Penicillin-binding proteins in Streptococcus agalactiae: a novel mechanism for evasion of immune clearance

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia and meningitis in the USA despite CDC-recommended chemoprophylaxis strategies for preventing infection. To cause infection pathogens such as GBS must evade recognition and clearance by the host's immune system. Strategies for avoidance of opsonization and phagocytic killing include elaboration of antiopsonophagocytic capsules and surface proteins. During screening for mutants of GBS that were attenuated for virulence in a neonatal rat sepsis model, we identified a mutant with a transposon insertion in the ponA gene. ponA encodes an extra-cytoplasmic penicillin-binding protein PBP1a, a newly identified virulence trait for GBS that promotes resistance to phagocytic killing independent of capsular polysaccharide. Complementation analysis in vivo and in vitro confirmed that the altered phenotypes observed in the mutant were due to the transposon insertion in ponA. Deletion of PBP1a does not affect C3 deposition on GBS suggesting that mechanism by which PBP1a protects GBS from phagocytic killing is distinct from the antiopsonic activity of capsular polysaccharide. This is the first report describing expression of an antiphagocytic surface protein by GBS and represents a novel mechanism for evasion of immune recognition and clearance that may explain the decreased virulence observed in Gram-positive bacterial species for penicillin-binding protein mutants.     

Studies on the possible application of molecular methods in diagnosing carriers and in similarity analysis of group B streptococci (Streptococcus agalactiae)

The most popular method of GBS identification in Poland currently is by culturing on enriched agar and verifying the Lancefield Group using special latex agglutination kits. However, the classical methods are time-consuming and their sensitivity is insufficient therefore it is becoming more common to try and apply molecular methods which are characterized by high sensitivity and rapid results. Moreover, molecular methods give us the possibility to carry out epidemiological investigations and gene detection, for instance for antibiotic resistance. It was confirmed that PCR and FISH procedures may be effective in rapid detection of GBS. Thanks to RAPD methods we showed that newborns born to colonized mothers were colonized by GBS strains which originated from the mother, irrespective of the way and the course of labour. Additionally, we detected GBS colonization in children who were born to mothers who were not colonized by GBS. These children were probably colonized with strains coming from hospital environment. More studies are needed to elucidate the route of transmission and the role of colonization of the medical staff. Using multiplex PCR we showed the presence of ermA, ermB and ermC genes in phenotypically confirmed MLS, GBS strains.     

Diagnosis and seroepidemiology of Neospora caninum-associated bovine abortion

A round table was conducted at the VIIIth International Coccidiosis Conference on Neospora diagnosis with particular emphasis on strategies to diagnose bovine abortion. The strength and weakness of different assays for Neospora caninum infection and whether these methods have resulted in the overdiagnosis of neosporosis was discussed. It was evident that each diagnostic method, namely histology, immunohistochemistry, molecular detection and serological assays were, under certain circumstances, valuable in assessing the role N. caninum in abortion. Histological, immunohistochemical and molecular detection assays are of outstanding importance for the examination of tissues of aborted foetuses. While histology and immunohistochemistry allow direct assessment of pathomorphological changes caused by infection, molecular detection assays such as PCR are superior because of higher sensitivity and specificity in identifying N. caninum in foetal tissues. Serological tests, such as ELISA, are useful in determining whether an animal has been infected with N. caninum. Seroepidemiological approaches allow one to assess an abortion problem at a herd level and when used in conjunction with certain statistical methods are able to confirm a suspected N. caninum-associated abortion.     

PCR-Based Techniques for Leprosy Diagnosis: From the Laboratory to the Clinic

In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.     

Characterization and evaluation of an arbitrary primed Polymerase Chain Reaction (PCR) product for the specific detection of Brucella species

Laboratory detection of Brucella is based largely on bacterial isolation and phenotypic characterization. These methods are lengthy and labor-intensive and have been associated with a heightened risk of laboratory-acquired infection. Antibody based indirect detection methods also suffer from limitations in proper diagnosis of the organism. To overcome these problems, nucleic acid amplification has been explored for rapid detection and confirmation of the presence of Brucella spp. PCR-based diagnostics is useful for screening large populations of livestock to identify infected individuals and confirms the presence of the pathogen. Random Amplification of Polymorphic DNA (RAPD) was performed and identified a 1.3 kb PCR fragment specifically amplifiable from DNA isolated from Brucella. A BLAST search revealed no significant homology with the reported sequences from species other than the members of Brucella. The isolated fragment seems to be a part of d-alanine–d-alanine ligase gene in Brucella sp. Translational BLAST revealed certain degree of homology of this sequence with orthologs of this gene reported from other microbial species at the deduced amino acid level. The sequence information was used to develop PCR based assays to detect Brucella sp. from various samples. The minimum detection limit of Brucella from blood and milk samples spiked with Brucella DNA was found to be 1 ng/ml and 10 ng/ml, respectively. In conclusion, we demonstrated that the PCR based detection protocol was successfully used for the detection of Brucella from various organs and spiked samples of diseased sheep. Diagnosis of Brucellosis by PCR based method reported in this study is relatively rapid, specific and simple.     

