Wolbachia pipientis growth kinetics and susceptibilities to 13 antibiotics determined by immunofluorescence staining and real-time PCR

Wolbachia spp. are strict intracellular bacteria that infect a wide range of arthropods and filarial nematodes. Filarial nematodes are important causes of human diseases. There is increasing evidence that Wolbachia spp. influence important functions in the biology of the hosts, specifically, infertility. Preliminary experiments with humans and animals have suggested that antibiotics with activity against Wolbachia may help to treat filariasis. In this study, we determined using a real-time quantitative PCR assay the growth kinetics of a strain of Wolbachia pipientis from a mosquito grown in Aa23 cells. The doubling time was estimated to be 14 h. We then determined the susceptibilities of this strain to 13 antibiotics by two methods: an immunofluorescent-antibody test and a real-time quantitative PCR assay. Both techniques gave similar results. Doxycycline and rifampin were the most effective compounds, with MICs of 0.125 and 0.06 to 0.125 micro g/ml, respectively. Fluoroquinolones were less effective, with MICs of 2 to 4 micro g/ml for ciprofloxacin, 2 micro g/ml for ofloxacin, and 1 micro g/ml for levofloxacin. beta-Lactams (penicillin G, amoxicillin, ceftriaxone) were not effective at concentrations up to 128 micro g/ml. The MIC of erythromycin was >32 micro g/ml, whereas that of telithromycin was 8 micro g/ml. Other antibiotic compounds were bacteriostatic only at high concentrations, including gentamicin, co-trimoxazole, and thiamphenicol. The real-time PCR assay was a convenient and reliable technique for determination of the antibiotic susceptibilities of WOLBACHIA: It may help in the future to simplify antibiotic susceptibility testing of strict intracellular pathogens.     

Direct antibiotic susceptibility testing of blood cultures of gram-negative bacilli using the Drug Susceptibility Testing Microfluidic (DSTM) device

Proper treatment of bloodstream infections requires rapid, early determination of appropriate antibiotic agents, emphasizing the need for more rapid drug susceptibility testing. The Drug Susceptibility Testing Microfluidic (DSTM) device represents a novel method in which a small amount of bacterial suspension is injected into the microchip-like device and cultured for 3 h. However, it remains unknown whether the DSTM method can directly determine antibiotic susceptibilities from positive blood cultures. Here, we developed a new approach to directly assess drug susceptibility, using the DSTM method for positive blood cultures. We compare the utility and accuracy of DSTM with those of conventional susceptibility testing methods. Fifty positive blood cultures identified as gram-negative bacilli were used herein. The outcomes of drug susceptibility and resistance assays for positive blood cultures were compared to those of conventional susceptibility testing methods to evaluate their utility and accuracy. Method agreement rates between DSTM and standard methods often exceed 90%, suggesting a high positive correlation with conventional methods. Furthermore, our results show that a combination of multiple drugs in the DSTM device helps identify extended-spectrum β-lactamase (ESBL)- and AmpC-β-lactamase (AmpC-)-producing microorganisms. In conclusion, DSTM method enables effective drug susceptibility and resistance screening within 3 h from positive blood cultures and is suitable for the rapid and personalized determination of the antimicrobial regimen.     

Evaluation of antibiotic susceptibilities of Ehrlichia canis, Ehrlichia chaffeensis, and Anaplasma phagocytophilum by real-time PCR

We determined MICs of antibiotics against Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis by real-time quantitative PCR. The doubling times of the organisms were established: 19 h for E. chaffeensis, 26 h for A. phagocytophilum, and 28 h for E. canis. In comparison to the reference method for determining sensitivities, which uses Diff-Quick staining, our PCR assay was very sensitive and specific. We confirmed that doxycycline and rifampin are highly active against these bacteria and found variable susceptibilities to fluoroquinolones; A. phagocytophilum was susceptible, but E. canis and E. chaffeensis were only partly susceptible. Beta-lactam compounds, cotrimoxazole, macrolide compounds, and telithromycin showed no activity against any of the three organisms. Thiamphenicol was found to be more active than chloramphenicol. For the first time, we showed that these three species have numerous point mutations in their 23S RNA genes, with those at positions 754, 2057, 2058, 2059, and 2611 (Escherichia coli numbering) known to confer resistance to macrolide compounds in other bacteria. The role of each of these mutations in resistance to these drugs should be investigated in the future. Our study confirms previous reports that quantitative PCR is a reliable method for determining antibiotic susceptibility; therefore, it might be useful for screening new drugs.     

