B亚型中Bx新等位基因的鉴定

目的对ABO血型中B亚型分子遗传背景的研究,发现并鉴定ABO新等位基因。方法对5份血型血清学鉴定为B亚型的样本,采用PCR-SSP、ABO基因第6及第7外显子直接测序方法进行基因定型;并对目的B亚型样本进行RT-PCR、巢式PCR扩增、测序,分析其cDNA结构的差异。结果5份血清学为B亚型的样本中,血清学鉴定为Bx的个体发现了一个新的B等位基因。该等位基因与B101等位基因相比,差异仅在于第7外显子的B基因序列中nt721位C>T突变。其余4份B亚型样本的ABO基因第6、7外显子都未发现新的点突变。结论首次在中国汉族人中发现一个721C>T新变异的B等位基因,该等位基因nt721位由C转变为T,241位氨基酸由精氨酸转变为色氨酸,可导致糖基转移酶活性的降低,表明ABO基因的第241位氨基酸对决定糖基转移酶活性是至关重要的。     

自贡市非结核分枝杆菌临床分离株的菌种鉴定结果分析

目的 对自贡市35株疑似非结核分枝杆菌（NTM）临床分离株进行菌种鉴定。方法 从自贡市各区（县）及市结核病防治获得的临床分枝杆菌分离株,通过罗氏培养基（L-J）、对硝基苯甲酸（PNB）和噻吩-2-羧酸肼（TCH）鉴别培养基初步鉴定为NTM,再经多位点PCR方法确定为NTM后,对rpoB、hsp64及its基因进行测序及分析鉴定。结果 35株疑似NTM临床分离株,通过初步鉴别,多位点PCR,rpoB、hsp64及its基因测序及分析鉴别出32株NTM,分别为脓肿分枝杆菌（M. abscessus）11株、胞内分枝杆菌（M. intracellulare）7株、堪萨斯分枝杆菌（M. kansasii）5株、鸟分枝杆菌（M. avium）3株、脓毒分枝杆菌（M. septicum）1株、马赛分枝杆菌（M. masseillense）1株、戈登分枝杆菌（M. gordonae）2株、副瘰疬分枝杆菌（M. parascrofulaceum）1株和Mycobacterium peregrinum 1株。结论 抗酸染色阳性、PNB/TCH鉴定为NTM的临床分离株中包含不同种类的NTM,自贡市NTM肺病的病原种类较多,以快生长脓肿分枝杆菌为主,其次为慢生长不产色菌的胞内分枝杆菌和缓慢生长光产色菌的堪萨斯分枝杆菌。实验室应开展进一步菌种鉴定,以指导临床正确诊断和合理治疗。     

Combination of real-time PCR and sequencing to detect multiple clinically relevant genetic variations in the lactase gene

Background: Lactase persistence is an autosomal dominant trait commonly distributed in Europe as well as some parts of east Africa and the Arabian Peninsula. Using real-time PCR to detect the ?13910C?>?T variant common in the European population is a reliable analysis although other variants in the probe-binding site may cause errors in analysis. The aim of this study was to determine the prevalence of the variants in a Danish cohort examined for lactose intolerance as well as to improve the real-time PCR analysis for detection of the different variants.

Methods: We genotyped 3395 routine samples using real-time PCR for the ?13910C?>?T-variant. All consecutive samples identified as ?13910CC were sequenced using Sanger Sequencing. Using the SDS software we examined various quality value settings to improve on the genetic analysis.

Results: Using real-time PCR resulted in 100% successful genotyping of the ?13910C?>?T variant. By using a quality value of 99% and sequencing the undetermined samples we improved the ability of the assay to identify variants other than ?13910C?>?T. This resulted in a reduction of the diagnostic error rate by a factor of 2.4 while increasing the expenses only 3%.

Conclusions: We conclude that using a quality value of 99% in the SDS software significantly improves the diagnostic efficiency of the real-time PCR assay for detecting variants associated to lactase persistence.     

