hospital with pain in the right lower abdomen. His condition was diagnosed as acute appendicitis. An emergency operation was performed, and histopathological examination revealed an actinomycete-related organism in the excised appendicitis specimen. On 16S rRNA gene sequence analysis, “Candidatus Actinobaculum timonae” was identified, which is the first known case in a pediatric patient.     

罕见豚鼠耳炎奴卡菌的鉴定及其药物敏感性分析

目的探讨罕见豚鼠耳炎奴卡菌的鉴定方法并评价其药物敏感性。方法采用形态、生理生化表型鉴定与16S rRNA序列分析相结合的方法鉴定菌株,使用微量肉汤法检测其药物敏感性。结果临床分离的奴卡菌被鉴定为豚鼠耳炎奴卡菌,其对哌拉西林、红霉素、头孢他啶、头孢哌酮、庆大霉素、头孢西丁、哌拉西林/他唑巴坦耐药,万古霉素、左氧氟沙星、亚胺培南、依替沙星、环丙沙星、阿米卡星、复方磺胺甲基异噁唑敏感。结论 16SrRNA基因序列分析法可用以鉴定豚鼠耳炎奴卡菌,磺胺类药物治疗有效。     

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。