Traditional Microbiological Methods Research Articles

Microbiological Test Data-Assuring Data Integrity.

Marketed drugs and devices possess specifications including critical microbiological quality attributes purposed to assure efficacy and patient safety. These attributes are legislated requirements intended to protect the recipient patient. Sampling, microbiological testing, interpretation of data for final products, raw materials, and intermediates all contribute to a cohesive assessment in the assurance of finished product quality. Traditional culture-based microbiological methods possess inherent and unavoidable variability, recognized by the compendia and which might lead to erroneous conclusion pertaining to product quality. Such variability has been associated and intrinsically linked with data integrity issues; manufacturers have subsequently been encouraged by regulatory authorities to introduce multiple microbiologists or checks to prevent such issues. Understanding microbiological variability is essential such that genuine data integrity issues are identified. Furthermore, a range of meaningful preventative strategies are feasible beyond increasing the capacity of the quality control microbiological laboratory. This short review describes the legislative requirements, inherent microbiological variability, and realistic actions and activities that genuinely assure patient safety.LAY ABSTRACT: Marketed drugs and devices possess specifications including critical microbiological quality attributes purposed to assure efficacy and patient safety. These attributes are legislated requirements intended to protect the recipient patient. Sampling, microbiological testing, interpretation of data for final products, raw materials, and intermediates all contribute to a cohesive assessment in the assurance of finished product quality. Traditional culture-based microbiological methods possess inherent and unavoidable variability, recognized by the compendia and which might lead to erroneous conclusion pertaining to product quality. Such variability has been associated and intrinsically linked with data integrity issues; manufacturers have subsequently been encouraged by regulatory authorities to introduce multiple microbiologists or checks to prevent such issues. Understanding microbiological variability is essential such that genuine data integrity issues are identified. Furthermore, a range of meaningful preventative strategies are feasible beyond increasing the capacity of the quality control microbiological laboratory. This short review describes the legislative requirements, inherent microbiological variability, and realistic actions and activities that genuinely assure patient safety.

Electrochemical Biosensors for Rapid Detection of Foodborne Salmonella: A Critical Overview.

Salmonella has represented the most common and primary cause of food poisoning in many countries for at least over 100 years. Its detection is still primarily based on traditional microbiological culture methods which are labor-intensive, extremely time consuming, and not suitable for testing a large number of samples. Accordingly, great efforts to develop rapid, sensitive and specific methods, easy to use, and suitable for multi-sample analysis, have been made and continue. Biosensor-based technology has all the potentialities to meet these requirements. In this paper, we review the features of the electrochemical immunosensors, genosensors, aptasensors and phagosensors developed in the last five years for Salmonella detection, focusing on the critical aspects of their application in food analysis.

Detection and quantification of Alicyclobacillus acidoterrestris by electrical impedance in apple juice

Alicyclobacillus acidoterrestris is a thermotolerant bacterium able to grow in fruit juices and drinks, as the spoilage by Alicyclobacillus in the final product does not product any gas, but leads to a “medicine flavor” due to the formation of guaicol. Also, its detection is a challenge for the quality control departments, because it takes several days to get the results of traditional microbiology methods. This study aimed at developing a more accurate electrical impedance technique for the detection of A. acidoterrestris in concentrated apple juice. Samples of apple juice were inoculated with A. acidoterrestris spores isolated from a peach and grape juice. For the spore germination, several heat-shock treatments were tested (80 °C/10 min, 70 °C/20 min and 60 °C/30 min). Direct and indirect electrical impedance was applied to detect and quantify the microorganism in the inoculated apple juice, using BAT broth and Bimedia 002A (pH 4). The 80 °C/10 min treatment was selected for spore activation. The valid electrical impedance technique was the indirect method in BAT broth, which measured the changes in the impedance through the formation of CO2. In addition, a positive correlation (r = 0.98, R2 = 0.97) was observed between the classical microbiology (BAM agar) and the indirect impedance method.

Rapid antibiotic sensitivity testing in microwell arrays.