Whole-Genome Sequencing in Healthy People

Recent technological advances have radically changed genetic testing from an expensive and burdensome undertaking to a rapid and less costly option for many purposes. The utility of “next-generation” sequencing has been found to establish the diagnosis for hundreds of genetic disorders, to assess pharmacogenomic variants, and to identify treatable targets within malignant neoplasms. The ready availability of genomic information has led to the question of whether there would be clinical benefit of sequencing the genome of individuals who are not seeking a diagnosis, that is, genomic screening in generally healthy people, to provide anticipatory insights for their health care. Little research has been conducted in this area. We examine the considerable unresolved scientific and ethical issues encountered when considering whole-genome sequencing of healthy people.     

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.     

中国人群中发现的主要弱D型——弱D15个体的分子背景研究

为了探讨中国汉族人群弱D15型个体的Rh血型系统血型血清学表型及分子背景，采用常规血型血清学技术检测RhD抗原弱阳性个体RhD、C、c、E、e抗原表型；采用序列特异性引物-聚合酶链反应（SSP—PCR）方法同时检测RHD基因和RHCE基因；标本测序分析RHD基因全长编码区序列；同时通过特异性PCR技术测定RHD合子型或RHD基因数目。结果表明，血型血清学试验证实为D抗原弱阳性表型的人群中，有18例为弱D15型（占D抗原弱阳性表型56％），RHD基因全长编码区序列分析发现其第6外显子有一处碱基突变：845G〉A，编码区序列其它部分与正常RHD序列相同；检测Rh小因子有3种表型CcEe（2例）、CcEe（2例）、ccEe（14例），分别占弱D15型的11％、11％、78％，血清学检测结果与分子生物学检测结果一致。RHD杂合性试验鉴定显示仅表型为CcEe的2例标本为纯合型RHD^＋／RHD^＋，提示基因型为DCe／DcE；其余为杂合型RHD^＋／RHD^-，提示基因型分别是DCe／dce和DcE／dce。结论：弱D15型是中国人群中最主要的弱D型，其中大部分为杂合型。     

Long-read sequencing revealed cooccurrence,host range,and potential mobility of antibiotic resistome in cow feces

While it is well recognized that the environmental resistome is global, diverse, and augmented by human activities, it has been difficult to assess risk because of the inability to culture many environmental organisms, and it is difficult to evaluate risk from current sequence-based environmental methods. The four most important criteria to determine risk are whether the antibiotic-resistance genes (ARGs) are a complete, potentially functional complement; if they are linked with other resistances; whether they are mobile; and the identity of their host. Long-read sequencing fills this important gap between culture and short sequence-based methods. To address these criteria, we collected feces from a ceftiofur-treated cow, enriched the samples in the presence of antibiotics to favor ARG functionality, and sequenced long reads using Nanopore and PacBio technologies. Multidrug-resistance genes comprised 58% of resistome abundance, but only 0.8% of them were plasmid associated; fluroquinolone-, aminoglycoside-, macrolide-lincosamide-streptogramin (MLS)-, and β-lactam–resistance genes accounted for 2.7 to 12.3% of resistome abundance but with 19 to 78% located on plasmids. A variety of plasmid types were assembled, some of which share low similarity to plasmids in current databases. Enterobacteriaceae were dominant hosts of antibiotic-resistant plasmids; physical linkage of extended-spectrum β-lactamase genes (CTX-M, TEM, CMY, and CARB) was largely found with aminoglycoside-, MLS-, tetracycline-, trimethoprim-, phenicol-, sulfonamide-, and mercury-resistance genes. A draft circular chromosome of Vagococcus lutrae was assembled; it carries MLS-, tetracycline- (including tetM and tetL on an integrative conjugative element), and trimethoprim-resistance genes flanked by many transposase genes and insertion sequences, implying that they remain transferrable.