Antibiotic susceptibilities of Legionella pneumophila strain Paris in THP-1 cells as determined by real-time PCR assay

OBJECTIVES: Legionella species are facultative intracellular bacteria. Evaluation of the activity of antibiotics against intracellular L. pneumophila is more predictive of their in vivo efficacy than MICs as determined in axenic medium. However, current methodologies are based on cfu count determination, and are tedious because of the slow growth of Legionella spp. We investigated antibiotic susceptibilities of L. pneumophila strain Paris in THP-1-derived macrophages, using a real-time PCR assay for evaluation of bacterial growth. METHODS: Intracellular activities of seven antibiotic compounds against two human isolates of L. pneumophila strain Paris were determined in THP-1-derived macrophages in vitro. Bacterial growth was evaluated using either cfu methodology or a real-time PCR protocol targeting the mip gene. RESULTS: Bacterial titres as determined using real-time PCR were well correlated with cfu counts. Antibiotic susceptibilities for the two L. pneumophila isolates tested were comparable when using either of the two techniques. MICs were also similar to those previously reported for other L. pneumophila serogroup 1 strains. In particular, rifampicin and the fluoroquinolones were the most active compounds, both in extracellular medium and in THP-1 cells. Real-time PCR, however, was much less laborious than the traditional cfu method. CONCLUSIONS: Real-time PCR is better adapted than cfu-based methods to evaluating the antibiotic susceptibilities of large series of Legionella strains to newer antibiotic compounds.     

Susceptibility of Staphylococcus aureus growing on fibronectin-coated surfaces to bactericidal antibiotics.

Several recent studies have shown that bacteria either grown in vitro as adherent biofilms or recovered from infected prosthetic devices have decreased susceptibilities to antimicrobial killing. To further study the microbial and environmental factors responsible for this decreased antibiotic susceptibility, we developed an in vitro model of surface-adherent Staphylococcus aureus growing on polymethylmethacrylate coverslips coated with pure fibronectin. After exponential growth for 4 h, the population of fibronectin-attached S. aureus remained constant for a further 48-h period, as evaluated by CFU counts of organisms quantitatively removed from the coverslips. At selected time points, surface-bound organisms were exposed to bactericidal concentrations of either oxacillin, vancomycin, fleroxacin, or gentamicin in short-term (0.5 to 2 h) or long-term (24 h) killing assays. Whereas at 2 h surface-growing organisms were still optimally killed by all antimicrobial agents, at 4 and 24 h attached bacteria expressed markedly altered susceptibilities to these agents. The decrease in susceptibility was moderate for fleroxacin, more important for oxacillin and vancomycin, and extensive for gentamicin. When surface-attached S. aureus was compared with bacteria grown in a fluid phase, both populations showed a parallel time-dependent decrease in their susceptibilities to either oxacillin, vancomycin, or fleroxacin. In contrast, attached organisms became considerably more resistance to gentamicin than suspended bacteria did. Subpopulations of organisms spontaneously released from coverslips during antibiotic exposure also showed markedly reduced susceptibilities to antimicrobial killing. This simple model of S. aureus colonization of in vitro fibronectin-coated surfaces might represent a useful approach to the study of the physiological and biochemical changes that underlie the decreased antibiotic susceptibilities of biomaterial-attached organisms.     

Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR

BACKGROUND: Broad-range PCR provides valuable information for detecting bacterial infections. This study assesses the combined use of broad-range real-time PCR and high-resolution melting analysis for rapid detection and identification of clinically important bacteria. METHODS: We subjected 46 bacterial culture colonies representing 25 clinically important bacterial species to LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye. We performed high-resolution melting analysis of the PCR products with the HR-1 instrument and used melting profiles as molecular fingerprints for bacterial species identification. We validated this method via assessment of 54 consecutive bacteria culture colonies obtained from a clinical microbiology laboratory. RESULTS: The 16S rRNA gene of all 25 bacterial species was amplifiable by this method, with PCR product lengths of 216 or 217 bp. Of the 25 bacterial species, we identified 11 via a 1-step post-PCR high-resolution melting analysis. The remaining bacterial species were identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species or by a 2nd real-time PCR targeting a different region of the 16S rRNA gene. A high-resolution melting database and a working protocol were established for identifying these 25 bacterial species. In the validation assay, a 94% accuracy rate was achieved when the bacterial species were in the high-resolution melting database. CONCLUSIONS: This assay requires no multiplexing or hybridization probes and provides a new approach for bacterial species identification in a molecular diagnostic laboratory.     

Real-time PCR detection and frequency of 16S rDNA mutations associated with resistance and reduced susceptibility to tetracycline in Helicobacter pylori from England and Wales

OBJECTIVES: To investigate the occurrence of 16S rDNA mutations associated with resistance or reduced susceptibility to tetracycline in Helicobacter pylori isolated in England and Wales, and to develop a real-time PCR assay to detect these DNA polymorphisms from culture and gastric biopsies. METHODS: Tetracycline susceptibility was determined by disc diffusion. The MIC of isolates with reduced susceptibility was determined by Etest and agar dilution methods. The 16S rDNA of these isolates was sequenced and resistance-associated mutations identified. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture and gastric biopsies. RESULTS: From 1006 isolates of H. pylori examined, 18 showed reduced susceptibility to tetracycline. Of these, three were resistant (>or=4 mg/L). Mutations in 16S rDNA were detected in 10 of the reduced susceptibility isolates: one double base mutation of A926T/A928C, one A926C, one A928C; and seven A926G. The first two polymorphisms were novel and had not been reported from clinical isolates previously. The LightCycler assay identified each of the 10 isolates with 16S rDNA mutations, but did not detect polymorphisms in 100 tetracycline-susceptible H. pylori isolates. The assay correctly determined the tetracycline susceptibility of H. pylori in 20 gastric biopsy samples. CONCLUSIONS: Mutations in 16S rDNA were detected in H. pylori isolated in England and Wales with reduced susceptibility to tetracycline, but resistance to this antibiotic was uncommon. We show molecular-based susceptibility testing for tetracycline is possible direct from biopsy material.     

Development of a rapid assay methodology for antimicrobial susceptibility testing of Staphylococcus aureus

Development of a new phenotypic technique for rapid antimicrobial susceptibility testing (AST) of methicillin-resistant Staphylococcus aureus is presented. The new technique combines bacterial culturing and specific immunometric detection in a single separation-free process. The technique uses dry chemistry reagents and the recently developed two-photon excitation detection technology, which allows online detection of bacterium-specific growth. The performance of the new technique was evaluated by monitoring the growth of S. aureus reference strains and determining their susceptibility to oxacillin. In the direct analysis of clinical specimens, method specificity and tolerance to interferences caused by other bacteria present in the sample are pivotal. Other bacteria can compete with the bacteria of interest for nutrients, for example. Specificity and tolerance were studied against Staphylococcus epidermidis reference strains. The results suggest that the new technique could allow rapid AST directly from clinical samples within 6 to 8 h. Such a rapid and simple testing methodology would be a valuable tool in clinical microbiology because it would shorten the turnaround times of microbiologic analyses. Advantages of the new approach in relation to conventional methods are discussed.     

Use of molecular techniques for the detection of antibiotic resistance in bacteria

The introduction of antibiotics in the 1930s revolutionized the treatment of previously fatal infections. Unfortunately, bacteria adapted quickly to this threat and antibiotic resistance soon developed. Both penicillin and methicillin resistance in Staphylococcus aureus were noted almost immediately following the introduction of penicillin and the penicillinase-resistant penicillins respectively. Antibiotic susceptibility testing rapidly became an integral part of the management of patients with infectious disease and remains the basis for clinical decision-making and antibiotic selection. However, the fundamental principles underlying antibiotic susceptibility testing methods have remained largely unchanged over the last few decades. Due to the inherent time delay imposed by bacterial growth rates, culture-based systems have traditionally provided results several hours to days after initial isolation. Better understanding of the genetic basis of resistance has resulted in the development of molecular methods to detect the genetic changes or mutations present in resistant bacterial phenotypes. Microbiologists and clinicians are faced with increasing options for the use of genotypic or phenotypic methods. This review provides an insight into the current molecular methods available for the detection of antibiotic resistance and to provide a basis for evaluating the choice of testing method to be used.     