The widespread bacterial resistance to a broad range of antibiotics necessitates rapid antibiotic susceptibility testing before effective treatment could start in the clinic. Among resistant bacteria, Staphylococcus aureus is one of the most important, and Methicillin-resistant (MRSA) strains are a common cause of life threatening infections. However, standard susceptibility testing for S. aureus is time consuming and thus the start of effective antibiotic treatment is often delayed. To circumvent the limitations of current susceptibility testing systems, we designed an assay that enables measurements of bacterial growth with higher spatial and temporal resolution than standard techniques. The assay consists of arrays of microwells that confine small number of bacteria in small spaces, where their growth is monitored with high precision. These devices enabled us to investigate the effect of different antibiotics on S. aureus growth. We measured the Minimal Inhibitory Concentration (MIC) in less than 3 hours. In addition to being significantly faster than the 48 hours needed for traditional microbiological methods, the assay is also capable of differentiating the specific effects of different antibiotic classes on S. aureus growth. Overall, this assay has the potential to become a rapid, sensitive, and robust tool for use in hospitals and laboratories to assess antibiotic sensitivity.

Uropathogenic Escherichia coli pathogenicity islands and other ExPEC virulence genes may contribute to the genome variability of enteroinvasive E. coli

BackgroundEnteroinvasive Escherichia coli (EIEC) may be the causative agent of part of those million cases of diarrhea illness reported worldwide every year and attributable to Shigella. That is because both enteropathogens have many common characteristics that difficult their identification either by traditional microbiological methods or by molecular tools used in the clinical laboratory settings. While Shigella has been extensively studied, EIEC remains barely characterized at the molecular level. Recent EIEC important outbreaks, apparently generating more life-threatening cases, have prompted us to screen EIEC for virulence traits usually related to extraintestinal pathogenic E. coli (ExPEC). That could explain the appearance of EIEC strains presenting higher virulence potential.ResultsEIEC strains were distributed mainly in three phylogroups in a serogroup-dependent manner. Serogroups O124, O136, O144, and O152 were exclusively classified in phylogroup A; O143 in group E; and O28ac and O29 in group B1. Only two serogroups showed diverse phylogenetic origin as follows: O164 was assigned to groups A, B1, C, and B2 (one strain each), and O167 in groups E (five strains), and A (one strain) (Table 1). Eleven of 20 virulence genes (VGs) searched were detected, and the majority of the 19 different VGs combinations found were serogroup-specific. Uropathogenic E. coli (UPEC) PAI genetic markers were detected in all EIEC strains. PAIs IJ96 and IICFT073 were the most frequent (92.1 and 80.4%, respectively). PAI IV536 was restricted to some serogroups from phylogroups A, B1 and E. PAI ICFT073 was uniquely detected in phylogroups B2 and E. A total of 45 (88%) strains presented multiple PAI markers (two to four). PAIs IJ96 and IICFT073 were found together in 80% of strains.ConclusionsEIEC is a DEC pathovar that presents VGs and pathogenicity island genetic markers typically associated with ExPEC, especially UPEC. These features are distributed in a phylogenetic and serogroup-dependent manner suggesting the existence of stable EIEC subclones. The presence of phylogroups B2 and E strains allied to the presence of UPEC virulence-associated genes may underscore the ongoing evolution of EIEC towards a hypervirulent pathotype.

Comparison between Flow Cytometry and Traditional Culture Methods for Efficacy Assessment of Six Disinfectant Agents against Nosocomial Bacterial Species.

The present study was undertaken to compare the use of flow cytometry (FCM) and traditional culture methods for efficacy assessment of six disinfectants used in Quebec hospitals including: two quaternary ammonium-based, two activated hydrogen peroxide-based, one phenol-based, and one sodium hypochlorite-based. Four nosocomial bacterial species, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Vancomycin-resistant Enterococci faecalis, were exposed to minimum lethal concentrations (MLCs) and sublethal concentrations (1/2 MLCs) of disinfectants under study. The results showed a strong correlation between the two techniques for the presence of dead and live cell populations, as well as, evidence of injured populations with the FCM. The only exception was observed with sodium hypochlorite at higher concentrations where fluorescence was diminished and underestimating dead cell population. The results also showed that FCM can replace traditional microbiological methods to study disinfectant efficacy on bacteria. Furthermore, FCM profiles for E. coli and E. faecalis cells exposed to sublethal concentrations exhibited distinct populations of injured cells, opening a new aspect for future research and investigation to elucidate the role of injured, cultural/noncuturable/resuscitable cell populations in infection control.