Antibiotic resistance is one of the greatest global public health crises, as more than 700,000 people die from antibiotic resistance-associated disease every year, and the number is predicted to rise dramatically if radical actions are not taken (1). Studies have shown that many clinical antibiotic-resistant genes (ARGs), such as the extended-spectrum β-lactamase (ESBL) CTX-M and the quinolone-resistance gene qnr, have direct links to the environmental resistome (2, 3). The rapid emergence of ARGs in Acinetobacter, making it one of the most difficult gram-negative bacteria to treat, likely acquired its resistance from Pseudomonas, Salmonella, or Escherichia coli mediated by environmental reservoirs (4). In accord with the “One Health” concept, it’s critical to understand the mobility risk of environmental ARGs, as well as their transmission routes to clinical settings (5).The diversity and abundance of ARGs have been well documented in various contexts, such as soils (6), animal feces (7, 8), wastewater sludge (9), and aquatic ecosystems (10), and so forth. ARGs in livestock and poultry manures are particularly noticeable due to the use of antibiotics in animal farming industry (11). Studies have demonstrated that antibiotics and some metals can increase abundance of antibiotic-resistant bacteria (ARB), ARGs, and metal-resistance genes (MRGs) (12). For example, oxytetracycline treatment significantly boosted tetracycline-resistant coliform bacteria in pig manure (13). The long-term presence of antibiotics in animal manure imposes selection on microbes, which likely means functional antibiotic resistance and higher risk of horizontal gene transfer (HGT) compared to other contexts (14). Moreover, large populations of human commensals in animal manure also increases the possibilities for ARG transfer to human-associated pathogens (15).In recent years valuable insights have been gained about environmental resistomes with qPCR and the next-generation sequencing. For example, diverse and high abundance of ARGs were identified in pig manure from three distantly located farms in China with high-throughput qPCR, and further analysis showed positive correlation between ARG abundance and heavy metal concentrations (16). Furthermore, metagenomic sequencing improved our understanding of the antibiotic resistome, mobile genetic elements (MGEs), and their microbial contexts in environmental samples. Widespread cooccurrence patterns of different classes of ARGs and MRGs have been revealed by metagenomic and network analyses (17, 18). However, most studies have estimated ARG risk by abundance quantification of ARG fragments and not the whole gene, nor whether other resistance genes are linked or their potential for transfer, features central to risk.It is now well recognized that risk of ARGs relies on their mobility and host contexts rather than simply their high diversity or abundance (19). MGEs—including plasmids, transposons, and integrative conjugative elements (ICEs)—play key roles in ARG propagation through HGT. ARGs on plasmids suggests higher transfer potential to pathogens, thus posing a larger threat to public health (20). Besides antibiotics, heavy metals, and biocides can also facilitate the enrichment and spread of ARGs and ARBs (21), hence their cooccurrence pattern can provide insights into coselection risks. The third-generation sequencing technologies, such as PacBio and Nanopore, can produce up to hundreds of kilobase reads and provide new opportunities for estimating ARG cooccurrence, mobility, and host range. For example, the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58 was resolved using Nanopore sequencing (22). The transfer of a tetracycline-resistance–carrying plasmid was observed from Staphylococcus haemolyticus to Staphylococcus aureus by PacBio sequencing (23). Most studies on ARG mobility, however, are conducted on cultured isolates. An approach is needed to assess the mobility of environmental resistomes and their potential risk.Ceftiofur is a third-generation cephalosporin antibiotic approved by the US Food and Drug Administration for therapeutic use in food animals and is widely used in cattle to treat respiratory diseases, foot rot, metritis, and mastitis (24). Resistance to ceftiofur also causes ineffectiveness to many clinically important antibiotics, which are critical to human health (24). Cattle manure is usually recycled to agricultural fields, representing a potential path of ARGs from manure to soils, ground water, and food systems (25, 26). Here, we collected feces from a dairy cow treated with ceftiofur for a uterine infection after calving, a standard treatment for metritis and known to increase the proportion of ceftiofur-resistant E. coli in the manure (27). Our objectives were to: 1) determine the genetic location (chromosomal, ICE-associated, and plasmidic) and hosts of different classes of ARGs; 2) define complete/near-complete plasmids in manure and infer transferability; 3) determine the linkage pattern of ARGs, MRGs, and biocide-resistance genes (BRGs); and 4) define a long-read sequencing method for environmental resistome evaluation. This approach fills the gap between culture-based isolate analyses and sequencing-based surveys and hence improves the ability to assess risk of environmental resistomes.