Rapid Polymerase Chain Reaction-based Screening Assay for Bacterial Biothreat Agents

Objectives:  To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens.
Methods:  The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria.
Results:  The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus .
Conclusions:  A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.     

细菌rDNA分类鉴定的方法学研究

目的 研究细菌rDNA快速鉴定方法.方法 以细菌16S rDNA、16S-23S rDNA基因间隔区（ISR）和常见细菌耐药基因为对象,通过改进标本中细菌DNA提取方法,指纹分析、直接测序与多重聚合酶链反应（PCR）技术,建立快速细菌分子鉴定方法.结果 细菌属种不同表现出独特的16S-23S rDNA ISR指纹,在分析软件指导下进行初步细菌鉴定和亲缘关系研究;16S rDNA和16S-23SrDNA ISR序列可确定细菌种与型;序列分析与生化鉴定一致.多重PCR可解决葡萄球菌16S rDNA与mecA基因、产超广谱β内酰胺酶菌16S rDNA与TEM和SHV基因同步检测.结论 细菌rDNA分类方法,提高了非培养细菌的鉴定能力.     

Detection of bacteria in platelet concentrates: comparison of broad-range real-time 16S rDNA polymerase chain reaction and automated culturing

BACKGROUND: Based on real-time polymerase chain reaction (PCR) technology, a broad-range 16S rDNA assay was validated and its performance was compared to that of an automated culture system to determine its usefulness for rapid routine screening of platelet concentrates (PCs). STUDY DESIGN AND METHODS: The presence of bacteria in pooled PCs was routinely assessed in an automated culturing system (BacT/ALERT, bioMerieux). The PCR assay was performed with DNA extracted from the same samples as used for culturing. DNA extraction was performed with a automated extraction system (MagNA Pure, Roche Diagnostics). PCR amplification was performed with a set of universal primers and probe targeting eubacterial 16S rDNA. RESULTS: A total of 2146 PCs were tested. Eighteen (0.83%) samples were found to be contaminated. These samples were positive for the presence of bacteria by both methods. All contaminants were identified as bacteria belonging to the common human skin flora. These included Propionibacterium spp. (n = 7), Staphylococcus spp. (n = 6), Bacillus spp. (n = 2), Micrococcus spp. (n = 2), and Peptostreptococcus spp. (n = 1). Estimation of the bacterial load in PCs by real-time PCR showed that the initial levels of contamination varied between 13.6 and 9 x 10(4) colony-forming unit equivalents per PCR procedure. CONCLUSIONS: Compared to culture in the BacT/ALERT system, the PCR assay had a sensitivity of 100 percent and a specificity of 100 percent. This real-time PCR assay has a much shorter turnaround time of 4 hours, which offers the possibility to test and obtain results on PCs before release or the day they are transfused. This would permit the withdrawal of contaminated PCs before transfusion.     

Détection génotypique des résistances bactériennes : de la phénotypie à la génotypie,deux méthodes complémentaires

The problem of emerging resistance urges clinicians to prescribe narrow-spectrum antibiotic targeted to the specific causative pathogen. In hospital, a high level of antibiotic use and a propensity for genetic exchange between bacteria provide a perfect environment for multiresistant micro-organisms. Detection of antimicrobial resistance is important to optimise decisions about antimicrobial therapy. In order to prevent or to slow the spread of resistance among bacterial strains, clinicians must know as soon as possible which bacteria they are dealing with and to which antibiotic the strain is susceptible. This essential information can take several days up to weeks using traditional culture-based methods. In most cases such phenotypic susceptibility tests continue to be useful and get improve. Now automated systems are available to determine the infectious strain and its susceptibility to antibiotics within hours. In the near future, genetic methods as DNA-based solutions will be able to identify an infectious agent and its resistance pattern in less than an hour. Applied DNA technology progresses and will lead to the development and application of sophisticated new strategies for the analysis of antibiotic resistant bacterial strains. In the present knowledge, both methods for the determination of antimicrobial resistance, the phenotypic and the genotypic tests, are complementary.     