Detection and Enumeration of Spore-Forming Bacteria in Powdered Dairy Products.

With the abolition of milk quotas in the European Union in 2015, several member states including Ireland, Luxembourg, and Belgium have seen year on year bi-monthly milk deliveries to dairies increase by up to 35%. Milk production has also increased outside of Europe in the past number of years. Unsurprisingly, there has been a corresponding increased focus on the production of dried milk products for improved shelf life. These powders are used in a wide variety of products, including confectionery, infant formula, sports dietary supplements and supplements for health recovery. To ensure quality and safety standards in the dairy sector, strict controls are in place with respect to the acceptable quantity and species of microorganisms present in these products. A particular emphasis on spore-forming bacteria is necessary due to their inherent ability to survive extreme processing conditions. Traditional microbiological detection methods used in industry have limitations in terms of time, efficiency, accuracy, and sensitivity. The following review will explore the common spore-forming bacterial contaminants of milk powders, will review the guidelines with respect to the acceptable limits of these microorganisms and will provide an insight into recent advances in methods for detecting these microbes. The various advantages and limitations with respect to the application of these diagnostics approaches for dairy food will be provided. It is anticipated that the optimization and application of these methods in appropriate ways can ensure that the enhanced pressures associated with increased production will not result in any lessening of safety and quality standards.

ELIME assay vs Real-Time PCR and conventional culture method for an effective detection of Salmonella in fresh leafy green vegetables

The detection of Salmonella according to EC regulation is still primarily based on traditional microbiological culture methods that may take several days to be completed.The purpose of this work is to demonstrate the applicability of an Enzyme-Linked-Immuno-Magnetic-Electrochemical (ELIME) assay, recently developed by our research group for the detection of salmonella in irrigation water, in fresh (raw and ready-to-eat) leafy green vegetables by comparison with Real-Time PCR (RTi-PCR) and ISO culture methods.Since vegetables represent a more complex matrix than irrigation water, preliminary experiments were carried out on two leafy green vegetables that resulted negative for salmonella by the ISO method. 25g of these samples were experimentally inoculated with 1–10 CFU of S. Napoli or S. Thompson and pre-enriched for 20h in two different broths. At this time aliquots were taken, concentrated at different levels by centrifugation, and analyzed by ELIME and RTi-PCR. Once selected the best culture medium for salmonella growth, and the optimal concentration factor suitable to reduce the sample matrix effect, enhancing the out-put signal, several raw and ready-to-eat leafy green vegetables were artificially inoculated and pre-enriched. Aliquots were then taken at different incubation times and analyzed with both techniques. Results obtained showed that 20 and 8h of pre-enrichment were required to allow the target salmonella (1–10 CFU/25g) to multiply until reaching a detectable concentration by ELIME and RTi-PCR assays, respectively. A confirmation with the ISO culture method was carried out. Based on the available literature, this is the first report of the application of an ELISA based method for the detection of Salmonella in vegetables.

Gram-Negative Polymicrobial Bloodstream Infections and Clinical Decision Making with a Microarray Testing System

BackgroundMolecular rapid diagnostic tests, such as the microarray-based Verigene Gram-Negative Blood Culture Test (BC-GN), when performed in conjunction with antimicrobial stewardship, are associated with improved clinical outcomes in bloodstream infections (BSIs). Optimal use of Verigene BC-GN to aid in clinical decision-making, however, can be hindered by a lack of confidence in results if patients have polymicrobial GN BSIs.MethodsBlood culture data from patients tested by Verigene BC-GN from June 2015 – July 2016 from the Detroit Medical Center and University of Maryland Medical Center were retrospectively reviewed and evaluated for incidence of missed GNs in GN BSIs. Missed GNs entailed those not detected in the tested sample and those that grew in a separate complementary positive blood culture bottle collected but not tested using Verigene BC-GN. For blood culture sets with missed GNs, potential clinical significance was evaluated.ResultsA total of 1,003 sets of GN blood cultures were reviewed. Fifty-seven (5.6%) were determined to be polymicrobial GN BSIs by traditional microbiological culture methods. Verigene BC-GN did not identify one or more GNs in 37 cases (65% of polymicrobial GN BSIs; 3.7% of total GN BSIs.); 25 were missed by the probe in the tested culture and 12 where missed due to isolation in the complementary blood culture bottle that was not tested by Verigene BC-GN. While potentially inappropriate de-escalation of antibiotic therapy could have occurred in 18 (31.5%) of polymicrobial GN BSIs, this represented only 1.8% of total GN BSIs.ConclusionCurrent practices utilizing Verigene BC-GN can result in the omission of undetected GNs in over half of polymicrobial GN BSIs. These misses were a combination of technology and workflow limitations. The potential for inappropriate de-escalation of antibiotic therapy in the entire cohort, however, was infrequent (<2% of total cases).Disclosures P. Lephart, Nanosphere: Grant Investigator, Grant recipient; J. K. Johnson, Nanosphere: Grant Investigator, Grant recipient

Diazotrophic bacteria associated to sugarcane varieties cropped at Northeast Region of Brazil

The objective of this work was to study the diversity of diazotrophic bacteria associated to 10 varieties of sugarcane cultivated in two states of the Northeast of Brazil and to identify the isolates by morphological and molecular methods. Diversity was studied by the application of traditional growth-depending microbiological methods: Gram-staining, comparison of colony morphology and growth on several culture media. Phylogenetic taxonomy was performed using 16S rRNA gene sequencing data. Almost all isolates obtained in semi-solid nitrogen free media were classified as Gram-negative and seven colony morphological groups were distinguished after growth on Potato medium. Among the 10 varieties evaluated, RB72454 resulted in higher number of isolates and diversity. The great majority of isolates from most of the genotypes tested showed similar characteristic to the genus Gluconacetobacter based on its peculiar colony morphology and growth characteristics in semi-solid media. Comparison of the 16S rRNA sequence of each isolate to the Genbank showed that they are closely related to Gluconacetobacter, Burkholderia, Kosakonia, Klebsiella and Stenotrophomonas.

Molecular Detection of the Three Major Pathogenic Vibrio Species from Seafood Products and Sediments in Tunisia Using Real-Time PCR

A rapid, sensitive, and highly reproducible real-time PCR assay was developed to detect the three major Vibrio spp. pathogenic for humans in Tunisian seafood products and sediments. A conventional culture method found 102 (41.3%) of 247 analyzed samples positive for Vibrio spp.; a conventional PCR method found 126 (51%) of the 247 samples positive. Real-time PCR assay found 126 (51.1%) samples positive; V. alginolyticus toxR was the most common, found in 99 (78.57%) of samples, followed by V. parahaemolyticus in 26 (20.63%) and V. cholerae in 1 (0.7%). All culture-positive samples were PCR positive. However, 24 samples that were positive by conventional PCR and real-time PCR were culture negative. Our findings indicate that retail seafood is commonly contaminated with Vibrio spp. and presents a potential risk to human health in Tunisia. These data also indicate that real-time PCR can provide sensitive species-specific detection of Vibrio spp. in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.

An Overview on Novel Microbial Determination Methods in Pharmaceutical and Food Quality Control.

Traditional microbiological methods tend to be labor-intensive and time-consuming. Rapid and novel methods in microbiological tests provide more sensitive, precise and reproducible results compared with conventional methods. In microbiology, the most rapid testing methods belong to the field of biotechnology such as PCR, ELISA, ATP bioluminescence and etc. Nevertheless impedance microbiology, biosensors and analytical procedures to determine microbial constituents are of significance. The present review article was conducted using internet databases and related scientific literatures and articles that provide information on developments in the rapid methods in microbiology. The main focus is on the application of rapid methods in microbial quality control of pharmaceutical products. Reviewed literature showed that rapid methods and automation in microbiology is an advanced area for studying and applying of improved methods in the early detection, and characterization of microorganisms and their products in food, pharmaceutical and cosmetic industrials as well as environmental monitoring and clinical applications. It can be concluded that rapid methods and automation in microbiology should continue as potent and efficient technologies to develop the novel tests to be performed in the future because of the ever-increasing concerns about the safety of food and pharmaceutical products. However the main issues to be considered are the scale up of developed methods and the regulatory requirements.

Molecular and antimicrobial susceptibility profiling of atypical Streptococcus species from porcine clinical specimens

The Streptococcus species present broad phenotypic variation, making identification difficult using only traditional microbiological methods. Even though Streptococcus suis is the most important species for the worldwide swine industry, other Streptococcus species appear to be able to cause disease in swine and could represent a higher underestimated risk for porcine health. The aim of this study was to identify Streptococcus-like isolates by MALDI-TOF MS and 16S rRNA sequencing and further molecular and antibiotic susceptibility characterization of the atypical Streptococcus species capable of causing disease in swine. Fifty presumptive Streptococcus isolates from diseased pigs isolated from different Brazilian States between 2002 and 2014 were evaluated. Among the studied isolates, 26% were identified as Streptococcus hyovaginalis, 24% as Streptococcus plurianimalium, 12% as Streptococcus alactolyticus, 10% as Streptococcus hyointestinalis, and the remaining isolates belonged to Streptococcus henryi (6%), Streptococcus thoraltensis (6%), Streptococcus gallolyticus (6%), Streptococcus gallinaceus (4%), Streptococcus sanguinis (4%), and Streptococcus mitis (2%). The Streptococcus isolates were successfully identified by spectral cluster analysis and 16S rRNA sequencing with 96% of concordance between the techniques. The SE-AFLP analysis also supported Streptococcus species distinction and enabled further observation of higher genetic heterogeneity intra-species. The identified Streptococcus species presented variable MIC values to β-lactams, enrofloxacin and florfenicol, and high resistance rates to tetracyclines and macrolides, which appear to be directly related to the industry's antimicrobial usage and resistance selection.

Microbial succession in an inflated lunar/Mars analog habitat during a 30-day human occupation.

BackgroundFor potential future human missions to the Moon or Mars and sustained presence in the International Space Station, a safe enclosed habitat environment for astronauts is required. Potential microbial contamination of closed habitats presents a risk for crewmembers due to reduced human immune response during long-term confinement. To make future habitat designs safer for crewmembers, lessons learned from characterizing analogous habitats is very critical. One of the key issues is that how human presence influences the accumulation of microorganisms in the closed habitat.ResultsMolecular technologies, along with traditional microbiological methods, were utilized to catalog microbial succession during a 30-day human occupation of a simulated inflatable lunar/Mars habitat. Surface samples were collected at different time points to capture the complete spectrum of viable and potential opportunistic pathogenic bacterial population. Traditional cultivation, propidium monoazide (PMA)–quantitative polymerase chain reaction (qPCR), and adenosine triphosphate (ATP) assays were employed to estimate the cultivable, viable, and metabolically active microbial population, respectively. Next-generation sequencing was used to elucidate the microbial dynamics and community profiles at different locations of the habitat during varying time points. Statistical analyses confirm that occupation time has a strong influence on bacterial community profiles. The Day 0 samples (before human occupation) have a very different microbial diversity compared to the later three time points. Members of Proteobacteria (esp. Oxalobacteraceae and Caulobacteraceae) and Firmicutes (esp. Bacillaceae) were most abundant before human occupation (Day 0), while other members of Firmicutes (Clostridiales) and Actinobacteria (esp. Corynebacteriaceae) were abundant during the 30-day occupation. Treatment of samples with PMA (a DNA-intercalating dye for selective detection of viable microbial population) had a significant effect on the microbial diversity compared to non-PMA-treated samples.ConclusionsStatistical analyses revealed a significant difference in community structure of samples over time, particularly of the bacteriomes existing before human occupation of the habitat (Day 0 sampling) and after occupation (Day 13, Day 20, and Day 30 samplings). Actinobacteria (mainly Corynebacteriaceae) and Firmicutes (mainly Clostridiales Incertae Sedis XI and Staphylococcaceae) were shown to increase over the occupation time period. The results of this study revealed a strong relationship between human presence and succession of microbial diversity in a closed habitat. Consequently, it is necessary to develop methods and tools for effective maintenance of a closed system to enable safe human habitation in enclosed environments on Earth and beyond.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0167-0) contains supplementary material, which is available to authorized users.

Rapid Identification and Characterization of Microbial Pathogens by Molecular Biology Techniques.

Microbial infections including bacterial and viral infections are potentially life-threatening and require rapid identification and characterization of the microorganisms. Rapid and accurate detection of foodborne microorganisms from food, environment and clinical samples requires characterization to the species and/or subspecies level as well as determining their antibiotic susceptibility. These steps are important for: 1) selecting appropriate antibiotics, 2) reducing morbidity and mortality, and 3) reducing health care costs. Traditional microbiological detection starts with an enrichment step usually using a nonselective enrichment that allows injured cells to repair and multiply. These enrichment cultures are then subcultured on selective and differential agars. Typical and suspected colonies are then stained by Gram staining and subsequent biochemical identification, and susceptibility testing are performed. These steps usually take 3-7 days. Identification of virulence factors requires additional time and involves expensive phenotypic and genotypic tests. Traditional microbiological identification methods often do not provide prompt results for optimal early patient diagnosis and treatment management. In recent years, more and more studies have demonstrated the value of molecular biologic techniques to rapidly identify and genotype bacteria or virus in food, environment, and clinical samples. Molecular biology techniques are methods which generally involve the analysis and handling of nucleic acid and other molecules. Compared to traditional microbial identification methods, molecular biology methods can be used to detect and identify microorganisms simultaneously, which results in quicker diagnoses and treatments. The other advantages of molecular methods are that the protocols are of higher sensitivity and specificity. The aim of this hot topic is to provide an overview of current exciting molecular biology techniques that are employed to rapidly identify and characterize important pathogenic bacteria and viruses. The topic would update knowledge on prompt bacterial and viral identification and characterization methods, which can speed up the diagnosis and treatment of infectious diseases, improve epidemiologic screening, and reduce their morbidity and mortality. Staphylococcus aureus causes a wide range of suppurative infections, food poisoning and toxic shock syndrome. Methicillin resistant S. aureus (MRSA) is resistant to most β-lactam antibiotics. MRSA infection is more difficult to treat with commonly used antibiotics and thus more dangerous. A recent study showed that about 47% of the meat and poultry in U.S. grocery stores were contaminated with S. aureus with about half of those bacteria being resistant to multiple antibiotics [1]. It is important to precisely and timely detect and identify the organism for epidemical and clinical investigation. Liu et al. outline different PCR-based approaches for MRSA detection in detailed and particular ways. Bacteroides fragilis is an anaerobic opportunistic pathogen, which cause bacteremia, abscess formation, diarrhea, and inflammatory bowel disease [2]. B. fragilis is the most commonly isolated bacteria in anaerobic infections, especially infections involving gastrointestinal flora. Next generation sequencing and analysis tools can be employed to detect quickly B. fragilis. The isolation, detection and characterization methods of Enterotoxigenic B. fragilis are clearly outlined by Drs. Fathi and Wu. Francisella tularensis is a small, nonmobile, Gram-negative bacterium. It is highly infectious, causing life-threatening tularemia in animals and humans [3]. It is considered by Center for Disease Control and Prevention to be a category A agent [4]. F. tularensis grows slowly, so it requests molecular biology assays to detect rapidly. A review article written by Lai et al. detailed summarize the up-to-date developments of these assays. Noroviruses are now recognized as the most common cause of nonbacterial acute gastroenteritis and foodborne viral illness worldwide. Human noroviruses account for over 267 million infections worldwide annually [5]. Noroviruses are extremely infectious; a few viral particles can initiate an infection. A major impediment of the identification and diagnosis of norovirus infection is the lack of an effective cell culture system or small animal models. Molecular techniques have been developed to detect the viruses. The molecular identified and diagnostic methods of noroviruses are meticulously outlined by Drs. Chen and Hu. The value of molecular biology technique has been demonstrated in identifying and characterizing bacteria and viruses in food, environmental and clinical specimens, and in hastening diagnostic and epidemiological screening procedures, which circumvent the problems of traditional culture methods. Thus, these review articles will updated knowledge on microorganisms, and provide useful and practicable guides for rapid identification and characterization of the bacteria and viruses.

Validation Study of Rapid Assays of Bioburden, Endotoxins and Other Contamination.

Microbial testing performed in support of pharmaceutical and biopharmaceutical production falls into three main categories: detection (qualitative), enumeration (quantitative), and characterization/identification. Traditional microbiological methods are listed in the compendia and discussed by using the conventional growth-based techniques, which are labor intensive and time consuming. In general, such tests require several days of incubation for microbial contamination (bioburden) to be detected, and therefore management seldom is able to take proactive corrective measures. In addition, microbial growth is limited by the growth medium used and incubation conditions, thus impacting testing sensitivity, accuracy, and reproducibility. For more than 20 years various technology platforms for rapid microbiological methods (RMM) have been developed, and many have been readily adopted by the food industry and clinical microbiology laboratories. Their use would certainly offer drug companies faster test turnaround times to accommodate the aggressive deadlines for manufacturing processes and product release. Some rapid methods also offer the possibility for real-time microbial analyses, enabling management to respond to microbial contamination events in a more timely fashion, and can provide cost savings and higher efficiencies in quality control testing laboratories. Despite the many proven business and quality benefits and the fact that the FDA's initiative to promote the use of process analytical technology (PAT) includes rapid microbial methods, pharmaceutical and biopharmaceutical industries have been somewhat slow to embrace alternative microbial methodologies for several reasons. The major reason is that the bioburden counts detected by the incubation method and rapid assay are greatly divergent. The use of rapid methods is a dynamic field in applied microbiology and one that has gained increased attention nationally and internationally over time. This topic has been extensively addressed at conferences and in published documents around the world. More recently, the use of alternative methods for control of the microbiological quality of pharmaceutical products and materials used in pharmaceutical production has been addressed by the compendia in an attempt to facilitate implementation of these technologies by pharmaceutical companies. The author presents some of the rapid method technologies under evaluation or in use by pharmaceutical microbiologists and the current status of the implementation of alternative microbial methods.

Microbiological characterization and effect of resin composites in cervical lesions.

BackgroundNon carious cervical lesions associated to muscle hyperfunctions are increasing. Microhybrid resin composites are used to restore cervical abfractions. The purpose of this study was to investigate if resin composites modify tooth plaque, inducing an increment of cariogenic microflora and evaluate their effect, in vivo and in vitro, against S. mutans.Material and MethodsEight abfractions were restored with two microhybrid resin composites (Venus, Heraeus-Kulzer® and Esthet-X, Dentsply®), after gnatological therapy, in three patients with muscle hyperfunctions. For each abfraction three samples of plaque were taken from the cervical perimeter: before the restoration, one week and three months after restoration. The samples were evaluated both by traditional microbiological methods and by Polymerase Chain Reaction (PCR). In vitro, disk-shaped specimens of the two composites were prepared to estimate the effects against pre-cultured S. mutans, after incubation at 37°C for 24h and assessed by a turbidimetric technique.ResultsIn vivo no differences were found in plaque growth, for all samples, before and after restoration with both composites; in vitro, instead, a significant reduction of S. mutans growth was found between specimens of two composites (Mann-Whitney U-test p>0,06).ConclusionsIn this study a relevant consideration was elicited: composite materials, in vivo, do not modify plaque composition of non carious cervical lesions to a potential cariogenic plaque. Key words:Abfraction, restoration, S. mutans, composite, class V.

Capillary zone electrophoresis as alternative tool for rapid identification and quantification of viable Saccharomyces cerevisiae cells

Abstract The aim of this work was to evaluate the possibility of using capillary electrophoresis for rapid and sensitive detection of microorganisms, such as yeasts, in order to propose it as an alternative to traditional microbiology methods both in manufacturing and research areas. After optimization of condition for the electrophoretic separation, three Saccharomyces cerevisiae strains, cultured in YPD medium, were analyzed by capillary electrophoresis at different growth times (3, 7, 24 and 48 h). The analysis was carried out on undamaged and non-pretreated cells. For all the strains, the electrophoretic profile was characterised by two distinct peaks, at each time of growth. Nevertheless some differences in the electropherograms of each strain were evident, probably in consequence of changing in the surface charge of yeasts at various stage of growth. The results demonstrated the high efficacy of capillary electrophoresis for intact cells separation of yeast and support the application of this technique for the evaluation of the viability of cultured cells.

Next generation genome sequencing reveals phylogenetic clades with different level of virulence among Salmonella Typhimurium clinical human isolates in Hong Kong.

BackgroundSalmonella Typhimurium is frequently isolated from foodborne infection cases in Hong Kong, but the lack of genome sequences has hindered in-depth epidemiological and phylogenetic studies. In this study, we sought to reconstruct the phylogenetic relationship and investigate the distribution and mutation patterns of virulence determinants among local S. Typhimurium clinical isolates using their genome sequences.ResultsWe obtained genome sequences of 20 S. Typhimurium clinical isolates from a local hospital cluster using a 454 GS FLX Titanium sequencing platform. Phylogenetic analysis was performed based on single nucleotide polymorphism positions of the core genome against the reference strain LT2. Antimicrobial susceptibility was determined using minimal inhibitory concentration for five antimicrobial agents and analyses of virulence determinants were performed through referencing to various databases. Through phylogenetic analysis, we revealed two distinct clades of S. Typhimurium isolates and three outliers in Hong Kong, which differ remarkably in antimicrobial susceptibility and presentation and mutations of virulence determinants. The local isolates were not closely related to many of the previously sequenced S. Typhimurium isolates, except LT2. As the isolates in the two clades spanned over 10 years of isolation, they probably represent endemic strains. The outliers are possibly introduced from outside of Hong Kong. The close relatedness of members in one of the clades to LT2 and the Japanese stool isolate T000240 suggests the potential reemergence of LT2 progeny in regions nearby.ConclusionsOur study demonstrated the utility of next-generation sequencing coupled to traditional microbiological testing method in a retrospective epidemiological study involving multiple clinical isolates. The evolution of multidrug- and ciprofloxacin-resistant strains among the more virulent clade is also an increasing concern.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1900-y) contains supplementary material, which is available to authorized users.

Characterization of Dickeya and Pectobacterium species by capillary electrophoretic techniques and MALDI-TOF MS.

Dickeya and Pectobacterium species represent an important group of broad-host-range phytopathogens responsible for blackleg and soft rot diseases on numerous plants including many economically important plants. Although these species are commonly detected using cultural, serological, and molecular methods, these methods are sometimes insufficient to classify the bacteria correctly. On that account, this study was undertaken to investigate the feasibility of three individual analytical techniques, capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), for reliable classification of Dickeya and Pectobacterium species. Forty-three strains, representing different Dickeya and Pectobacterium species, namely Dickeya dianthicola, Dickeya dadantii, Dickeya dieffenbachiae, Dickeya chrysanthemi, Dickeya zeae, Dickeya paradisiaca, Dickeya solani, Pectobacterium carotovorum, and Pectobacterium atrosepticum, were selected for this purpose. Furthermore, the selected bacteria included one strain which could not be classified using traditional microbiological methods. Characterization of the bacteria was based on different pI values (CIEF), migration velocities (CZE), or specific mass fingerprints (MALDI-TOF MS) of intact cells. All the examined strains, including the undetermined bacterium, were characterized and classified correctly into respective species. MALDI-TOF MS provided the most reliable results in this respect.