新型冠状病毒主蛋白酶抑制剂的筛选方法研究进展

新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)席卷全球,具有较高的传染性和死亡率,但目前尚缺乏安全有效的COVID-19疫苗与治疗药物.新型冠状病毒主蛋白酶(main protease,Mpro)的进化高度保守,在调控新冠病毒RNA复制中具有重要的生物学功能,已成为新型广谱抗冠状...     

Rapid genetic characterization of poly(hydroxyalkanoate) synthase and its applications

Microorganisms containing short-chain-length (scl-) or medium-chain-length (mcl-) poly(hydroxyalkanoates) (PHAs) are commonly screened by applying rapid staining methods using lipophilic reagents. These methods provide powerful means for general screening of organisms actively producing and accumulating PHAs. The Southern blot hybridization method additionally allows the identification of potential PHA-producing microorganisms. Polymerase chain reaction (PCR)-based detection methods further afford rapid and sensitive means to screen for PHA biosynthesis genes. Specific PCR assays had been developed for the simultaneous or individual detection of the class II mcl-PHA synthase genes of Pseudomonas. The amplicons (approximately 0.54 kb) can be directly sequenced or used as probes for hybridization studies. The sequence information can further be used to initiate chromosome walking for an eventual cloning of the complete PHA biosynthesis operon. In addition, the amplification pattern and sequence data can be used to differentiate subgroups of organisms, as demonstrated for P. corrugata and P. mediterranea. Other researchers reported PCR methods for the detection of scl-PHA synthase genes and those of Bacillus spp., thus greatly expanding the types of PHA synthase gene and the organisms that can be characterized by this approach. The vast sequence information obtainable through PCR-based studies of various PHA synthase operons should facilitate the identification or construction of new PHA synthases capable of synthesizing novel PHAs.     

微生物高通量快速检测技术研究进展

微生物在大自然中广泛存在,特别是食源性致病菌是引发食源性疾病的主要因素,传统的检测技术已难以适应疾病预防控制和基层监管执法的需求,因此更加快速灵敏的高通量检测技术成为研究的热点。在对微生物快速准确识别方面,主要包括双功能抗体、核酸适配体筛选和噬菌体鉴定等方法。在多目标检测方面,主要包括多重PCR、基质辅助激光解吸电离飞行时间质谱和生物芯片等方法。本文对每种方法的原理、优缺点及应用研究现状进行了较为系统的介绍,以期为今后微生物高通量检测技术的快速发展提供参考。     

玉米细菌性枯萎病及其病原菌的检测技术

玉米细菌性枯萎病是影响玉米生产的重要病害 ,该病害通过带菌的种子进行远距离传播 ,对该病菌的检测成为防止和控制该病害的重要手段。介绍现有的检测该病菌的各种方法 ,即黑色素选择培养法、double sandwichELISA以及RAPD PCR ,LCR PCR ,Nested PCR ,multiplePCR和荧光实时PCR等分子生物学检测方法 ,并对这些方法的特性进行比较和探讨。     

Rapid diagnostics for methicillin-resistant Staphylococcus aureus: current status

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of healthcare- and community-associated infections, and its prevalence continues to increase. These infections are associated with morbidity and excessive mortality compared with infections caused by methicillin-susceptible S. aureus (MSSA). Numerous studies have cited the increased healthcare costs associated with MRSA infections. Infection control guidelines that combine active surveillance with aggressive patient management, such as patient isolation, decontamination, and other strategies, have been shown to reduce transmission and subsequent infections. The availability of rapid molecular diagnostics has strengthened infection control programs by providing results in hours rather than days, as the time required for culture-based methods. This review summarizes the current status of rapid diagnostic methods available for MRSA detection from nasal surveillance specimens, and assays available for rapid identification of MRSA from positive blood cultures containing Gram-positive cocci in clusters. Both amplification- and probe-based assays are highlighted and discussed in detail. Future technological advances are likely to see real-time assays that combine multiple gene targets for assessment of microbial identification, virulence detection, and mechanisms of resistance beyond mecA.     

产前B族链球菌筛查对妊娠结局及新生儿的影响

目的 研究妊娠期B族链球菌（GBS）感染对妊娠结局和新生儿的影响,探讨GBS筛查的临床意义。方法 选取2016年1月至2017年6月在我院进行GBS筛查的800名孕妇为受试对象,实时荧光定量PCR技术检测GBS携带情况,根据GBS筛查结果及是否愿意接受干预治疗分为治疗组、非治疗组和GBS阴性组,对比三组妊娠结局、新生儿情况。结果 800例受试对象中,GBS阳性136例（17.0%）;治疗组胎膜早破、早产、宫内感染、胎儿窘迫及新生儿肺炎、窒息、败血症、病理性黄疸发生率与GBS阴性组相比,差异均无统计学意义（Ps>0.05）;而与非治疗组相比,除新生儿脑膜炎差异无统计学意义（P>0.05）外,其他指标差异均有统计学意义（Ps<0.05）。结论 妊娠期进行GBS感染的筛查具有重要意义,及时采取干预措施可有效减少不良妊娠结局及新生儿感染的发生率。     

Detection, isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques, immunoassays and colony hybridization

AIMS: To provide information on detection of Shiga toxin-producing Escherichia coli (STEC) in retail-minced beef using an approach combining (i) PCR-based techniques and automated immunoassay for stx screening and detection of the five major serogroups associated with human infection, and (ii) immunomagnetic separation (IMS) and colony hybridization assays for bacterial strain isolation. METHODS AND RESULTS: Twenty-seven out of 164 minced beef samples were stx-positive by PCR-ELISA, nine of which were also positive by real-time PCR for at least one marker of the five main serogroups tested (O26, O103, O111, O145 and O157). Two E. coli O103 stx-negative strains were isolated from two out of 10 IMS and nine STEC strains that did not belong to the five main serogroups were isolated by colony hybridization. CONCLUSIONS: PCR techniques are applicable for rapid screening of samples containing both an stx gene and an O-group marker of the five main pathogenic STEC serogroups. Isolation of STEC strains belonging to the main non-O157 serogroups remains difficult. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an evaluation of a multi-faceted approach for the detection of the most frequently reported human pathogenic STEC serogroups. The advantages and limits of this strategy are presented.     

Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay

Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. Escherichia coli is the most common cause of UTI accounting for up to 70?% and a variable contribution from Proteus mirabilis, Pseudomonas aeruginosa and Klebsiella pneumoniae. To establish a complete diagnostic system, we have developed a single-tube multiplex PCR assay (mPCR) for the detection of the above-mentioned four major uropathogens. The sensitivity of the assay was found to be as low as 10(2)?cfu/ml of cells. The mPCR evaluated on 280 clinical isolates detected 100?% of E. coli, P. aeruginosa, P. mirabilis and 95?% of K. pneumonia. The assay was performed on 50 urine samples and found to be specific and sensitive for clinical diagnosis. In addition, the mPCR was also validated on spiked urine samples using 40 clinical isolates to demonstrate its application under different strain used in this assay. In total, mPCR reported here is a rapid and simple screening tool that can compete with conventional biochemical-based screening assays that may require 2-3?days for detection.     

Fluorescent detection techniques for real-time multiplex strand specific detection of Bacillus anthracis using rapid PCR

Speed is a key area in our development of PCR assays for Bacillus anthracis. We believe that the strand specific detection of amplicons within 10 min is a realistic goal and that this will be achieved through fluorescent in-tube assays. We have used the Idaho LightCycler to study and develop candidate assays for B. anthracis. New strand specific fluorescent methods have been developed and a number of formats have been studied for speed and sensitivity. Internal controls have been developed as a method of improving our assay confidence. In this communication we will introduce the field of rapid PCR whilst discussing previous work in the areas described above, the development of our own rapid assay and a novel internal control system for B. anthracis. This work used PCR assays and hardware that are either commercially available, or have been previously described in open literature publications.     

Differential identification of Ascogregarina species (Apicomplexa: Lecudinidae) in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) by polymerase chain reaction

We report 2 polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar gregarine species based on amplification of variable regions of the internal transcribed spacer region of ribosomal DNA. The gregarines we investigated were Ascogregarina barretti (Vavra), A. culicis (Ross), and A. taiwanensis (Lien and Levine), parasites of the mosquitoes Ochlerotatus triseriatus (Say), Aedes aegypti (Linnaeus), and Ae. albopictus (Skuse), respectively. These 3 important vector mosquitoes often utilize the same container habitats, where larval development and infection by the parasite occurs, leaving ample opportunity for cross-species gregarine infection. Because previous studies have shown that the parasites A. culicis and A. taiwanensis variably affect fitness in both normal and abnormal mosquito hosts, distinguishing parasite infection and species is important. The task is complicated by the fact that these 2 parasite species are virtually identical in morphology, whereas A. barretti is morphologically distinct. Of the 2 PCR-based assays reported here, the first provides a rapid, sensitive, and straight-forward means of general ascogregarine detection based on a single PCR amplification. The second method provides a means of differentiation between A. culicis and A. taiwanensis based on a species-specific PCR assay. Together, these assays allow whole mosquitoes to be tested for the presence of Ascogregarina species as well as identification of both A. culicis and A. taiwanensis singly or in dual infections.     

Detection of Epstein Barr virus in formalin-fixed paraffin tissues by fluorescent direct in situ PCR

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. However, molecular detection is also considered the reference standard assay for diagnosis of central nervous system infections and of most cases of nasopharyngeal carcinoma (NPC). One-step or nested polymerase chain reaction (PCR) has rapidly replaced immunological assays based on virus-specific Ig antibodies for the laboratory diagnosis of Herpesvirus infections, even if serological methods are considered an additional tool for defining clinical diagnosis. In this article, we will present a rapid, sensitive and robust molecular tool for the viral detection of EBV (EBNA-1) within tissue specimens by making use of in situ PCR (IS-PCR).     

Identification of Streptococcus agalactiae virulence genes in the neonatal rat sepsis model using signature-tagged mutagenesis

Group B streptococcal (GBS) infections are the most common cause of bacterial sepsis in the immediate newborn period. Apart from the capsule, the factors required for survival of GBS in the host are not well defined. In this study, signature-tagged transposon mutagenesis (STM) was used to identify genes required for growth and survival of GBS in a neonatal rat sepsis infection model. Approximately 1600 transposon mutants were screened in pools of 80 mutants, and approximately 120 mutants defective for survival in the animal host were identified. We successfully cloned and sequenced DNA flanking the transposon insertions from 92 of the mutants. Fifty per cent of the mutants had transposon insertions in genes with homologues in the public databases, whereas the remaining 50% had transposon insertions in genes with unknown function. A significant proportion of the avirulent mutants had transposon insertions in genes encoding transport-associated or regulatory proteins or in genes involved in cell surface metabolism, emphasizing the significance of these functions for in vivo survival of GBS. Overall, STM analysis revealed GBS genomic loci that encode a wide variety of functional gene classes, underscoring the diversity of bacterial processes required for the infection process. Currently, the function of the genes identified during the screening can only be inferred by homology to previously described genes. However, a number of the genes identified in this study have been shown to correlate with virulence in other pathogens. A virulence of a subset of mutants identified during the screening was confirmed by performing competitive index assays and lethal dose assays. This represents the first report of a genome-wide scan for virulence factors in GBS. The identified genes will further our understanding of the pathogenesis of GBS infections and may represent targets for intervention or lead to the development of novel therapies.     

Development of specific and rapid detection of bacterial pathogens in dairy products by PCR

A simple and specific method for direct detection of bovine mastitis pathogens (Streptococcus agalactiae (GBS), Staphylococcus aureus and Escherichia coli) in milk products, bacterial samples from milk and isolated bacterial DNA was developed. The method is based on polymerase chain reaction (PCR) using sequence-specific primers only for GBS and species-specific primers derived from 16S and 23S rRNA for all chosen species. The presence of the gene of surface immunogenic protein (Sip) in bovine GBS isolates, described previously only in human GBS isolates was confirmed. The GBS detection was performed with the sequence coding for surface immunogenic protein from GBS human isolates designated as Sip specific sequence (SSS); this sequence was selected for specific primer design. The sequence is unique for GBS and was designed from a consensus of all known sip genes. The specific identification was shown on a collection of 75 GBS bovine isolates from different localities in Slovakia. All isolates were positive to SSS, 16S and 23S rRNA sequence. The 16S and 23S rRNA PCR detection was also performed with S. aureus and E. coli isolates and specific PCR products were also detected. The detection limit of this assay for milk products was 6 CFU/microL (i.e. 6000 CFU/mL) for GBS and E. coli, and 16 CFU/microL for S. aureus. This rapid, sensitive and specific diagnostic method can be performed within hours and represents an innovative diagnostic tool for the detection of milk pathogens in dairy products.     

Comparative evaluation of Strepto B ID chromogenic medium and Granada media for the detection of Group B streptococcus from vaginal samples of pregnant women

Two types of selective media, the chromogenic medium Strepto B ID® and two non-chromogenic media Strepto B agar® and the Granada® medium, were tested and compared to blood agar plates (BAP) for screening of Group B streptococcus vaginal colonization in pregnant women. All tested media were comparable in terms of sensitivity however, their use in routine laboratories may markedly facilitate the rapid detection of GBS in vaginal samples.