Conventional culture for water quality assessment: is there a future?

Problems of deleterious effects in food are mainly caused by two groups of microbial agents including food pathogenic or spoilage microorganisms. The presence and contamination of those microorganisms in food can lead to the adverse impacts for both consumer health and food business operators around the world. To overcome such microbial risk in food or food-processing environments, one of control measures which food business operators can apply is to monitor or evaluate those relevant microorganisms by using specific, sensitive, and reliable detection, identification, or enumeration methods. Therefore, this chapter aims to basically emphasize on the brief principles and applications of standard culturing methods (reference methods) and rapid methods (alternative methods) as analysis tools for detection, identification, or enumeration of foodborne microorganisms. These methods are generally classified into two major categories: culture-dependent and culture-independent assays. Additionally, their major advantages and disadvantages are described.KeywordsFoodborne microorganismsContaminationStandard methodsRapid methodsDetection

uring the past 10 to 15 years, we have seen expansive growth of the use of molecular technology in the clinical laboratory for diagnosing infectious diseases. As a result, many laboratories are able to offer more sensitive testing, faster turnaround times, and ultimately improved patient care. The gold standard in bacteriology largely remains culture, primarily due to cost accounting and the potential complex nature of associated infections (ie, urine, wound, and respiratory cultures). However, in circumstances in which there may be minute quantities of a specific pathogen present, the patient may have received antibiotics prior to specimen collection, or the etiologic agent may require unusual culture conditions, molecular detection offers a great advantage to culture techniques. In many virology laboratories, molecular detection has supplanted cell culture techniques for the identification of several viral pathogens and in many cases has become the new gold standard. Though molecular techniques can offer an abundance of added benefits when used to augment current gold standards such as culture and/or serology, the optimal use of molecular methodologies in microbiology resides with specimens in which a limited number of pathogenic organisms are sought and in cases where the enhanced sensitivity and faster turnaround time of molecular methods far outweighs the increased cost. Applications in Bacteriology A classic example of successful nucleic acid amplification (NAA) testing in microbiology is the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) from vaginal, cervical, urethral, and first-void urine specimens. Sexually transmitted infections such as those caused by CT and NG can be rapidly and accurately identified using NAA, thus improving treatment and transmission prevention. Implementation of routine screening for CT has lowered the prevalence rates of CT and associated pelvic inflammatory disease. 1 The increased sensitivity offered by NAA detection of CT and NG is important not only for the diagnosis of symptomatic patients, but also for the asymptomatic individuals that account for more than 70% of positive cases. Until implementation of NAA testing for CT and NG, culture was the gold standard, although it has subsequently been shown to have only 60% to 75% sensitivity compared to NAA. 2 A further disadvantage of culture is that organism viability must be preserved during transport. The implementation of routine confirmatory testing should be considered when using NAA for a low prevalence population that results in a positive predictive value below 90%. 2 Another prime example of NAA results positively impacting patient care is the laboratory diagnosis of tuberculosis. Using

The problems associated with culture-based methods for detection and identification of microorganisms in clinical and environmental samples have motivated the development of alternative methods which do not require cultivation of the target organisms. Molecular genetic methods, which specifically target microbial nucleic acids, have become important tools for the identification of both cultured and uncultured microorganisms. The sensitivity of these methods approaches that of culture-based methods and it is arguable that the specificity of these methods generally exceeds that of culture-based methods. Indeed, molecular genetic methods have become a powerful set of tools used by many investigators to detect and identify culturable microorganisms in their viable but nonculturable state, as well as organisms which have yet to be brought into culture.

Rapid detection and identification of microorganisms is a challenging and important aspect in a wide range of fields, from medical to industrial, affecting human lives. Unfortunately, classical methods of microorganism identification are based on time-consuming and labor-intensive approaches. Screening techniques require the rapid and cheap grouping of bacterial isolates; however, modern bioanalytics demand comprehensive bacterial studies at a molecular level. Modern approaches for the rapid identification of bacteria use molecular techniques, such as 16S ribosomal RNA gene sequencing based on polymerase chain reaction or electromigration, especially capillary zone electrophoresis and capillary isoelectric focusing. However, there are still several challenges with the analysis of microbial complexes using electromigration technology, such as uncontrolled aggregation and/or adhesion to the capillary surface. Thus, an approach using capillary electrophoresis of microbial aggregates with UV and matrix-assisted laser desorption ionization time-of-flight MS detection is presented.

Quantitative spectroscopy analysis of prokaryotic cells: vegetative cells and spores

Routine identification of pathogenic microorganisms predominantly based on nutritional and biochemical tests is a time-consuming process, but delay may lead to fatal consequences at times. In this work, nanoparticle-induced nanoSPR enhanced IR spectroscopy was used in conjunction with a background elimination data processing algorithm to directly identify microorganisms in mixed cultures. It was demonstrated that the microbial composition of mixtures of different E. coli strains could be accurately identified using our method, at a concentration level of 103 CFU mL -1 . The procedure was also applied to determine the presence or absence of pathogenic microorganisms in a simple but real food matrix (apple juice). Results indicated that microorganisms in a cocktail of up to eight different species suspended in an apple juice matrix could be accurately identified for its presence or absence.

Fast detection and identification of microorganisms is a challenging and significant feature from industry to medicine. Standard approaches are known to be very time-consuming and labor-intensive (e.g., culture media and biochemical tests). Conversely, screening techniques demand a quick and low-cost grouping of bacterial/fungal isolates and current analysis call for broad reports of microorganisms, involving the application of molecular techniques (e.g., 16S ribosomal RNA gene sequencing based on polymerase chain reaction). The goal of this review is to present the past and the present methods of detection and identification of microorganisms, and to discuss their advantages and their limitations.

Diagnosis of prosthetic joint infection with culture technique can be problematic since the causative agent(s) are not possible to cultivate in all cases. Molecular methods had been evaluated in many studies but their inclusion in routine diagnostics is still controversial. The purpose of our prospective study was to compare the diagnostic accuracy of broad-range (BR)-PCR and culture technique. Intraoperative samples of periprosthetic tissue were retrieved in 67 patients undergoing revision arthroplasty. Samples were analyzed with culture technique, immunohistochemistry and BR 16S rRNA gene PCR. Bacteria in PCR-positive samples were identified using two different methods: direct sequencing of PCR products and specific TaqMan assays. In 63 cases, full concordance was found between BR-PCR and culture technique. Specific TaqMan assays failed to identify bacteria in four culture- and BR-PCR-positive cases and therefore had a lower sensitivity in comparison with BR-PCR. Molecular methods detected bacteria with the same accuracy as culture; however, identification of bacteria was inferior to culture. Further development of species-recognition techniques is required to improve identification of causative microorganisms.

The aim of this research was to use a polyphasic approach to differentiate Penicillium verrucosum from Penicillium nordicum, to compare different techniques, and to select the most suitable for industrial use. In particular, (1) a cultural technique with two substrates selective for these species; (2) a molecular diagnostic test recently set up and a RAPD procedure derived from this assay; (3) an RP-HPLC analysis to quantify ochratoxin A (OTA) production and (4) an automated system based on fungal carbon source utilisation (Biolog Microstation™) were used. Thirty strains isolated from meat products and originally identified as P. verrucosum by morphological methods were re-examined by newer cultural tests and by PCR methods. All were found to belong to P. nordicum. Their biochemical and chemical characterisation supported the results obtained by cultural and molecular techniques and showed the varied ability in P. verrucosum and P. nordicum to metabolise carbon-based sources and to produce OTA at different concentrations, respectively.

BackgroundMolecular amplification techniques are suggested to be a useful adjunct in early detection of pathogens in septic patients. The aim was to study the feasibility of a polymerase chain reaction (PCR) assay compared to the standard microbiological culture (MC) technique in identification of pathogenic microorganisms from blood and non-blood samples in septic patients.MethodsSamples for pathogen identification were taken during febrile septic episodes (SE) in 54 patients with sepsis and analyzed using both MC and PCR. Semi-automated multiplex PCR, provided by Philips Medical Systems, was able to detect nine different pathogens. The accuracy of pathogen identification using PCR vs. MC as well as the time-saving effect of PCR on the potential decision-making process for antimicrobial therapy was evaluated.ResultsIn a total of 258 samples taken during 87 SE, both methods yielded more pathogens from the non-blood than blood samples (87 % vs. 45 %; p = 0.002). PCR identified more pathogens than MC in the blood samples (98 vs. 21; p < 0.0001), but not in other body fluids. In 35 SE, the potential decision on appropriate antimicrobial therapy based on PCR results could have been made 50 (median; interquartile range 35–87) hours earlier than decisions based on standard MC.ConclusionsIn septic patients, multiplex PCR identified more pathogenic microorganisms isolated from the blood samples than the standard MC technique. In the non-blood samples, PCR was comparable to that of MC.Electronic supplementary materialThe online version of this article (doi:10.1186/s40560-015-0116-1) contains supplementary material, which is available to authorized users.

Application of a digital signal enhancement processor to investigate improvements in sensitivity for capillary electrophoresis and capillary electrochromatography applications

Rapid identification of microorganisms from positive blood cultures in pediatric patients by MALDI-TOF MS: Sepsityper kit versus short-term subculture

Brucellosis is caused by members of the Brucella spp. and remains one of the world's major zoonotic diseases. Brucella melitensis (B. melitensis) as the most contagious Brucella species cannot be ignored as an essential source of infection for brucellosis, especially in countries/regions dominated by animal husbandry. Thus, the identification of the Brucella genus and B. melitensis is crucial for rapid diagnosis of brucellosis to control disease transmission and clinical treatment. Here, we developed the CRISPR/Cas12b nuclease combined with a multiple cross displacement amplification (MCDA) assay (CRISPR-MCDA) for highly specific and sensitive detection of Brucella genus and B. melitensis in clinical applications. Two sets of specific primers were designed targeting the specific gene of Brucella genus (Bcsp31) and B. melitensis (BMEII0466), respectively. The CRISPR-MCDA assay showed high specificity and sensitivity in 28 non-Brucella isolates and 64 clinical samples. The detection limit of CRISPR-MCDA assay was 2 copies/μL in the plasmid dilution template, and the whole detection process took within 90 minutes with nanoparticle-based lateral flow biosensor (LFB) to validate experimental results. Taken together, the CRISPR-MCDA-LFB assay is a visual, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Brucella genus and B. melitensis.IMPORTANCEThe prevention and control of Brucellosis urgently require rapid and accurate diagnostic methods. This work validates a new method for the simultaneous detection of Brucella genus and B. melitensis. The method can effectively reduce the chances of contamination and provides a more rapid, sensitive, and specific on-site detection of Brucella. It also offers a solution for the rapid screening of Brucellosis in resource-limited environments, which is crucial for effective disease prevention and control. This technology can also be widely applied to the rapid detection of other pathogens beyond Brucella.

Synthesis and evaluation of novel chromogenic aminopeptidase substrates for microorganism detection and identification

BackgroundStrongyloides stercoralis infection is a neglected tropical disease which can lead to severe symptoms and even death in immunosuppressed people. Unfortunately, its diagnosis is hampered by the lack of a gold standard, as the sensitivity of traditional parasitological tests (including microscopic examination of stool samples and coproculture) is low. Hence, alternative diagnostic methods, such as molecular biology techniques (mostly polymerase chain reaction, PCR) have been implemented. However, there are discrepancies in the reported accuracy of PCR.MethodologyA systematic review with meta-analysis was conducted in order to evaluate the accuracy of PCR for the diagnosis of S. stercoralis infection. The protocol was registered with PROSPERO International Prospective Register of Systematic Reviews (record: CRD42016054298). Fourteen studies, 12 of which evaluating real-time PCR, were included in the analysis. The specificity of the techniques resulted high (ranging from 93 to 95%, according to the reference test(s) used). When all molecular techniques were compared to parasitological methods, the sensitivity of PCR was assessed at 71.8% (95% CI 52.2–85.5), that decreased to 61.8% (95% CI 42.0–78.4) when serology was added among the reference tests. Similarly, sensitivity of real-time PCR resulted 64.4% (95% CI 46.2–77.7) when compared to parasitological methods only, 56.5% (95% CI 39.2–72.4) including serology.ConclusionsPCR might not be suitable for screening purpose, whereas it might have a role as a confirmatory test.