A quantitative LightCycler PCR to detect Streptococcus pneumoniae in blood and CSF

A quantitative real-time PCR targeting the Pneumolysin (ply) gene of Streptococcus pneumoniae was developed for the LightCycler instrument using Fluorescence Resonance Energy Transfer (FRET) probes. All common S. pneumoniae serotypes were detected while other bacteria and viruses were not. The sensitivity was determined to be between one and ten target copies per reaction. The PCR was applied to six CSF and 16 whole blood specimens from 17 patients with laboratory proven invasive pneumococcal disease. One hundred percent of CSF specimens and 69% of whole blood specimens were PCR positive. The bacterial loads were determined to be 7.6 to 6.01 x 10(5) copies/microL for the six CSF specimens, and 0.08 to 5.4 x 10(2) copies/microL for the 16 whole blood specimens. Ninety-seven percent of 30 culture and Gram's stain negative CSF specimens and 100% of 50 normal whole blood specimens were PCR negative. This highly sensitive and specific PCR assay has the potential to provide sufficiently rapid results to improve antibiotic treatment of S.pneumoniae infections, while bacterial load quantitation has opened up exciting possibilities for patient management.     

TaqMan实时荧光定量PCR检测艰难梭菌

目的 建立特异敏感的实时荧光定量PCR方法,用于艰难梭菌的快速检测;评价基于纯培养艰难梭菌和粪便中艰难梭菌的2种标准曲线;并应用该荧光定量PCR法对急性艰难梭菌感染（CDI）的小鼠进行粪便含菌量检测评价。方法 针对艰难梭菌基因组中16S rRNA序列设计特异性引物和探针,建立一套快速检测艰难梭菌含量的实时荧光定量PCR方法,验证方法的特异性、灵敏性;绘制艰难梭菌纯菌浓度梯度稀释标准曲线和粪便中同浓度梯度艰难梭菌的标准曲线,比较两者的差异;用艰难梭菌高毒株NAP1/027感染用抗生素处理的C57BL/6小鼠,建立CDI小鼠模型,同时应用该荧光定量PCR和活菌培养法定量检测小鼠粪便中的艰难梭菌含量变化。结果 建立的TaqMan实时荧光定量PCR具有较高的灵敏性和特异性,生成标准曲线的相关系数为0.999 8,斜率为-3.400 4;用纯培养艰难梭菌和粪便中艰难梭菌分别制备标准曲线,结果表明2种标准曲线定量检测结果差异无统计学意义。建立CDI小鼠模型,应用该荧光PCR能有效、准确的检测出粪便中艰难梭菌的含量,可替代费时费力的活菌培养计数法。结论 用纯培养艰难梭菌来制备标准曲线不影响对含菌粪便标本的准确定量检测,荧光定量PCR能准确快捷地检测CDI小鼠粪便中的艰难梭菌含量,比活菌计数更快速和方便,可用于艰难梭菌感染小鼠模型中小鼠肠道内艰难梭菌定植的定量检测。     

重症急性胰腺炎合并细菌感染的快速诊断——附17例检测分析

目的:探讨以16S rRNA基因为基础的细菌PCR法诊断重症急性胰腺炎早期合并细菌感染的价值.方法:17例疑有细菌感染的重症急性胰腺炎病人,术前B超引导下行胰周渗液细针穿刺检查,分别行以16S rRNA基因为基础的细菌PCR法检测和细菌培养.比较细菌PCR法检测结果和细菌培养结果.结果:17例中,PCR检测9例阳性,细菌培养10例阳性,该10例病人行手术治疗,术中所取胰周渗液行细菌培养证实为阳性.PCR检测重症急性胰腺炎合并细菌感染的敏感度为90%,特异度为100%.PCR法和细菌培养法分别需时5 h和3 d.结论:以16S rRNA基因为基础的细菌PCR法能快速、准确地诊断重症急性胰腺炎早期合并细菌感染,为手术治疗提供可靠依据.     

Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry

OBJECTIVES: To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. METHODS: Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic. RESULTS: In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays. CONCLUSIONS: This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis.